Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications [1st ed.] 9783030487973, 9783030487980

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Food Bioactive Ingredients

Mohamed Fawzy Ramadan Editor

Black Cumin (Nigella sativa) Seeds: Chemistry, Technology, Functionality, and Applications

Food Bioactive Ingredients Series Editor Seid Mahdi Jafari Food Materials Process Design Gorgan University Gorgan, Iran

The Food Bioactive Ingredients Series covers recent advances and research on the science, properties, functions, technology, engineering and applications of food bioactive ingredients and their relevant products. The series also covers health-­ related aspects of these bioactive components, which have been shown to play a critical role in preventing or delaying different diseases and to have many health-­ improving properties. The books in this series target professional scientists, academics, researchers, students, industry professionals, governmental organizations, producing industries and all experts performing research on functional food development, pharmaceuticals, cosmetics and agricultural crops. More information about this series at http://www.springer.com/series/16307

Mohamed Fawzy Ramadan Editor

Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications

Editor Mohamed Fawzy Ramadan Deanship of Scientific Research Umm Al-Qura University Makkah, KSA Agricultural Biochemistry Department Faculty of Agriculture Zagazig University Zagazig, Egypt

ISSN 2661-8958     ISSN 2661-8966 (electronic) Food Bioactive Ingredients ISBN 978-3-030-48797-3    ISBN 978-3-030-48798-0 (eBook) https://doi.org/10.1007/978-3-030-48798-0 © Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Dedicated to my beloved family

Preface

Nigella sativa (black cumin) is one of the most admired medicinal plants in history. Recently, black cumin has become an important topic for scientific research worldwide. Nigella sativa seeds are rich in bioactive phytochemicals (i.e., thymoquinone, tocols, sterols, polar lipids, and amino acids) with diverse biological and health-­ promoting traits. Extracts, essential oils, and fixed oils from Nigella sativa seeds have been used in pharmaceuticals, functional foods, and nutraceuticals. Nigella sativa is evident to promote health and it might serve to be a novel source for modern phytomedicine. This book project aims to build a multidisciplinary discussion on the advances in Nigella sativa chemistry, technology, cultivation practices, functional properties, health-promoting activities, as well as food and non-food applications. Upon kind invitation from the Springer Nature, this book was edited. The book contains chapters that describe cultivation, composition, and applications of N. sativa seeds as well as the chemistry, technology, functionality, and applications of its extracts, fixed oil, and essential oil. Aiming to provide a major reference work for those involved in pharmaceuticals, nutraceuticals, and oil industry as well as undergraduate and graduate students, this volume presents a comprehensive review of the results that have led to the advancements in Nigella sativa chemistry, technology, and applications. I hope this book will be a valuable source for people involved in medicinal plants and functional foods. I sincerely thank all authors for their valuable contributions and for their cooperation during book preparation. I highly acknowledge the support from Deanship of Scientific Research (Umm Al-Qura University, KSA). The help and support given to me by the Springer Nature staff, especially Daniel Falatko and Arjun Narayanan, was essential for the completion of my task and is appreciated. “Let food be your medicine and medicine be your food” (Hippocrates)

Makkah, Saudi Arabia

Mohamed Fawzy Ramadan

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Description

Nigella sativa seeds have an increasing number of applications in food and pharmaceutical industries. Black cumin is used worldwide in traditional medicine for treatment of several diseases. Bioactive phytochemicals with pharmacological properties have been identified in black cumin, including thymoquinone, t-anethol, alkaloids, and saponins. Black Cumin (Nigella sativa) Seeds: Chemistry, Technology, Functionality, and Applications covers several specific topics with a focus on cultivation, composition, and applications of Nigella sativa seeds as well as the chemistry, technology, functionality, and applications of Nigella sativa extracts, fixed oil, and essential oil. Edited by a team of experts, Black Cumin (Nigella sativa) Seeds: Chemistry, Technology, Functionality, and Applications brings together diverse developments in food science to chemists, nutritionists, and students of food science, nutrition, lipids chemistry and technology, agricultural science, pharmaceuticals, cosmetics, and nutraceuticals. Black Cumin (Nigella sativa) Seeds: Chemistry, Technology, Functionality, and Applications is a key textbook for pharmaceutical and functional food developers as well as research and development (R&D) managers working in all sector using medicinal plants and vegetable oils. It is a useful reference work for companies reformulating their products or developing new products. Key Features • Broad coverage encompasses chemistry, technology, functionality, and applications of Nigella sativa • Authored by international academics and industry experts • Addresses growing application areas including pharmaceuticals, functional foods, nutraceuticals, and cosmetics

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Description

Readership –– Academics and students with a research interest in the area (pharmacologists, food chemists, lipid scientists, food scientists, and agronomists) –– Pharmaceutics, functional food developers, and R&D managers working in all sectors using medicinal plants and specialty oils

Contents

1 Introduction to Black Cumin (Nigella sativa): Chemistry, Technology, Functionality and Applications������������������������������������������    1 Mohamed Fawzy Ramadan Part I  Nigella sativa Seeds: Cultivation, Composition and Applications 2 Effect of Cultivation, Ferfztilization and Irrigation Practices on Nigella sativa Yield and Quality��������������������������������������������������������   11 Enas Mohamed Wagdi Abdel-Hamed 3 Morphological Characters of Nigella sativa������������������������������������������   23 Mohamed Helmy El-Morsy and Hanan El-Sayed Osman 4 Micro and Macroscopic Characterization of Traded Nigella sativa Seeds Using Applied Systematics Techniques����������������   31 Sofia Rashid, Muhammad Zafar, Mushtaq Ahmad, Shazia Sultana, Sidra Nisar Ahmed, and Omer Kilic 5 Composition of Nigella sativa Seeds ������������������������������������������������������   45 Hamid Mukhtar, Muhammad Waseem Mumtaz, Tooba Tauqeer, and Syed Ali Raza 6 Nigella sativa Seed Peptides (Thionins)��������������������������������������������������   59 Ali Osman 7 Black Cumin Polysaccharides����������������������������������������������������������������   67 Ines Trigui, Salma Cheikh-Rouhou, Hamadi Attia, and Mohamed Ali Ayadi 8 Thymoquinone: Chemistry and Functionality��������������������������������������   81 Amr E. Edris

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9 Novel Prospective of N. sativa Essential Oil Analysis, Culinary and Medicinal Uses����������������������������������������������������������������������������������   97 Doaa M. Abo-Atya, Mohamed F. El-Mallah, Hesham R. El-Seedi, and Mohamed A. Farag 10 Rediscovering Nigella Seeds Bioactives Chemical Composition Using Metabolomics Technologies����������������������������������������������������������  131 Mohamed A. Farag, Hamada H. Saad, and Nesrine M. Hegazi 11 Health Promoting Activities of Nigella sativa Seeds������������������������������  153 Ebru Aydin and Arzu Kart 12 Nigella sativa Seed Extract in Green Synthesis and Nanocomposite����������������������������������������������������������������������������������  179 Raya Soltane, Dalila Mtat, Amani Chrouda, Noof Alzahrani, Youssef O. Al-Ghamdi, Hussam El-Desouky, and Khaled Elbanna 13 Food Applications of Nigella sativa Seeds����������������������������������������������  191 Omar Bashir, Nusrat Jan, Gousia Gani, H. R. Naik, Syed Zameer Hussain, Monika Reshi, and Tawheed Amin 14 Nutraceutical Importance and Applications of Nigella sativa Seed Flour ������������������������������������������������������������������������������������������������  209 Jasmeet Kour, Adil Gani, Vishal Sharma, and Sajad Ahmad Sofi 15 Nigella sativa Seed Cake: Nutraceutical Significance and Applications in the Food and Cosmetic Industry��������������������������  223 Jasmeet Kour and Adil Gani 16 Nigella sativa Seeds in Cosmetic Products: Shedding the Light on the Cosmeceutical Potential of Nigella sativa and its Utilization as a Natural Beauty Care Ingredient ����������������������������������������������������  231 Fadia S. Youssef 17 Nigella sativa Supplementation in Ruminant Diets: Production, Health, and Environmental Perspectives��������������������������  245 Yasmina M. Abd El-Hakim, Adham A. Al-Sagheer, Asmaa F. Khafaga, Gaber E. Batiha, Muhammad Arif, and Mohamed E. Abd El-Hack 18 Nigella sativa Seeds and Its Derivatives in Poultry Feed����������������������  265 Mohamed E. Abd El-Hack, Abdel-Moneim E. Abdel-Moneim, Noura M. Mesalam, Khalid M. Mahrose, Asmaa F. Khafaga, Ayman E. Taha, and Ayman A. Swelum 19 Nigella sativa Seeds and Its Derivatives in Fish Feed����������������������������  297 Mohamed E. Abd El-Hack, Sameh A. Abdelnour, Asmaa F. Khafaga, Ayman E. Taha, and Hany M. R. Abdel-Latif

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Part II  Nigella sativa Fixed Oil: Chemistry, Technology, Functionality and Applications 20 Composition and Functionality of Nigella sativa Fixed Oil������������������  319 Mustafa Kiralan, Sündüz Sezer Kiralan, Gulcan Ozkan, and Mohamed Fawzy Ramadan 21 Effect of Processing on the Composition and Quality of Nigella sativa Fixed Oil������������������������������������������������������������������������  335 Yeganeh Mazaheri, Mohammadali Torbati, and Sodeif Azadmard-Damirchi 22 Food Applications of Nigella sativa Fixed Oil����������������������������������������  349 Mustafa Kiralan, Sündüz Sezer Kiralan, Gulcan Ozkan, and Mohamed Fawzy Ramadan 23 Health-Promoting Activities of Nigella sativa Fixed Oil ����������������������  361 Mahmoud Balbaa, Marwa El-Zeftawy, Shaymaa A. Abdulmalek, and Yasmin R. Shahin 24 Micro- and Nano-encapsulation of Nigella sativa Oil ��������������������������  381 Amr E. Edris 25 Biodiesel Production Potential of Nigella sativa Oil������������������������������  389 Muhammad Sajjad Iqbal Part III  Nigella sativa Essential Oil: Chemistry, Technology, Functionality and Applications 26 Composition and Functionality of Nigella sativa Essential Oil������������  409 Mehmet Aksu, Gulcan Ozkan, Sündüz Sezer Kiralan, Mustafa Kiralan, and Mohamed Fawzy Ramadan 27 Effect of Processing on the Composition and Quality of Nigella sativa Essential Oil������������������������������������������������������������������  421 Faten M. Ibrahim and S. F. El Habbasha 28 Food Applications of Nigella sativa Essential Oil����������������������������������  433 Wei Liao, Waisudin Badri, Amani H. Alhibshi, Emilie Dumas, Sami Ghnimi, Adem Gharsallaoui, Abdelhamid Errachid, and Abdelhamid Elaissari 29 Health-Promoting Activities of Nigella sativa Essential Oil ����������������  457 Mahmoud Alagawany, Shabaan S. Elnesr, Mayada R. Farag, Mohamed E. Abd El-Hack, Asmaa F. Khafaga, Khan Sharun, Gopi Marappan, and Kuldeep Dhama

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Contents

Part IV  Nigella sativa Seed Extracts: Chemistry, Technology, Functionality and Applications 30 Composition and Functionality of Nigella sativa Seed Extracts����������  481 Songul Kesen 31 Nigella sativa Seed Extracts in Functional Foods and Nutraceutical Applications��������������������������������������������������������������  501 Ranga Rao Ambati and Mohamed Fawzy Ramadan 32 Health Promoting Activities of Nigella sativa Seed Extracts����������������  521 Arzu Kart and Ebru Aydın Index������������������������������������������������������������������������������������������������������������������  539

Contributors

Mohamed  E.  Abd  El-Hack  Department of Poultry, Faculty of Agriculture, Zagazig University, Zagazig, Egypt Yasmina M. Abd El-Hakim  Department of Forensic Medicine and Toxicology, Zagazig University, Zagazig, Egypt Enas  Mohamed  Wagdi  Abdel-Hamed  Soil Science Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt Hany  M.  R.  Abdel-Latif  Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Alexandria University, Edfina, Behera, Egypt Abdel-Moneim  E.  Abdel-Moneim  Biological Application Department, Nuclear Research Center, Atomic Energy Authority, Abu-Zaabal, Egypt Sameh A. Abdelnour  Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig, Egypt Shaymaa  A.  Abdulmalek  Biochemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt Doaa  M.  Abo-Atya  Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Koom, Egypt Mushtaq  Ahmad  Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan Sidra  Nisar  Ahmed  Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan Department of Botany, The Women University Multan, Multan, Pakistan Mehmet  Aksu  Isparta Provincial Coordination Unit, Agriculture and Rural Development Support Institution, Isparta, Turkey Mahmoud  Alagawany  Department of Poultry, Faculty of Agriculture, Zagazig University, Zagazig, Egypt xv

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Contributors

Youssef O. Al-Ghamdi  Department of Chemistry, College of Al-Zulfi, Majmaah University, Al-Majmaah, Saudi Arabia Amani H. Alhibshi  Department of Neuroscience Research, Institute of Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia Adham A. Al-Sagheer  Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig, Egypt Noof Alzahrani  Department of Basic Sciences, Adham University College, Umm Al-Qura University, Adham, Saudi Arabia Ranga  Rao  Ambati  Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research University (VFSTRU) (Deemed to Be University), Guntur, Andhra Pradesh, India Tawheed  Amin  Division of Food Science and Technology, SKUAST-Kashmir, Srinagar, Jammu and Kashmir, India Muhammad  Arif  Department of Animal Sciences, College of Agriculture, University of Sargodha, Sargodha, Pakistan Hamadi  Attia  Valuation, Security, and Food Analysis Laboratory, National Engineering School of Sfax, Sfax, Tunisia Mohamed Ali Ayadi  Valuation, Security, and Food Analysis Laboratory, National Engineering School of Sfax, Sfax, Tunisia Ebru Aydin  Faculty of Engineering, Department of Food Engineering, Suleyman Demirel University, Isparta, Turkey Sodeif  Azadmard-Damirchi  Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran Waisudin Badri  Univ Lyon, University Claude Bernard Lyon-1, CNRS, LAGEP-­ UMR 5007, Lyon, France Mahmoud  Balbaa  Biochemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt Omar  Bashir  Division of Food Science and Technology, SKUAST-Kashmir, Srinagar, Jammu and Kashmir, India Gaber  E.  Batiha  National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan Department of Pharmacology and Therapeutics, Faculty of Veterinary Medicine, Damanhour University, Damanhour, AlBeheira, Egypt Salma  Cheikh-Rouhou  Valuation, Security, and Food Analysis Laboratory, National Engineering School of Sfax, Sfax, Tunisia

Contributors

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Amani  Chrouda  Department of Chemistry, College of Al-Zulfi, Majmaah University, Al-Majmaah, Saudi Arabia Laboratory of Interfaces and Advanced Materials, Faculty of Sciences, University of Monastir, Monastir, Tunisia Laboratory of Analytical Sciences UMR CNRS-UCBL-ENS 5280, Villeurbanne Cedex, France Kuldeep  Dhama  Division of Pathology, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India Emilie Dumas  Univ Lyon, University Claude Bernard Lyon-1, CNRS, LAGEP-­ UMR 5007, Lyon, France Amr E. Edris  Aroma & Flavor Chemistry Department, Food Industries & Nutrition Division, National Research Center, Cairo, Egypt Abdelhamid  Elaissari  Univ Lyon, University Claude Bernard Lyon-1, CNRS, LAGEP-UMR 5007, Lyon, France Khaled Elbanna  Department of Agricultural Microbiology, Faculty of Agriculture, Fayoum University, Fayoum, Egypt Department of Biology, Faculty of Applied Science, Umm Al-Qura University, Makkah, Saudi Arabia Hussam  El-Desouky  Chemistry Department, Faculty of Science, Helwan University, Helwan, Egypt Chemistry Department, Jamoum University College, Umm Al-Qura University, Makkah, Saudi Arabia S. F. El Habbasha  Field Crops Research Department, National Research Centre, Dokki, Giza, Egypt Mohamed F. El-Mallah  Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Koom, Egypt Mohamed  Helmy  El-Morsy  Deanship of Scientific Research, Umm Al-Qura University, Makkah, Saudi Arabia Range Management Unit, Desert Research Center, Cairo, Egypt Shabaan  S.  Elnesr  Department of Poultry Production, Faculty of Agriculture, Fayoum University, Fayoum, Egypt Hesham  R.  El-Seedi  Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Koom, Egypt Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden Marwa  El-Zeftawy  Biochemistry Department, Faculty of Veterinary Medicine, The New Valley University, New Valley, Egypt

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Contributors

Abdelhamid  Errachid  Univ Lyon, University Claude Bernard Lyon-1, CNRS, ISA- UMR 5280, CNRS, Villeurbanne, France Mayada  R.  Farag  Forensic Medicine and Toxicology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt Mohamed  A.  Farag  Pharmacognosy Department, College of Pharmacy, Cairo University, Cairo, Egypt Department of Chemistry, School of Sciences & Engineering, The American University in Cairo, New Cairo, Egypt Adil Gani  Department of Food Science and Technology, University of Kashmir, Srinagar, Jammu and Kashmir, India Gousia  Gani  Division of Food Science and Technology, SKUAST-Kashmir, Srinagar, Jammu and Kashmir, India Adem  Gharsallaoui  Univ Lyon, University Claude Bernard Lyon-1, CNRS, LAGEP-UMR 5007, Lyon, France Sami  Ghnimi  Univ Lyon, University Claude Bernard Lyon-1, CNRS, LAGEP-­ UMR 5007, Lyon, France Nesrine  M.  Hegazi  Department of Pharmaceutical Biology, Pharmaceutical Institute, Eberhard Karls University of Tübingen, Tübingen, Germany Syed  Zameer  Hussain  Division of Food Science and Technology, SKUAST-­ Kashmir, Srinagar, Jammu and Kashmir, India Faten M. Ibrahim  Medicinal and Aromatic Plants Research Department, National Research Centre, Dokki, Giza, Egypt Muhammad  Sajjad  Iqbal  Department of Botany, University of Gujrat, Gujrat, Pakistan Nusrat  Jan  Division of Food Science and Technology, SKUAST-Kashmir, Srinagar, Jammu and Kashmir, India Arzu Kart  Faculty of Engineering, Department of Food Engineering, Suleyman Demirel University, Isparta, Turkey Songul  Kesen  Naci Topcuoglu Vocational High School, Gaziantep University, Gaziantep, Turkey Asmaa  F.  Khafaga  Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, Edfina, Egypt Omer  Kilic  Department of Basic Science of Pharmacy, Adıyaman University, Adıyaman, Turkey Mustafa  Kiralan  Faculty of Engineering, Department of Food Engineering, Balıkesir University, Balikesir, Turkey

Contributors

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Sündüz Sezer Kiralan  Faculty of Engineering, Department of Food Engineering, Balıkesir University, Balikesir, Turkey Jasmeet Kour  Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Longowal, Punjab, India Wei Liao  Univ Lyon, University Claude Bernard Lyon-1, CNRS, LAGEP-UMR 5007, Lyon, France Khalid  M.  Mahrose  Animal and Poultry Production Department, Faculty of Technology and Development, Zagazig University, Zagazig, Egypt Gopi Marappan  Division of Avian Physiology and Reproduction, ICAR-Central Avian Research Institute, Bareilly, Uttar Pradesh, India Yeganeh  Mazaheri  Department of Food Science and Technology, Faculty of Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran Noura M. Mesalam  Biological Application Department, Nuclear Research Center, Atomic Energy Authority, Abu-Zaabal, Egypt Dalila  Mtat  Laboratory of Asymmetric Organic Synthesis and Homogeneous Catalysis (UR11ES56), Faculty of Sciences of Monastir Avenue of the Environment, Monastir, Tunisia Hamid  Mukhtar  Institute of Industrial Biotechnology, Government College University, Lahore, Pakistan Muhammad Waseem Mumtaz  Department of Chemistry, University of Gujrat, Gujrat, Pakistan H. R. Naik  Division of Food Science and Technology, SKUAST-Kashmir, Srinagar, Jammu and Kashmir, India Ali Osman  Agricultural Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt Hanan El-Sayed Osman  Biology Department, Faculty of Applied Science, Umm Al-Qura University, Makkah, Saudi Arabia Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt Gulcan  Ozkan  Faculty of Engineering, Department of Food Engineering, Suleyman Demirel University, Isparta, Turkey Mohamed  Fawzy  Ramadan  Deanship of Scientific Research, Umm Al-Qura University, Makkah, KSA Agricultural Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt Sofia Rashid  Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan Department of Bio Sciences, Comsats University Islamabad, Islamabad, Pakistan

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Contributors

Syed Ali Raza  Department of Chemistry, Government College University, Lahore, Pakistan Monika  Reshi  Division of Food Science and Technology, SKUAST-Kashmir, Srinagar, Jammu and Kashmir, India Hamada H. Saad  Phytochemistry and Plant Systematics Department, Division of Pharmaceutical Industries, National Research Centre, Cairo, Egypt Department of Pharmaceutical Biology, Pharmaceutical Institute, Eberhard Karls University of Tübingen, Tübingen, Germany Yasmin  R.  Shahina  Biochemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt Vishal  Sharma  Department of Industries and Commerce, District Industries Centre, Kathua, Jammu and Kashmir, India Khan  Sharun  Division of Surgery, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India Sajad  Ahmad  Sofi  Division of Food Science and Technology, Sher-e-Kashmir University of Agricultural Science & Technology, Srinagar, Jammu and Kashmir, India Raya  Soltane  Department of Basic Sciences, Adham University College, Umm Al-Qura University, Adham, Saudi Arabia Department of Biology, Faculty of Sciences, Tunis El Manar University, El Manar, Tunis, Tunisia Shazia  Sultana  Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan Ayman  A.  Swelum  Department of Theriogenology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt Ayman  E.  Taha  Department of Animal Husbandry and Animal Wealth Development, Faculty of Veterinary Medicine, Alexandria University, Edfina, Egypt Tooba Tauqeer  Department of Chemistry, University of Gujrat, Gujrat, Pakistan Mohammadali Torbati  Department of Food Science and Technology, Faculty of Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran Ines  Trigui  Valuation, Security, and Food Analysis Laboratory, National Engineering School of Sfax, Sfax, Tunisia Fadia S. Youssef  Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt Muhammad  Zafar  Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan

About the Editor

Mohamed  Fawzy  Ramadan  is a Professor in the Department of Agricultural Biochemistry, Faculty of Agriculture, at Zagazig University, Zagazig, Egypt. Since 2013, Prof. Ramadan has also been a Professor of Biochemistry and Consultant of International Publishing at the Deanship of Scientific Research, Umm Al-Qura University, Makkah, Saudi Arabia. Prof. Ramadan obtained his Ph.D. (Dr.rer.nat.) in Food Chemistry from Berlin University of Technology (Germany, 2004). He continued his postdoctoral research at ranked universities in different countries, such as the University of Helsinki (Finland), MaxRubner Institute (Germany), Berlin University of Technology (Germany), and the University of Maryland (USA). In 2010, he was appointed as Visiting Professor (100% research) at King Saud University in Saudi Arabia. In 2012, Prof. Ramadan was appointed as Visiting Professor (100% teaching) in the School of Biomedicine at Far Eastern Federal University in Vladivostok, Russian Federation. Prof. Ramadan has published more than 250 research papers and reviews in international peerreviewed journals as well as several books and book chapters (Scopus h-index is 40 and more than 4300 citations). He was an invited speaker at several international conferences. Since 2003, Prof. Ramadan has been a reviewer and editor of several highly cited international journals such as Journal of Medicinal Food and Journal of Advanced Research. xxi

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About the Editor

Prof. Ramadan received Abdul Hamid Shoman Prize for Arab Researcher in Agricultural Sciences (2006), Egyptian State Prize for Encouragement in Agricultural Sciences (2009), European Young Lipid Scientist Award (2009), AU-TWAS Young Scientist National Awards (Egypt) in Basic Sciences, Technology and Innovation (2012), TWAS-ARO Young Arab Scientist (YAS) Prize in Scientific and Technological Achievement (2013), and Atta-ur- Rahman Prize in Chemistry (2014).

Chapter 1

Introduction to Black Cumin (Nigella sativa): Chemistry, Technology, Functionality and Applications Mohamed Fawzy Ramadan

Abstract  Nigella sativa L. (botanical family, Ranunculaceae) is one of the most admired medical oilseeds in history. Nigella sativa seeds have been mentioned in the words of the Prophet Mohammed. Nigella sativa seeds contain active phytochemicals (i.e., phenolics, thymoquinone, fatty acids, tocols, sterols, polar lipids, amino acids…etc) with diverse biological effects. Functional extracts, essential oil, and fixed oil from Nigella sativa have been used in novel foods, nutraceuticals and pharmaceuticals. Nigella sativa is evident to promote health and it might serve to be a novel source for modern phytomedicine. Recently, black cumin has become an important topic for research worldwide. This book project aims to build a multidisciplinary discussion on the development and advances in Nigella sativa phytochemistry, cultivation practices, technology, functional characteristics, health-promoting activities as well as the food and non-food applications. Keywords  Ranunculaceae · Black seeds · UNSDG · Phyto-medicine · Functional properties · Essential oil · Fixed oil · Coronavirus (CoV) · Lipid technology · Lipid chemistry

M. F. Ramadan (*) Deanship of Scientific Research, Umm Al-Qura University, Makkah, KSA Agricultural Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_1

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1  N  igella sativa: Chemistry, Technology, Functionality and Applications The United Nations Sustainable Development Goals (UNSDG) were announced in 2015 (https://sustainabledevelopment.un.org). UNSDG offer a vision of a fairer, peaceful, more prosperous, and sustainable world. In foods, the way it is grown, processed, transported, stored, marketed, and consumed, lies the fundamental connection between people and the path to sustainable economic development. The third goal of UNSDG which called “Good Health and Well-Being” is aimed to promote human well-being and healthy life which is related to the use of health-­ promoting medical plants and herb as well as the environmental-friendly techniques in food processing. Scientists are searching for new foodstuffs with novel traits that could be designed to improve their healthfulness. Current research being carried out, will have a great influence on the way we eat in the near future (McClements 2019). Aromatic and medical plants have been used to formulate nutraceuticals and pharmaceuticals. According to World Health Organization (WHO), about 80% of world population depends on conventional medicine, which uses plant extracts or phytochemicals to treat several diseases. With the developments in the field of nutrition, there is an increasing interest in herbs and medicinal plants as phytochemicals-rich sources for functional foods, nutraceuticals and drugs. The demand for extracting plants phytochemicals, oils, and bioactive compounds has recently increased due to the beneficial roles played by different bioactive phytochemicals. Research is recently focusing on studying the bioactive compounds and therapeutic traits as well as investigating the toxicological impacts and the mode of action of plant extracts, oils and bioactive phytochemicals (Ramadan and Moersel 2002a, b; Ramadan 2007; Ramadan and Wahdan 2012; Kiralan et al. 2014; Hassanien et al. 2015). The WHO is giving importance on the exploration of medical plants for the benefits of human health care. Emphasis have been provided on scientific results, on the quality assurance, quality control, safety, efficacy, toxicity of the species, dosage, clinical trials, therapeutic applications, and drug interactions. Medical plants, especially those showing multiple biological effects, are of great interest. Black seeds or black cumin (Nigella sativa, family Ranunculaceae), is of importance due to its widespread food and medical applications (Ramadan et  al. 2003; Ramadan and Moersel 2004; Hassanien et  al. 2014; Kiralan et  al. 2016). Nigella sativa is used worldwide for the treatment of several diseases. These findings were stimulated by the talks of Prophet of Islam religion (Prophet Mohammad) who said that the black seeds contain all kinds of remedies except death (Ahmad et al. 2013; Islam et al. 2019). In the traditional medicine, Nigella sativa seeds have been used to manage dispiritedness and fatigue, and chronic headache. Roasted Nigella sativa seeds with honey or butter are prescribed for colic and cough and considered a novel lactagogue and antiseptic agents to treat eye infection. World attentions has been directed to the outbreak of coronavirus disease (COVID-19) that was first reported from Wuhan, China, on 31 December 2019. The effect of Nigella

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sativa extract on the replication of coronavirus (CoV) and on the expression of TRP-gene during CoV infection was evaluated (Ulasli et al. 2014). Nigella sativa extract exhibited an impact virus load and TRP-gene expression after CoV infection. Effective using of black cumin in therapeutic applications and for trade is markedly depend on yield (raw product, oils, seeds, and active compounds) and quality (Yimam et al. 2015). Numerous bioactive phytochemicals that possess many pharmacological properties have been identified in black cumin including thymoquinone (TQ), dithymoquinone, thymohydroquinone, t-anethol, alkaloids (nigellicines and nigelledine), saponins (α-hederin), flavonoids, and monoterpenes such as p-cymene and α-piene (Ramadan and Moersel 2003; Ramadan et al. 2012; Ramadan 2016). Nigella sativa also contains important ingredients including oils, essential fatty acids, vitamins, carbohydrates, minerals, proteins, and essential amino acids. These bioactive ­compounds exhibited cardiovascular supportive, antidiabetic, anticancer, analgesic, anti-inflammatory, antiepileptogenic, antioxidant, anti-schistosomiasis, immunomodulatory, gastroprotective, hepatoprotective and nephron-protective activities. Black seed’s antimicrobial properties included those on gram-positive bacteria, gram-negative bacteria, parasites, viruses, and fungi pathogens (Ramadan et  al. 2012; Ramadan 2016). Nigella sativa seed oil (fixed or volatile) is widely used in many foodstuffs, cosmetics and pharmaceutical products (Kiralan et al. 2017). Black seed oil is a popular natural painkiller and used as an antiseptic and analgesic remedy and for treatment of joint’s pain and stiffness. Nigella sativa seed oil and sesame oil blend is used for abdominal disorders, jaundice, dermatosis, cough, fever, liver ailments, headache, sore eyes and hemorrhoids. Thymoquinone (TQ) is the main active constituent in N. sativa essential oil, and most of the Nigella sativa traits are attributed to it (Kiralan et al. 2018). Thymoquinone protects the human cells from oxidation and gives recovery to cells by inhibiting from harmful impacts. Thymoquinone exerts high potential on carcinogenesis, eicosanoids production, and membrane lipid oxidation. In addition, TQ working as an effective chemo-protective agent with a hyperproliferative action in the experimental animals. Thymoquinone shows anti-­ proliferative impacts on cancer cell lines of colon, ovary, larynx, breast, lung, myeloblastic leukemia, and osteosarcoma. Moreover, TQ protects from several diseases and prevents from weakening the immune system (Ramadan 2016).

2  Nigella sativa Market Increasing demand for pharmaceuticals with natural phytochemicals owing to their health benefits is likely to propel Nigella sativa market size in the near future (www. gminsights.com). Nigella sativa fixed seed oil is a source of bioactive lipids, tocols, sterols, vitamins, folate, phosphorous, calcium, copper, zinc and essential fatty acids, which helps in improving digestion and boosting immune system. This product is usually organic (cold pressed extract), contain low calorie and could be used

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in flavoring and cooking, and stimulating industry size. In addition, increasing interest for novel natural products in bakery products is likely to stimulate Nigella sativa seed oil market size. On the other side, Nigella sativa essential oil have been used in novel foods, and nutraceuticals. Nigella sativa essential oil (in form of oil or capsules) contain thymoquinone (TQ), p-cymene, β-pinene, α-pinene, longifolene, α-thujene and carvacrol, which are essential in the nutraceutical formulation. Nutraceuticals market is expected to surpass 550 billion USD by 2025, owing growing e-commerce and consumer awareness, which is anticipated to drive the market size of Nigella sativa. Due to the consumption needs of the rapidly increasing world population, Nigella sativa has a large international market. Nigella sativa seed oil market size was more than USD 15 million in 2018, and by 2025 industry expects to surpass USD 25 million. Moreover, at the end of 2025, Nigella sativa oil market size from cosmetic applications and personal care is projected to surpass 700 tons (https://www.gminsights.com/industry-analysis/black-seed-oil-market). Recently, the global market size, sales, share, and growth analysis report of Nigella sativa oil industry was released (www.marketwatch.com).

3  Nigella sativa in the International Literature Being a spacious habitual and rich in bioactive phytochemicals, black cumin is considered as a weapon to the drug discovery and drug development. Several studies have been done on Nigella sativa and its oils as well as its bioactive compounds, especially on TQ and its derivatives (Islam et al. 2019). A search with the keyword “Nigella sativa” in PubMed database (February 2020) showed 1334 published contributions belonging to Nigella sativa extracts (water/organic/water-organic solvents), essential oil, fatty acids, seed oil, and isolated bioactive constituents. When Nigella sativa was used as a keyword to search in Scopus or ISI Web of knowledge databases, about 2890 articles and reviews have been found (till February 2020). A careful search on Nigella sativa (as keywords) in the title, abstract and keywords among contributions in the Scopus database (www.scopus.com) revealed that the total number of scholarly outputs published is high (ca. 2890 till February 2020). Apart from the total published scholarly outputs ca. 2550 were research articles, ca. 227 reviews, and 14 book chapters. Figure 1.1 shows the scholarly output on Nigella sativa since 2000. It is clear that the scholarly outputs published annually on Nigella sativa are markedly increased from 20 contributions in 2000 to 252 article in 2019. These measurable indicators reflect the importance of Nigella sativa as a topic in the international scientific community. The publications were mainly related to the subject areas of pharmacology, toxicology and pharmaceutics, agricultural and biological sciences, medicine, biochemistry, genetics and molecular biology, chemical engineering, chemistry, and engineering. Scienticts form Iran, Egypt, India, Saudi Arabia, Turkey, Pakistan and United States have emerged as main contributors. Most prolific journals were

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Fig. 1.1  Scholarly output on Nigella sativa since 2000. (www.scopus.com)

Journal of Ethnopharmacology, Phytotherapy Research, Pakistan Journal of Pharmaceutical Sciences, International Journal of Pharmacy and Pharmaceutical Sciences, Pharmaceutical Biology, Evidence Based Complementary and Alternative Medicine, Industrial Crops and Products, and BMC Complementary and Alternative Medicine. Several books already published on the composition and functional properties of herbs, oilseeds, and medicinal plants. However, it is hard to find a book focused on the cultivation, composition and functionality of Nigella sativa. This book is planned to contain comprehensive chapters focusing on Nigella sativa, which contain unique bioactive components that have led to their being considered health-promoting seeds. The main goal of editing this book was to discuss the phytochemical composition, therapeutic properties and functionality of high value oils, phytochemicals, nutrients, extracts and volatiles of Nigella sativa seeds, to explore their useful uses in pharmaceuticals, nutraceuticals, novel foods, natural drugs, and feed. Nigella sativa seeds have unique phytochemical profile and characteristics that make them a novel source for nutraceuticals, pharmaceuticals and functional foods. Book chapters are designed to have the following main sections: 1. Cultivation, Composition and Applications of N. sativa seeds 2. Chemistry, Technology, Functionality and Applications of N. sativa fixed oil 3. Chemistry, Technology, Functionality and Applications of N. sativa essential oil 4. Chemistry, Technology, Functionality and Applications of N. sativa extracts

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References Ahmad, A., Husain, A., Mujeeb, M., Khan, S. A., Najmi, A. K., Siddique, N. A., Damanhouri, Z.  A., & Anwar, F. (2013). A review on therapeutic potential of Nigella sativa: A miracle herb. Asian Pacific Journal of Tropical Medicine, 3(5), 337–352. https://doi.org/10.1016/ s2221-1691(13)60075-1. Hassanien, M. F. R., Mahgoub, S. A., & El-Zahar, K. M. (2014). Soft cheese supplemented with black cumin oil: Impact on food borne pathogens and quality during storage. Saudi Journal of Biological Sciences, 21, 280–288. Hassanien, M. F. R., Assiri, A. M. A., Alzohairy, A. M., & Oraby, H. F. (2015). Health-promoting value and food applications of black cumin essential oil: An overview. Journal of Food Science and Technology, 52, 6136–6142. Islam, M.  T., Khan, M.  R., & Mishra, S.  K. (2019). An updated literature-based review: Phytochemistry, pharmacology and therapeutic promises of Nigella sativa L. Oriental Pharmacy and Experimental Medicine, 19, 115–129. https://doi.org/10.1007/s13596-019-00363-3. Kiralan, M., Özkanb, G., Bayrak, A., & Ramadan, M. F. (2014). Physicochemical properties and stability of black cumin (Nigella sativa) seed oil as affected by different extraction methods. Industrial Crops and Products, 57, 52–58. Kiralan, M., Ulaş, M., Özaydin, A. G., Özdemir, N., Özkan, G., Bayrak, A., & Ramadan, M. F. (2016). Changes in hexanal, thymoquinone and tocopherols levels in blends from sunflower and black cumin oils as affected by storage at room temperature. La Rivista Italiana Delle Sostanze Grasse, XCIII, 229–236. Kiralan, M., Ulaş, M., Özaydin, A. G., Özdemir, N., Özkan, G., Bayrak, A., & Ramadan, M. F. (2017). Blends of cold pressed black cumin oil and sunflower oil with improved stability: A study based on changes in the levels of volatiles, tocopherols and thymoquinone during accelerated oxidation conditions. Journal of Food Biochemistry, 41, e12272. Kiralan, M., Çalik, G., Kiralan, S., & Ramadan, M. F. (2018). Monitoring stability and volatile oxidation compounds of cold pressed flax seed, grape seed and black cumin seed oils upon photo-oxidation. Journal of Food Measurement and Characterization, 12, 616–621. McClements, D. J. (2019). The science of foods: Designing our edible future. In D. J. McClements (Ed.), Future foods: How modern science is transforming the way we eat. Cham: Springer. Ramadan, M. F. (2007). Nutritional value, functional properties and nutraceutical applications of black cumin (Nigella sativa L.) oilseeds: An overview. International Journal of Food Science and Technology, 42, 1208–1218. Ramadan, M. F. (2016). Black cumin (Nigella sativa) oils. In V. R. Preedy (Ed.), Essential oils in food preservation, flavor and safety (pp.  269–275). Academic Press (Elsevier). ISBN: 9780124166417. Ramadan, M. F., & Moersel, J.-T. (2002a). Characterization of phospholipid composition of black cumin (Nigella sativa L.) seed oil. Nahrung/Food, 46, 240–244. Ramadan, M. F., & Moersel, J.-T. (2002b). Neutral lipid classes of black cumin (Nigella sativa L.) seed oils. European Food Research and Technology, 214(3), 202–206. Ramadan, M. F., & Moersel, J.-T. (2003). Analysis of glycolipids from black cumin (Nigella sativa L.), coriander (Coriandrum sativum L.) and niger (Guizotia abyssinica Cass.) oilseeds. Food Chemistry, 80, 197–204. Ramadan, M. F., & Moersel, J.-T. (2004). Oxidative stability of black cumin (Nigella sativa L.), coriander (Coriandrum sativum L.) and niger (Guizotia abyssinica Cass.) upon stripping. European Journal of Lipid Science and Technology, 106(1), 35–43. Ramadan, M. F., & Wahdan, K. M. M. (2012). Blending of corn oil with black cumin (Nigella sativa) and coriander (Coriandrum sativum) seed oils: Impact on functionality, stability and radical scavenging activity. Food Chemistry, 132, 873–879. Ramadan, M. F., Kroh, L. W., & Moersel, J.-T. (2003). Radical scavenging activity of black cumin (Nigella sativa L.), coriander (Coriandrum sativum L.) and niger (Guizotia abyssinica Cass.) crude seed oils and oil fractions. Journal of Agricultural and Food Chemistry, 51, 6961–6969.

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Ramadan, M. F., Asker, M. M. S., & Tadros, M. (2012). Antiradical and antimicrobial properties of cold-pressed black cumin and cumin oils. European Food Research and Technology, 234, 833–844. Ulasli, M., Gurses, S. A., Bayraktar, R., Yumrutas, O., Oztuzcu, S., Igci, M., Igci, Y. Z., Cakmak, E.  A., & Arslan, A. (2014). The effects of Nigella sativa (Ns), Anthemis hyalina (Ah) and Citrus sinensis (Cs) extracts on the replication of coronavirus and the expression of TRP genes family. Molecular Biology Reports, 41(3), 1703–1711. Yimam, E., Nebiyu, A., Ibrahim, A.  M., & Getachew, M. (2015). Effect of nitrogen and phosphorus fertilizers on growth, yield and yield components of black cumin (Nigella sativa L.) at Konta District, South West Ethiopia. Journal of Agronomy, 14, 112–120.

Part I

Nigella sativa Seeds: Cultivation, Composition and Applications

Chapter 2

Effect of Cultivation, Fertilization and Irrigation Practices on Nigella sativa Yield and Quality Enas Mohamed Wagdi Abdel-Hamed

Abstract  Nigella sativa (black cumin) seeds are used as a food additive, and in medical proposes. Considering the importance of Nigella sativa plant, large-scale production is important. Phytochemical profile changes during germination because biochemical activities produce energy and essential constituents, wherein some phytochemicals transform into active constituents. Few studies reported on the impacts of cultivation conditions on the growth, yield and biochemical constituents of Nigella sativa. This chapter summarizes and highlights the impact of different cultivation, fertilization and irrigation regimes on the yield and quality of Nigella sativa seeds. Keywords  Water-use efficiency · Salinity · Drought stress · Geometrical properties · Plant growth characters · Germination · Seed treatment · Gibberellic acid

Abbreviations AET Actual evapotranspiration Cd Cadmium DAE Days after emergence EO Essential oil G × E Genotype × Environment GA3 Gibberellic acid K Potassium KIN Kinetin MAD Maximum allowable depletion N Nitrogen E. M. W. Abdel-Hamed (*) Soil Science Department, Faculty of Agriculture Zagazig University, Zagazig, Egypt e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_2

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P Phosphorous SFA Saturated fatty acids WUE Water use efficiency

1  Introduction Nigella sativa (family Ranunculaceae), known also as black cumin, is an annual herbaceous medical plant cultivated around the Mediterranean countries (Gharibzahedi et al. 2010; Espanany et al. 2016). Nigella sativa has been used as a remedy for several diseases such as tumor, hypertension, gastrointestinal disturbances, diabetes, asthma, inflammation, and gynecological disorders (Ramadan 2007). Nigella sativa seed yield is of great importance (Shahbazi 2019). Nigella sativa seed yield was generally less than 1000 kg/ha, wherein the plant has a potential of cultivation and diversification of cropping systems. Some problems such as the lack of recommended fertilizer levels, improved seeds, lack of enhanced agriculture practices and extension system, lack of know-how on postharvest processing as well as marketing systems are the reasons for Nigella sativa low productivity (Yosef 2008; Yimam et al. 2015). The production of Nigella sativa could be increased using new high-yielding varieties with proper management practices and production technologies (Agha et al. 2014). In addition, knowledge of Nigella sativa seeds properties is needed for construction of the handling devices, to facilitate seed processing and transportation. For example, seed bulk density influences storage and transport capacity, and porosity influences the resistance to airflow during seed drying and aeration (Kachru et al. 1994; Zapotoczny et al. 2019). There has been a growing interest in the effects of germination methods on composition, antinutritional factors, in-vitro digestibility and naturally occurring compounds of seeds. Seeds biochemical profile changes during germination because biochemical activities produce essential constituents and energy, wherein some nutrients transform into active compounds (Moongngarm and Saetung 2010). During germination carbohydrates, lipids, and proteins in the seeds are broken down to produce amino acids and energy important for the plant development (Ferreira et al. 1995). Germination process has been used for softening the kernel structure, improving its nutritional potential, reducing anti-nutritional impacts and improving the functionality of seed protein (Suliburska et al. 2009; Ijarotimi and Keshinro 2012; Mariod et al. 2012). There are studies on the impact of germination on nutrient amounts of several seeds, but few research is reported on Nigella sativa seeds. In addition, most of investiagtions have been carried out using a single set germination condition, while studies are scarce on the impact of processing conditions (Mariod et al. 2012). In semi-arid and arid regions, there was a decline in seed yield due to delayed planting and drought stress. Thus, genotypes having good stability in different con-

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ditions with high seed yield must be selected. The phenotypic value is due to the impacts of the genotype (G), environment (E) and the interaction of G × E. Strong G  ×  E interaction could affect the yields of genotypes in different environments (Gauch and Zobel 1996). On the other side, stability has been investgated by geneticists and biometricians (Huehn 1996). Two methods (parametric and non-­parametric) to analyze G × E interaction under different growth conditions are utilized to select the compatible genotypes. When their seed yields are stabilized, enhanced genotypes could show an ideal adaptation to variable conditions (Shahbazi 2019). The plant sensitivity to management factors and E varies upon the developmental stage. The main step towards maximizing plant productivity is to ensure that the plant development stage is matched to the production E and the resources. According to FAO (1999), plants rich in essential oils are more abundant in arid zone than in humid habitat. In cultivation, the use of fertilizers is of important for production. Nitrogen (N) has been reported to be an important factor for the development in crop production, including medical plants (Shah 2004, 2008, 2011). On the other side, research has been performed on responses of Nigella sativa to several irrigation regimes (Mozzafari et  al. 2000; Bannayan et  al. 2008; Ghamarnia et  al. 2010; Al-Kayssi et al. 2011). They mentioned that the Nigella sativa production on fertile silty clay soil was decreased by increasing soil moisture at the seed formation stage, wherein the seeds oil recovery was not affected. To the best of knowledge, few investigations studied the impacts of cultivation conditions on the growth, yield and bioactive compounds of Nigella sativa seeds. The current chapter highlights the impacts of fertilization, cultivation practices and irrigation regimes on the yield and quality of Nigella sativa seeds.

2  E  ffects of Salinity and Drought Stress on Nigella sativa Yield and Quality The influences of salinity on morphological traits and chemical accumulation in Nigella sativa seeds were investigated. Khalid and Ahmed (2017) studied the growth and biochemical composition of Nigella sativa cultivated under salinity stress. Plants were subjected to different NaCl levels (0.4–6.3 dSm−1). Plant growth characters (PGC) including number of leaves, NL (plant1), plant height, PH (cm), number of branches, NB (plant1), dry weight of herb (DWH) number of capsules, NC (plant1), and yield of seeds, (YS) (plant1) were decreased when salinity levels increased. The chemical profile including essential oil (EO), total soluble sugars (TSS) main EO constituents (p-cymene, α-thujene, and γ-terpinene), and proline were promoted under salinity, while N and protein levels were decreased. The maximum PGC was detected in the 0.4 dSm−1 treatment. The greatest amounts of EO (0.4%), α-thujene (7%), p-cymene (60%), γ-terpinene (1.5%), TSS (11.8%), and PRO content (23 μm/g) were found under 6.3 dSm−1. The highest values of protein

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and N uptake (16.5% and 0.4 μm/g) were obtained under 0.4 dSm−1. It was concluded that Nigella sativa could grow under low salinity levels. Shahbazi (2019) studied the yield performance and the stability of different N. sativa genotypes. A randomized complete-block design was applied with non-­ parametric and parametric methods to analyze the stability under experiment environments (combination of data of planting, year, and moisture parameters). The study showed that drought stress and delay in cultivation and markedly decreased N. sativa seed yield.

3  Effects of Fertilization on Nigella sativa Yield and Quality The optimal and deficient levels of fertilization cause adverse impacts on the plant growth. For instant, a high N level decreases the activities of phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase, thus decreasing the photosynthetic level (Greef 1994; Ashraf et al. 2006). On the other hand, N deficiency decreases photosynthesis as well as the growth and yield of the plant (Dietz and Harris 1997). Triple superphosphate (10–30 kg/ha) increased Nigella sativa essential and fixed oils (El-Deen and Ahmed 1997; Ashraf et al. 2006). El-Sayed et al. (2000) reported that fertilization of Nigella sativa (100 kg/ha of P and N or 200 kg P and 50 kg/ha N) increased the thymoquinone (TQ) amount in Nigella sativa EO and 11,14-eicosadienoic acid in Nigella sativa fixed oil. Shah et al. (2006) studied, during Nigella sativa growth, the impact of gibberellic acid (GA3) spray on the nutrients uptake, growth, and yield. Field experiments were performed on N. sativa sprayed with water or 10−5 M GA3 at 40 or 60 days after sowing (DAS). Leaf area/plant, leaf number/plant, branch number/plant, dry weight/ plant, shoot length/plant, and accumulation of P, N and K were measured at 70 DAS.  Seed yield and NPK accumulation were maximal when GA3 sprayed at 40 DAS. Ashraf et al. (2006) studied the impact of N on the amounts and composition of minerals as well as the fixed oil, and EO in N. sativa seeds. Plants (63 day-olds) were treated with N (0–90 kg N/ha). Fixed oil content recorded 32.7–37.8%, while remained unchanged at both higher N treatments. Palmitic acid slightly increased at all N treatments, but stearic acid decreased when 60 kg N/ha applied. Linoleic acid followed by oleic acid were the predominant fatty acids. A reduction in α-linolenic acid was recorded at higher N treatments (60 and 90 kg N/ha). The EO content did not change with the changes in the supplied N amounts. p-Cymene, the main active component of Nigella sativa EO, level was increased when 30  kg  N/ha applied, while no changes in the amounts of α- or β-pinene were recorded at different N treatments. Increasing the N levels did not influence the amounts of P, Mg, Ca, K, or Cr in the seed, while a decrease in Zn, Mn, and Ni was noted with the increase in N amount.

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Shah (2008) studied the response of Nigella sativa to N and a kinetin (KIN) spray. Field experiments studied the impact of soil N (0–100 kg N/ha) alone and in combination with KIN (10−5 M) foliar spray at the vegetative stage on the physiology, growth, and yield of N. sativa. The fertilization (80  kg  N/ha) enhanced net photosynthetic rate, leaf N content, chlorophyll content, stomatal conductance, leaf area, and dry mass (30 days after KIN spray) as well as the number of 1000-seed weight, biological yield per ha, seed capsules/plant, seed yield per ha, and harvest index (130 days after KIN spray). Positive impacts of N treatment were noted by KIN spray at 40 days after seeding. Moreover, KIN-sprayed plants exploited the soil N more effectively, resulting in increased physiological and yield parameters. Goswami (2011) reported on the irrigation and N management impacts on N. sativa in alluvial plains. Irrigation upon soil moisture tension (ψ m = −0.03 at 20 cm depth) resulted in the highest seed yield (445 kg/ha), whereas crop treated with 4 irrigation intervals (18–20 days). The maximum seed yield, measured with 80  kg  N/ha treatment, was superior to 20–60  kg/ha. Actual evapotranspiration (AET) of Nigella sativa was low (181 mm) under limited water, but irrigation at ψ m = −0.03 MPa reached the highest AET. High amount (80 kg N/ha) increased AET value (225 mm), and increment in N amount increased AET. WUE reached the maximum (1.8 kg/ha/mm) level when the plant was under moderately moisture, wherein WUE decreased with the increased water supply. N management and irrigation increased the benefit: cost ratio of the plant. In Nigella sativa, treatment with 80 kg/ ha N dose, and irrigation at an interval of 18–20 days was equally economic and productive in the sandy loam soils. Abdel-Aziez et  al. (2014) evaluated the efficacy of Azotobacter chroococcum and their effect in compination with inorganic N on the rhizosphere yield, biological activity, yield components of Nigella sativa cultivated in sandy soil. Samples of rhizosphere soil were collected from sites planted with different standing crops and utilized for the isolation of N2-fixer Azotobacter sp. Purified Azotobacter chroococcum isolate was tested for its N2 fixation potential, exopolymer secretion, production of plant growth-promoting compounds, salicylic acid (SA) formation, phosphates dissolving ability, siderophores production, and enzymatic production. The isolates were tested for biochemical traits (enzymes production and hormonal activity) and utilized to prepare microbial inoculants for N. sativa seeds. The mixed inoculation with the biofertilizer and using a half of recommended N dose improved the densities of the phosphate dissolving bacteria, azotobacters colonization, total microbial microflora, CO2 evolution in the rhizosphere of the inoculated plants and plant growth parameters. The impact of N and phosphorus (P) fertilization on yield, growth, and yield components of N. sativa was studied (Yimam et  al. 2015). Different levels of P (0–40 kg ha−1 as TSP) and N (0–60 kg ha−1 as urea), were arranged in RCB design. Interaction of N and P was affected by the yield and growth parameters except for 1000-seed weight. The maximum seed yield (1336  kg  ha−1) was produced from 60/40 kg N P ha−1. The maximum number of branches (46.1) was produced from the interaction impact of 60/40 kg N P ha−1. The maximum number of pods per plant (45.9) was recorded from 40 kg P ha−1 and 60 kg N ha−1 interactions. The tallest

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plants (72.5 cm) were produced in plots treated with 60/40 kg N P ha−1. The maximum number of seeds/pod (91.6) was found at combination of 60/40 kg N P ha−1. The longest days (86.7 days) to 50% flowering were measured for 60/40 kg N P ha−1 treatment. The maximum harvest index (20.8%) was obtained at 60/40  kg  N P, while the minimum harvest index (15.1%) was recorded at 15 and 0 kg N P interaction. The both combinations of N P ha−1 were economically viable with the marginal rate of revenue beyond the minimum acceptable level (150%). The growers may use a combination of 45/40 kg N P ha−1 and 15/20 Kg N P2O5 ha−1 for N. sativa cultivation. Salehi et al. (2016) investigated the impact of combining urea and cattle manure on microbial biomass carbon, soil CO2 flux, and dry matter accumulation during N. sativa growth. Integrated application of urea and cattle manure increased total N by 3.27%, microbial biomass carbon by 10%, N at flowering stage by 7.57%, soil organic carbon by 2.45%, and CO2 flux by 9% over solitary urea fertilization. The soil characteristics and growth parameters of black cumin benefited more from the full-dose treatment than the split urea treatment. Cattle manure combined with urea and the full-dose treatment of urea increased the efficiency of fertilizer and increased soil biological traits as well as the plant growth. The reported combinations are useful for the environment and decreased the cost of top dressing urea. Zapotoczny et al. (2019) studied the physical properties of Nigella sativa seeds subjected to different cultivation conditions (sowing date, levels of N fertilization, row spacing, and integrated protection). The most effective impact on the physical characteristics of Nigella sativa seeds had N fertilization (100 kg/ha), row spacing of 15 or 30 cm, seeding delayed by 20 days, and the treatment with Penncozeb 80 WP.

4  Effects of Germination on Nigella sativa Yield and Quality The effects of pre-sowing seed with KIN (growth regulator) on the growth and photosynthetic characteristics of Nigella sativa were investigated (Shah 2011). Seeds were sterilized and soaked for 5–15 h in aqueous KIN solution (1–100 μM). Potted plants were analyzed at 30–70 days after emergence (DAE) for chlorophyll content, dry mass, stomatal conductance, carbonic anhydrase, leaf area, nitrate reductase activity, protein content, and photosynthetic rate. Seed yield and capsule number were measured at 90 DAE. KIN treatment enhanced all parameters, with the most prominent results recorded following 10-h soaking with KIN (10 μM). In another experiment, Shah (2007) reported on the photosynthetic and yield responses of N. sativa to pre-sowing seeds with GA3. Espanany et al. (2016) studied the impact of per-sowing on increasing the germination rate of Nigella sativa seeds under different levels of cadmium (Cd). Nigella sativa seeds were subjected to hormopriming and halopriming to investigate the impact of seed priming on biochemical and physiological traits. Germination percentage and rate were not impressed by Cd. Hormopriming (salicylic acid 100  mg/L) increased germination, dry weight and radicle elongation compared with the control under Cd (30 mg/L) stress. An increase

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in Cd levels increased malondialdehyde, electrical conductivity, and proline content. The reduction in soluble protein was noted in primed and unprimed seedlings with salicylic acid, KNO3 and KCl under the maximum Cd level. Seed priming with salicylic acid due to improving the germination performance of Nigella sativa could imporve seedlings tolerance to toxic metals in the irrigation water. Mariod et al. (2012) reported the impact of germination conditions on the amino acids and fatty acids composition of Nigella sativa seeds originated from Syria and Ethiopia. Germination increased oil and protein amounts, while other compounds were decreased. Little changes were detected in the fatty acids after germination as monounsaturated fatty acids (MUFA) and saturated fatty acids (SFA) decreased, while polyunsaturated fatty acids (PUFA) increased. Total amino acids was affected by germination conditions and increased during germination. Germinated seeds exhibited decrease in the Ca K, Na and Fe contents. It was concluded that germinated Nigella sativa seeds are novel food with nutritional traits. The responses of N. sativa to high zinc (Zn) levels in pot experiments was assessed (Marichali et al. 2016). Zinc excess did not influence the germination, but decreased radicle elongation. In addition, a reduction in yield, growth parameters and yield components was recorded. With the increase in Zn levels, total lipids decreased and changes in fatty acids profile toward SFA formation were noted. An increase in the total phenolics concomitant with improved antioxidant potential has been observed. Despite the decrease in the seed’s EO yield, a redirection of the terpene metabolism to oxygenated compounds synthesis was noted. Zn excess might represent an alternative to increase Nigella sativa nutritional value.

5  E  ffect of Irrigation Regime on Nigella sativa Yield and Quality Nigella sativa is generally cultivated in unirrigated conditions, therefore, the reaction of the plant to irrigation is necessary in terms of yield and the optimal water use (Şenyigit and Arslan 2018). Few studies performed on the effects of irrigation practices and water yield functions in deficit irrigation conditions on Nigella sativa. Ghamarnia et  al. (2010) obtained the seasonal plant water consumption within 414–1461 mm and WUE values within 0.49–1.39 kg ha−1 mm−1. Ghamarnia and Jalili (2013) determined the same values as 193–645 mm, and 0.11–1.87 kg ha−1 mm−1, respectively (Şenyigit and Arslan 2018). Bannayan et  al. (2008) exposed Nigella sativa to several irrigation practices, wherein weekly irrigation was the control. The practices based on the developmental stage were the termination of irrigation at blooming, flowering, and seed formation. The minimum seed yield was measured when irrigation stopped at the blooming stage, while the number of seeds per plant was the main affected yield factor. Nigella sativa exhibited tolerance to water deficit except when irrigation was stoped at seed formation. 1000-seed weight was stable across treatments, while the higher seed

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yield was consistent with higher plant height and straw yield. The study did not show reduction in oil recovery for all water-deficit treatments. Al-Kayssi et al. (2011) studied the effects of irrigation on Nigella sativa seed nigellone (dithymoquinone) content cultivated on gypsiferous soils. Over two growing seasons on soils with different gypsum contents, randomized complete block design experiments were carried out with 4 irrigation treatments and 3 replications. Soil gypsum treatments were 60.0, 137.6, 275.2, 314.2 and 486 g/kg. Treatments based on replenishing the deep root zone (0.60 m) to field capacity when the highest allowable depletion (MAD) of the available soil water holding capacity of 25%, 50% and 75% were maintained. The seasonal values of crop water stress index for the treatments were increased when the gypsum content increased. Liner relationships were noted between Nigella sativa seed yield, dithymoquinone content and seasonal crop water stress index values. Those relations might be applied to predict Nigella sativa seed yield, dithymoquinone amount, and irrigation timing in soils containing different gypsum levels.

Fig. 2.1  Schematic of drainable lysimeter. (Ghamarnia and Jalili 2014)

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Ghamarnia and Jalili (2014) conducted lysimeter experiments (Fig. 2.1) to study the groundwater contributions percentage made by N. sativa. Plants were cultivated in columns (0.4 m diameter) packed with silty clay soil. Factorial experiments were performed at different treatment combinations using 3 replicates with randomized complete block design. Treatments were applied via maintaining EC of groundwater with 1, 2 and 4 dS m−1 at different water table depths (0.6–1.10 m). The groundwater contribution was recorded as a part of plant evapotranspiration by recording of water levels in Mariotte tubes daily. The study shows that crop yield and groundwater use decrease as water-table depth increase and groundwater salinity. Şenyigit and Arslan (2018) determined, in a field experiment, the impact of different irrigation practices obtained by pan evaporation and water balance methods on the yield and vegetative parameters as well as the water consumption of Nigella sativa. The irrigation treatments were carried out with three intervals (SA3: 3 days, SA5: 5 days, and SA10: 10 days) and 4 water levels as 0% (I0: non-irrigated) 50%, 75% (I50, I75: deficit irrigation), and 100% (I100: full irrigation) of the cumulative evaporation amount measured from class A pan in the first year, and of the required water amount to replenish the available soil moisture to the field capacity in the 0.60  m soil depth in the second year. The highest seed yield (1700  kg/ha) was obtained in SA5-I100 and the lowest seed yield (722 kg/ha) was obtained in I0. Since the yield and vegetative parameters in the same irrigation programs formed by two methods were close, both methods might be used for the irrigation of Nigella sativa.

References Abdel-Aziez, S. M., Eweda, W. E., Girgis, M. G. Z., & Abdel Ghany, B. F. (2014). Improving the productivity and quality of black cumin (Nigella sativa) by using Azotobacter as N2 biofertilizer. Annals of Agricultural Sciences, 59(1), 95–108. Agha, Q., Ahmad, S., Muhammad, I., Gill, A., & Athar, M. (2014). Growth and production potential of five medicinal crops in highlands of Balochistan, Pakistan. Journal of Medicinal Plants Research, 4, 2159–2163. Al-Kayssi, A. W., Shihab, R. M., & Mustafa, S. H. (2011). Impact of soil water stress on Nigellone oil content of black cumin seeds grown in calcareous-gypsifereous soils. Agricultural Water Management, 100(1), 46–57. Ashraf, M., Ali, Q., & Iqbal, Z. (2006). Effect of nitrogen application rate on the content and composition of oil, essential oil and minerals in black cumin (Nigella sativa L.) seeds. Journal of the Science of Food and Agriculture, 86(6), 871–876. Bannayan, M., Nadjafi, F., Azizi, M., Tabrizi, L., & Rastgoo, M. (2008). Yield and seed quality of Plantago ovata and Nigella sativa under different irrigation treatments. Industrial Crops and Products, 27(1), 11–16. Dietz, K. J., & Harris, G. C. (1997). Photosynthesis under nutrient deficiency. In M. Pessarakli (Ed.), Handbook of Photosynthesis (pp. 951–971). New York: Marcel Dekker. El-Deen, E., & Ahmed, T. (1997). Influence of plant distance and some phosphorus fertilization sources on black cumin (Nigella sativa L.) plants. Assiut Journal of Agricultural Sciences, 28, 39–56.

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El-Sayed, K. A., Ross, S. A., El-Sohly, M. A., Khalafalla, M. M., Abdel Halim, O. B., & Ikegami, F. (2000). Effect of different fertilizers on the amino acid, fatty acid and essential oil composition of Nigella sativa seeds. Saudi Pharmaceutical Journal, 8, 175–182. Espanany, A., Fallah, S., & Tadayyon, A. (2016). Seed priming improves seed germination and reduces oxidative stress in black cumin (Nigella sativa) in presence of cadmium. Industrial Crops and Products, 79, 195–204. FAO. (1999). State of the worlds forests. Rome: FAO. Ferreira, R. B., Melo, T. S., & Teixeira, A. N. (1995). Catabolism of the seed storage proteins from Lupinus albus: Fate of globulins during germination and seedling growth. Australian Journal Plant Physiology, 22, 373–381. Gauch, H. G., & Zobel, R. W. (1996). AMMI analysis of yield trials. In M. S. Kang & H. G. Gauch (Eds.), Genotype-by-environment interaction (pp. 85–122). Boca Raton: CRC Press. Ghamarnia, H., & Jalili, Z. (2013). Water stress effects on different black cumin (Nigella sativa L.) components in a semi-arid region. International Journal of Agronomy and Plant Production, 4(4), 753–762. Ghamarnia, H., & Jalili, Z. (2014). Shallow saline groundwater use by black cumin (Nigella sativa L.) in the presence of surface water in a semi-arid region. Agricultural Water Management, 132, 89–100. Ghamarnia, H., Khosravy, H., & Sepehri, S. (2010). Yield and water use efficiency of (Nigella sativa L.) under different irrigation treatments in a semi arid region in the west of Iran. Journal of Medicinal Plants Research, 4(16), 1612–1616. Gharibzahedi, S.  M. T., Mousavi, S.  M., Moayedi, A., Garavand, A.  T., & Alizadeh, S.  M. (2010). Moisture-dependent engineering properties of black cumin (Nigella sativa L.) seed. Agricultural Engineering International CIGR Journal, 12, 1. Goswami, S. B. (2011). Effect of irrigation and nitrogen on growth, yield and water-use efficiency of black cumin (Nigella sativa) in lower Indo-Gangetic plains. Indian Journal of Agricultural Sciences, 81, 524–527. Greef, J. M. (1994). Productivity of maize in relation to morphological and physiological characteristics under varying amount of nitrogen supply. Journal of Agronomy and Crop Science, 172, 317–326. Huehn, M. (1996). Nonparametric analysis of genotype x environment interactions by ranks. In Genotype-by-environment interaction (pp. 213–228). Boca Raton: CRC Press. Ijarotimi, O. S., & Keshinro, O. O. (2012). Comparison between the amino acid, fatty acid, mineral and nutritional quality of raw, germinated and fermented African locust bean (Parkia biglobosa) flour. Acta Scientiarum Polonorum Technologia Alimentaria, 11(2), 151–165. Kachru, R. P., Gupta, R. K., & Alam, A. (1994). Physico-chemical constituents and engineering properties of food crops. Jodhpur: Scientific Publishers. Khalid, K. A., & Ahmed, A. M. A. (2017). Growth and certain biochemical components of black cumin cultivated under salinity stress factor. Journal of Materials and Environmental Science, 8, 7–13. Marichali, A., Dallali, S., Ouerghemmi, S., Sebei, H., Casabianca, H., & Hosni, K. (2016). Responses of Nigella sativa L. to zinc excess: Focus on germination, growth, yield and yield components, lipid and terpene metabolism, and Total Phenolics and antioxidant activities. Journal of Agricultural and Food Chemistry, 64(8), 1664–1675. Mariod, A. A., Edris, Y., Cheng, S. F., & Abdelwahab, S. (2012). Effect of germination periods and conditions on chemical composition, fatty acids and amino acids of two black cumin seeds. Acta Scientiarum Polonorum Technologia Alimentaria, 11, 401–410. Moongngarm, A., & Saetung, N. (2010). Studies on comparison of chemical compositions and bioactive compounds of germinated rough rice and brown rice Thailand. Food Chemistry, 122, 782–788. Mozzafari, F. S., Ghorbanli, M., Babai, A., & Faramarzi, M. (2000). The effect of water stress on the seed oil of Nigella sativa. Journal of Essential Oil Research, 12, 36–38.

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Ramadan, M.  F. (2007). Nutritional value, functional properties and nutraceutical applications of black cumin (Nigella sativa L.): An overview. International Journal of Food Science & Technology, 42(10), 1208–1218. Salehi, A., Fallah, S., & Surki, A. (2016). Organic and inorganic fertilizer effect on soil CO2 flux, microbial biomass, and growth of Nigella sativa L. International Agrophysics, 31, 103–116. Şenyigit, U., & Arslan, M. (2018). Effects of irrigation programs formed by different approaches on the yield and water consumption of black cumin (Nigella sativa L.) under transition zone in the West Anatolia conditions. Tarim Bilimleri Dergisi, 24, 22–32. Shah, S. H. (2004). Morphophysiological response of black cumin (Nigella sativa L.) to nitrogen, gibberellic acid, and kinetin application. Ph.D. thesis, Aligarh Muslim University, Aligarh, India. Shah, S. H. (2007). Photosynthetic and yield responses of Nigella sativa L. to pre-sowing seed treatment with GA3. Turkish Journal of Biology, 31, 103–107. Shah, S.  H. (2008). Synergistic responses of black cumin (Nigella sativa L.) to nitrogen and a kinetin spray. Journal of Herbs, Spices & Medicinal Plants, 13(4), 45–54. Shah, S. H. (2011). Growth and photosynthetic characteristics of Nigella sativa L. as affected by presowing seed treatment with kinetin. Photosynthetica, 49(1), 154–160. Shah, S. H., Ahmad, I., & Samiullah. (2006). Effect of gibberellic acid spray on growth, nutrient uptake and yield attributes during various growth stages of black cumin (Nigella sativa L.). Asian Journal of Plant Sciences, 5, 881–884. Shahbazi, E. (2019). Genotype selection and stability analysis for seed yield of Nigella sativa using parametric and non-parametric statistics. Scientia Horticulturae, 253, 172–179. Suliburska, J., Krejpcio, Z., Lampart-Szczapa, E., & Wójciak, R. W. (2009). Effect of fermentation and extrusion on the release of selected minerals from lupine grain preparations. Acta Scientiarum Polonorum Technologia Alimentaria, 8(3), 87–96. Yimam, E., Nebiyu, A., Ibrahim, A.  M., & Getachew, M. (2015). Effect of nitrogen and phosphorus fertilizers on growth, yield and yield components of black cumin (Nigella sativa L.) at Konta District, South West Ethiopia. Journal of Agronomy, 14, 112–120. Yosef, H. H. (2008). Effect of high levels of nitrogen and phosphorus fertilizer on growth, yield and yield components of Nigella sativa L.  Horticulture Department College of Agriculture, Duhok University, Iraq. Mesopotamia Journal Agriculture, 36, 2–11. Zapotoczny, P., Zuk-Golaszewska, K., & Ropelewska, E. (2019). Impact of cultivation methods on properties of black cumin (Nigella sativa L.) seeds. Journal of Central European Agriculture, 20, 353–364.

Chapter 3

Morphological Characters of Nigella sativa Mohamed Helmy El-Morsy and Hanan El-Sayed Osman

Abstract  Herbs are an important vital source of drugs from ancient times in several countries. Among these herbs is the Nigella sativa, which has been widely used in traditional medicine in different civilizations around the world as a treatment for humans and animals. Black seed is an annual medicinal plant belonging to the buttercup family (Ranunculaceae). Nigella sativa has many common names that vary from one language to another and from country to country. Black seed plants and seeds are known to all Arabian and Islamic countries and carry various colloquial names. It is known generally by the names Habbat Albarakah, Alhabahat Alsawda and Alkamoun Alaswad (black seeds). In some countries, it is known by the names Shuniz and Khodhira. Its English name is black cumin. This chapter reviews on the morphological characteristics of Nigella sativa. Keywords  Agroecology · Black seed · Morphology · Ranunculaceae

1  Introduction According to the data of the world health organization (WHO), more than 75% of the developing countries population depends upon phytomedicine for their health care needs because a lot of them are unable to have access to medication (WHO 2008; Jamshidi-Kia et al. 2018). Herbal medicines and remedies are more effective for certain ailments and low cost compared to the chemical prescribed by the doctor.

M. H. El-Morsy (*) Deanship of Scientific Research, Umm Al-Qura University, Makkah, Saudi Arabia Range Management Unit, Desert Research Center, Cairo, Egypt e-mail: [email protected] H. E.-S. Osman Biology Department, Faculty of Applied Science, Umm Al-Qura University, Makkah, Saudi Arabia Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_3

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In addition, the chemical medicine prescribed by pharmacists or doctors could have certain negative side effects. Among several medicinal plants, Nigella sativa (black seed) has been considered one of the most treated nutrient-rich herb in history around the world. Scientific studies are in progress to validate the traditionally claimed uses of a small seed of this species (Takruri and Dameh 1998; Ramadan 2007). Traditionally, seeds of N. sativa have been used to treat many diseases in several countries as well as in folk medicine. Currently, several investigations are carried out to focus on herbal plant properties, like, mechanism of action, evaluation of their safety, and toxicology. Black seeds are one of the greatest forms of healing medicine. The herb has been recommended for use on a regular basis in Tibb-e- Nabwi. In Holy Bible, the plant is known as the curative black cumin and described as Melanthion by Hippocrates. There are about 20 varieties (including unmatched seeds) of black seeds distributed and spread over many regions from the Mediterranean Sea to west Asia. Plants are cultivated and distributed in India, Syria, Lebanon, the Middle East, and Southern Europe.

1.1  Scientific Classification of the Plant

Domain: Kingdom: Subkingdom: Super division: Phylum: Class: Subclass: Order: Family: Genus: Species:

Eukarya Plantae (Plants) Tracheobionta (Vascular plants) Spermatophyta (Seed plants) Magnoliophyta (Flowering plants) Magnoliopsida (Dicotyledons) Magnoliidae Ranunculales Ranunculaceae (Buttercup family) Nigella N. sativa

2  Common Names English: fennel flower, nutmeg flower; French: nigelle de Crète, toute épice; German: Schwarzkümmel, Roman coriander, blackseed or black caraway, black sesame; India: Assamese  - kaljeera or kolajeera, Bengali-kalo jeeray, Kannada-­ Krishna Jeerige, Tamil-karum jeerakaml; Hindi/Urdu - kalaunji/mangrail; Russian: Chernushka; Hebrew: Ketzakh; Turkish: çörek out; Arabic: habbat al-barakah;

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Persian: siyâhdâne; Indonesian: jintan hitam; Bosnian: urekot; Portuguese: cominho-negro; Spanish: ajenuz, arañuel; Swedish: svartkummin.

3  Agroecology Nigella sativa is a hardy cool-season crop, with an optimum temperature of 15 °C and a range of 5–25 °C. It grows in full sun on a wide range of well-drained soils from pH 6–7, but prefers sandy loam soils. It is quite a drought-tolerant and can survive in dry soils, but requires regular watering during prolonged dry periods (Paarakh 2010).

4  Morphological Features Nigella sativa is an erect annual herb attending 30–68 cm (mean 52 cm) at maturity (Benkaci-Ali et al. 2007).

4.1  Root The species belonging to the Ranunculaceae family which characterized by tap root, adventitious or tuberous. The tap root system is in the initial stage but eventually replaced by the adventitious roots (Ramsey 1987).

4.2  Stem Stem very slender, erect sometimes-upper part branched, glabrous, 20–50 cm, sparingly branched (Fig. 3.1). The stem is becoming hollow with age and light to dark green (KÖkdil et al. 2006). The number of primary branches per plant ranges from 4 to 10.

4.3  Leaves The plant has shiny green, and tripartite leaves (Nergiz and Ötleş 1993). The leaves are pinnate sect 2–4 cm long cut into the linear segment (Fig. 3.1), which is oblong in shape. Leaf arrangement is alternate, and the number of pinna per rachis is 5–6 (Datta et al. 2012) (Fig. 3.2).

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Fig. 3.1  Nigella sativa plants in the field (https://rareplant.me/treesflowers/black-cumin)

Fig. 3.2  Nigella sativa plant has an erect, branching stem, bears linear green leaves

4.4  Flower Flowers are hermaphrodite with determinate flowering patterns, the main axis terminates with a solitary flower, delicate flower size 2.74 × 2.78 cm (Fig. 3.3). The flowers have a different colors (could be, white, yellow, pink, pale blue or pale

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Fig. 3.3  Some forms of Nigella sativa flowers (Nigella sativa has a hermaphrodite, solitary flower with a different color)

purple) with 5–10 petals (Ahmad et al. 2013; Kooti et al. 2016). Floral parts are depressed in a conical receptacle. The flowers have a variable number of sepals, ovate in shape, and characterized by the presence of nectarines. Also, the flowers without any involucre of bracts, and have a long peduncle (Benkaci-Ali et al. 2007).

4.5  Androecium The stamens in 3–4 whorls, numerous and shed their pollen as the filament bent outward during the male phase (Kumar 2009). Stamens are polyandrous, spirally arranged with long filaments and yellow anthers.

4.6  Gynoecium Gynoecium has 5–12 united carpels, each with a long indehiscent style composed of a variable number of the multi-ovule carpels, developing into a follicle after pollination (Kumar 2009).

4.7  Fruit The fruit is a single large and inflated capsule composed of 3–7 united follicle (Fig. 3.4), dehiscence of seed from fruit occur through suture. Fruits length ranges from 0.4 to 1.7  cm with numerous seeds (Hassanien et  al. 2015; Rajsekhar and Kuldeep 2011).

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Fig. 3.4  Nigella sativa matured fruit capsule contains seeds

Fig. 3.5  Nigella sativa seeds are three-cornered, with two sides flat and one convex, black or brown externally, and white and oleaginous within. Seeds have a strong, agreeable aromatic odor, like that of nutmeg, and a spicy, pungent taste

4.8  Seeds Numerous small, dicotyledonous seeds are found in the capsule. Each seed is ovate, tetrangular, angles sharp with more tapering end (Fig.  3.5). The seed size is 2.33 × 1.14 mm. the average seed production is 59.3 g/plant. The seeds are black externally, white inside, odour slightly, and taste bitter (Rajsekhar and Kuldeep 2011).

References Ahmad, A., Husain, A., Mujeeb, M., Khan, S.  A., Najmi, A.  K., Nasir Ali Siddique, N.  A., Damanhouri, Z. A., & Anwar, F. (2013). A review on therapeutic potential of Nigella sativa: A miracle herb. [J] Asian Pacific Journal of Tropical Biomedicine, 3(5), 337–352. Benkaci-Ali, F., Baaliouamer, A., Meklati, B. Y., & Chemat, F. (2007). Chemical composition of seed essential oils from Algerian Nigella sativa extracted by microwave and hydrodistillation. Flavour and Fragrance Journal, 22, 148–153. Datta, A. K., Saha, A., Bhattacharya, A., Mandal, A., Paul, R., & Sengupta, S. (2012). Black cumin (Nigella sativa L.)- A review. Journal of Plant Development Sciences, 4(1), 1–43.

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Hassanien, M. F. R., Assiri, A. M. A., Alzohairy, A. M., & Oraby, H. F. (2015). Health-promoting value and food applications of black cumin essential oil: An overview. Journal of Food Science and Technology, 52(10), 6136–6142. Jamshidi-Kia, F., Lorigooini, Z., & Amini-Khoei, H. (2018). Medicinal plants: History and future perspective. Journal of Herb Med Pharmacology, 7(1), 1–7. KÖkdil, G., Çim, A., ÖzbilGin, B., & Uygun, C. (2006). Morphology and stem anatomy of some species of genus Nigella L. in Turkey. Journal of Faculty of Pharmacy of Ankara, 35(1), 19–41. Kooti, W., Hasanzadeh-Noohi, Z., Sharafi-Ahvazi, N., Asadi-Samani, M., & Ashtary-Larky, D. (2016). Phytochemistry, pharmacology, and therapeutic uses of black seed (Nigella sativa). Chinese Journal of Natural Medicines, 14(10), 0732–0745. Kumar, A. (2009). A textbook of practical botany 2 (p. 646). Rastogi Publications. Nergiz, C., & Ötleş, S. (1993). Chemical composition of Nigella sativa L. seeds. Food Chemistry, 48, 259–261. Paarakh, P. M. (2010). Nigella sativa Linn.-A comprehensive review. [J] Indian Journal of Natural Products and Resources, 1, 409–429. Rajsekhar, S., & Kuldeep, B. (2011). Pharmacognosy and pharmacology of Nigella sativa. International Research Journal of Pharmacy, 2(11), 36–39. Ramadan, M.  F. (2007). Nutritional value, functional properties and nutraceutical applications of black cumin (Nigella sativa L.): An overview. International Journal of Food Science & Technology, 42(10), 1208–1218. Ramsey, G.  W. (1987). Morphological considerations in the North American Cimicifuga (Ranunculaceae). Castanea, 52(2), 129–141. Takruri, H. R. H., & Dameh, M. A. F. (1998). Study of the nutritional value of black cumin seeds (Nigella sativa L). Journal of the Science of Food and Agriculture, 76(3), 404–410. World Health Organization (WHO). (2008). Traditional medicine fact sheet No 134, 2017. http:// www.siav-itvas.org/images/stories/doc/.../WHOTraditionalmedicine2008.pdf/

Chapter 4

Micro and Macroscopic Characterization of Traded Nigella sativa Seeds Using Applied Systematics Techniques Sofia Rashid, Muhammad Zafar, Mushtaq Ahmad, Shazia Sultana, Sidra Nisar Ahmed, and Omer Kilic

Abstract  Black cumin (Nigella sativa L., family Ranunculaceae) has been used for many centuries in traditional systems of medicines as a health remedy for numerous diseases. Nigella sativa is an annual flowering plant producing small seeds with aromatic odor and taste. The seeds have great importance in different systems of medicine like Unani and Tibb, Ayurveda and Siddha. The seeds and oil obtained from this herb have been extensively used in food as a spice and condiment. Furthermore, they have medicinal worth and used as a diuretic, liver tonics, appetite stimulant, digestive, and analgesics. Nigella sativa is considered as one of the greatest forms of healing medicine in Islamic literature. In Tibb-e-Nabwi (Prophetic Medicine), Nigella sativa seeds have been recommended for use on a regular basis. The medicinal properties of N. sativa are due to the presence of thymoquinone (TQ), which is the main bioactive component of its essential oil. The miraculous healing power, N. sativa renders it among the top evidence-based herbal medicines. The present work is based on various techniques comprising morphological (qualitative and quantitative), palynological (LM & SEM), anatomical and pharmacognostic examination. Palynological features of N. sativa are characterized by the presence of tricolporate pollen with scan rate exine sculpturing pattern. However, S. Rashid Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan Department of Bio Sciences, Comsats University Islamabad, Islamabad, Pakistan e-mail: [email protected] M. Zafar (*) · M. Ahmad · S. Sultana Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan e-mail: [email protected] S. N. Ahmed Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan Department of Botany, The Women University Multan, Multan, Pakistan O. Kilic Department of Basic Science of Pharmacy, Adıyaman University, Adıyaman, Turkey © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_4

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anatomical studies exposed irregular epidermal cells, actinocytic stomata and non-­ glandular trichomes in N. sativa. The pharmacognostic examination includes fluorescence analysis and physicochemical parameters like moisture content, total ash, acid insoluble ash, water-soluble ash, and water-insoluble ash. The data is useful for micro and macroscopic characterization of herbal drug Nigella sativa. Keywords  Bioactive compounds · Pharmacological potentials · Prophetic Medicine · Morphology · Palynology · Anatomy

Abbreviations LM Light Microscopy SEM Scanning Electron Microscopy TQ Thymoquinone

1  Introduction The medicinal plants have gained immense popularity nowadays to be made into medicines as opposed to pharmaceutical medicines, due to their natural and safe characteristics. Nigella sativa is one of the emerging medicinal herbs recognized in markets as black cumin, fennel flower, Roman coriander and Kalonji (Weiss 2002). Other names of the herb are black caraway seeds (USA), kalonji (Urdu and Hindi), habba-tu sawda (Arabic), krishnajirika (Sanskrit), kala jira (Bangali), and shonaiz (Persian). N. sativa seeds are commonly known as black seed. This herb is native to Southern Europe, Southwest Asia, and North Africa and currently it is grown in many countries like Pakistan, India, Iran, Middle East, Turkey, Syria, and Saudi Arabia. As the trend of medicine shifts back to its ancient roots, significant research has been devoted throughout the world to investigate black cumin for its alleged medicinal properties. A number of bioactive compounds have been found in this herb with beneficial effects on health (Hussain et  al. 2006). Interestingly, black seeds and its oil play an important role in food as well as in medicine of historical Indian and Saudi Arabian civilizations (Yarnell and Abascal 2011). The seeds have been extensively used in south Asian and Middle Eastern cuisines as spices for preparing meals due to pungent bitter taste and aroma. They are perfect to add flavor in curries, vegetables, and pulses, as well as in bread and pickles. In Egypt, black cumin was historically used as a mummifying preservative. The Indians seemed to have figured out cumin’s medicinal properties long before anyone else and used it for Unani and Ayurveda (Sharma et  al. 2005). The active constituents of black

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cumin seeds have been known to cure respiratory problems like bronchitis, asthma, and other inflammatory diseases. N. sativa therapeutic potential makes it a consequential substance and possesses a wide spectrum of activities like anticancer, ­anti-­microbial, anti-inflammatory, gastroprotective, hepatoprotective, spasmolytic, bronchodilator and anti-diarrheal. It can be used as a liver tonic and parasitic infections could be relieved. Conventionally, black seeds are broadly used for diabetes, fever, hypertension, bronchitis, gastrointestinal disturbances, soreness and skin problems (Goreja 2003).

2  Micro and Macroscopic Characterization of N. sativa Micro and macroscopic characterization play a vital role to discriminate closely related species. Morphological characters can be used as an important tool for the safety, purity, and quality of traded drugs by showing variations in vegetative as well as floral morphology. Nigella sativa is an annual herb having linear, finely divided leaves. The seeds are triquetrous and rugose with white oily interior (Fig.  4.1a, b). A detailed morphological description of N. sativa is presented in Table 4.1. In some cases, morphological characters show resemblance which causes misidentification of traded drugs (Shaheen et al. 2014). In such cases, palynological characters can be used as an aid in establishing the systematic position of taxa within their respective classification by providing additional taxonomic characters. Palynological investigation revealed that N. sativa has tricolporate pollen and scan rate exine sculpturing (Fig.  4.1c, d). Palynological features of N. sativa are presented in Tables 4.2 and 4.3. Other valuable characters that can be used to discriminate alike taxa are foliar epidermal characters such as epidermal cells shape, stomata type, and presence of trichomes (Naz et al. 2009; Riaz et al. 2010). Their importance is fully supported by previous work which is helpful to define plant taxa (Ullah et al. 2019; Zaman et al. 2018). Valuable intergeneric and interspecific variations in foliar epidermal cells have been reported by Ahmad et al. (2010) that can be used as an important taxonomic tool for identification. The foliar epidermal features of N. sativa is characterized by the presence of irregular epidermal cells with normocytic stomata and non-glandular trichomes (Fig. 4.1e, f). Detailed foliar epidermal features of N. sativa are shown in Tables 4.4 and 4.5. Trichomes display an important role at a microscopic level to differentiate similar species (Adedeji 2004; Adedeji et al. 2007). The trichome type is one of the main characteristics required in the discrimination of species (Ashfaq et al. 2019). Their configuration and nature are of remarkable significance in systematics (Khokar 2009). Pharmacognostic parameters are useful for the quality and purity assessment of crude herbal drugs (Kadam et al. 2012). Fluorescence analysis is an essential parameter, which is unique to a specific plant and helpful in its standardization (Prasanth et  al. 2017). Fluorescence analysis and solubility tests of N. sativa with various chemical reagents are presented in Table 4.6. The authentication of various drugs having plant origin is carried out by pharmacognostic features (Kokoski et al. 1958;

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Fig. 4.1 (a) Nigella sativa flower, (b) dried seeds, (c) SEM of pollen (polar view), (d) pollen sculpturing (equatorial view), (e) epidermal cells and stomata (abaxial: 40X), (f) epidermal cells and stomata (adaxial: 40X)

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Table 4.1  Morphological characteristics of Nigella sativa Character English names Family Flowering period Phytogeography

Nigella sativa Black seed, black cumin, fennel flower Ranunculaceae February–March South Europe, South West Asia, North Africa, Arab countries, Pakistan, India, Bangladesh, Turkey, the Middle East, and the Mediterranean basin. Morphological Annual herb, short-lived, slender or stout, 20–60 cm tall, pubescent, hairs description are glandular. Stem erect, stiff, simple or branched and striate finely. Leaves are 2.5 cm wide, linear, acute, dark green, delicate and finely divided by thread-like and wispy lobes with 0.8–2 mm width. Flowers solitary, delicate and without an involucre. Sepals are ovate, ± obtuse, pale blue or whitish, five in number, puberulous and with a distinct, short stipe. Petals with a short, thick capitate appendix. Follicles are tuberculate, inflated and coherent over the entire length. Seeds triquetrous, 1–5 mm long, black or grayish black having an oily white interior, rugose, ends tapered and slightly curved. Seeds are traded at herbal shops. Organoleptography Aerial dried branches are rigid and light green in color. Leaves grayish-­ green, pinnatisect, segments oblong, 2–4 cm long. Floral parts are delicate, pale or bluish-white, star-shaped, terminal and solitary. Seeds are compressed, black in color, trigonous and angular, sides are smooth and one convex having aromatic odor and pungent spicy taste. Part used Seeds Ethno-medicinal Indigestion, nausea, diarrhea, stomach pains, spasms, dropsy, menstrual uses disorders, insufficient lactation, bronchial problems, intestinal worms, and skin rashes. Traditional folk Tincture of seed is used in diarrhea, indigestion, loss of appetite, dropsy, recipes worm treatment, and skin eruptions. The seeds are grounded and mixed with water to make a paste used to treat boils. Seeds are used to increases milk flow in nursing mothers. Roasted seeds are effective to stop vomiting. Powdered seeds are used to remove lice from the hairs. Grounded seeds mixed with sesame oil are externally applied for the treatment of abscesses and hemorrhoids. Oil of seed is used externally as an antiseptic and local anesthetic. Table 4.2  Qualitative palynological features of N. sativa Species name N. sativa

Shape (polar Pollen type view) Tricolporate Circular

Shape (equatorial Colpi (present / view) absent) Prolate-spheroidal Present

Sculpturing of exine Scabrate

Table 4.3  Quantitative palynological features of N. sativa Species name N. sativa

Polar diameter (μm) 45.5 (43.5–47.5)

Equatorial diameter (μm) 51.75 (47.7–56.5)

P/E ratio 0.87

Colpi length (μm) 1.25 (1.2–1.5)

Colpi width (μm) 3.5 (3.35– 3.85)

Exine thickness (μm) 3.22 (3.12–3.45)

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Table 4.4  Qualitative features of foliar epidermal anatomy of N. sativa Abaxial epidermis Species Cell name shape

Wall pattern

N. sativa Irregular Weakly undulate

Stomata type

Adaxial epidermis Trichome Cell Wall type shape pattern

Anom ocytic

Non-­ glandular

Irregular

Weakly undulate

Trichome Stomata type type Anomocytic

Non-­ glandular

Badami et al. 2002; Sunita et al. 2010). The physicochemical parameters also play a very important role in the quality assurance of herbal drugs. The physicochemical parameters of N. sativa evaluated in the study are presented in Table  4.7. The ­moisture content of herbal drugs should not be more than 14% w/w (Ilanchezhian et  al. 2011). During the storage of crude drugs, the growth of fungi and yeast is enhanced in excess moisture that may cause the breakdown of crucial bioactive compounds. Antara (2012) employed such physicochemical parameters for correct identification and standardization of powdered drugs.

3  High-Value Bioactive Compounds in N. sativa Seeds The high nutritional and therapeutic value of N. sativa seeds can be related to the significant amount of protein, fiber, minerals, and vitamins. Black seed contains: (1) Protein (26.7%) (2) Lipids (28.5%) (3) Carbohydrates (24.9%) (4) Crude fibre (8.4%) (5) Ash (4.8%) Glutamate, arginine, and aspartate were identified among various amino acids, while the major and minor amino acids were cysteine and methionine. The phytochemical analyses of N. sativa showed the presence of more than a hundred phytoconstituents primarily alkaloids, saponins, sterols, and essential oil. The most important active compounds of N. sativa as reported by Boskabady and Shirmohammadi (2002) and Ali and Blunden (2003) are thymoquinone (Fig. 4.2), dithymoquinone (nigellone), thymohydroquinone, carvacrol, p-cymene, t-anethole, 4-terpineol, sesquiterpene longifolene α-pinene and thymol. The therapeutic properties of N. sativa are mainly due to its quinine component wherein thymoquinone (TQ) is the most bioactive compound and a major constituent of the essential oil with a variety of pharmacological benefits.

Abaxial epidermis Species Cell size (μm) name L × W N. sativa 117.5 (85.2– 150.6) × 45.5 (40.5–50.3)

Stomata Size (μm) L × W 46.5 (45.3– 47.2) × 39.75 (37.5–40.2)

Stomatal index (%) 18.5

Trichomes (μm) L × W 217.5 (185.2– 250.6) × 24.5 (10.5–37.3)

Table 4.5  Quantitative features of foliar epidermal anatomy of N. sativa Adaxial epidermis Cell size (μm) L × W 121.2 (84.7– 147.3) × 46.7 (39.2–52.2)

Stomata size (μm) L × W 43.3 (41.5– 45.8) × 38.25 (35.5–39.5)

Stomatal index (%) 12.81

Trichomes (μm) L × W 225.2 (189.7– 247.3) × 27.7 (15.2–32.2)

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Table 4.6  Fluorescence analysis and solubility test of N. sativa powdered seeds

S. No. Treatment 1 Dried seeds powder 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Powder + 5% KOH

Under visible light Black

Grayish black Powder + 10% aq. Purplish FeCl3 black Powder + dH2O Grayish black Powder + HCL Conc. Reddish black Powder + HCL 50% Yellowish black Powder + H2SO4 Reddish Conc. black Powder + H2SO4 50% Yellowish black Powder + HNO3 Reddish Conc. black Powder + HNO3 50% Brownish black Grayish Powder + CH3OH Conc. black Powder + CH3OH Grayish 50% black Powder + CHCl3 Purplish Conc. black Powder + CHCl3 50% Brownish black Powder + C2H5OH Orion Conc. Powder + C2H5OH Light 50% chocolate Powder + CH3COOH Grayish Conc. black Powder + CH3COOH Brownish 50% black Powder + C6H6 Conc. Yellowish gray Powder + C6H6 50% Milky gray

Filter paper under Under UV visible light Purplish – black Brownish Antique black white Brownish Halo black Purplish Satin white gray Purplish Rose white black Purple Orchid shadow Purplish Antique brown white Purplish Halo brown Brownish Ash white black Brownish Antique purple white Ash white Satin white Sky gray

Onyx

Purplish brown Snow mountain Lavender white Ash white

Antique white Lavender white Satin white

Orion

Ash white

Light chocolate Halo

Greenish purple Satin white

Orion

Brilliant white

Sky gray

Filter paper under UV light Solubility – – Ash white Ash white Snow bell

Semi soluble Semi soluble Semi soluble Semi soluble Partially soluble Soluble

Light mulberry Lavender white Light chocolate Light brown Partially soluble Lavender Soluble white Snow bell Soluble Ash white Orchid shadow Snow bell Snow mountain Light mulberry Orchid shadow Halo Ash white Brilliant white Ash white

Partially soluble Partially soluble Slightly soluble Slightly soluble Partially soluble Partially soluble Soluble Slightly soluble Partially soluble Partially soluble

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Table 4.7  Physicochemical parameters of N. sativa Physicochemical parameter (%) Total ash Acid insoluble ash Water-soluble ash Water-insoluble ash Moisture content

Nigella sativa 4 0.67 2.66 1.34 6

Fig. 4.2  Structure of thymoquinone (TQ)

4  Pharmacological Potentials of N. sativa During the last few decades, N. sativa has been extensively studied and it was revealed that the herb exhibits several pharmacological actions and medicinal properties. The literature relevant to the pharmacological perspective of N. sativa was retrieved by using PubMed, Scopus, Science Direct, and Google Scholar. Some of the major pharmacological properties of the N. sativa are given below. Anti-inflammatory Activities The chronic or acute pain like arthritis, osteoarthritis, cystic fibrosis, allergies, asthma, and cancer are linked to inflammation, which has been known as the main cause in the growth of various tumors. The current anti-inflammatory drugs, unfortunately, might have adverse effects related to the bone marrow, depression, gastric ulcer, water, and salt retention (Das et al. 2014). This makes it more important to switch entirely to medicinal herbs, such as black cumin, to potentially combat any reaction. The oil extracted from N. sativa and TQ at varying quantities showed anti-­ inflammatory potential against carrageenan-induced hind paw edema in rats’ that is equivalent to indomethacin (Pise and Padwal 2017). The experimental study to explore the effects of N. sativa against inflammation in the patients with signs of allergic rhinitis resulted in a reduction in the nasal mucosal and irritation, sneezing, turbinate hypertrophy, and mucosal pallor (Nikakhlagh et al. 2011). Antidiabetic Activities Many limitations caused by some existing synthetic agents, hinders the exploration and advancement of managing diabetes (Daryabeygi-Khotbehsara et al. 2017). The seeds of N. sativa have been previously used to cure diabetes mellitus in indigenous systems of medicinal remedies. In a controlled experimental study of 99 patients with diabetes got the placebo and two sets were given oral administration of N. sativa seed oil. After 20 days of administration of 1.5 and 3 mL/day of black seed oil, a

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substantial decrease of random sugar amounts in blood and glycated hemoglobin A1c was observed (Rachman 2017). Antioxidant Activities There is a recent trend to exploit medicinal plants as natural antioxidants. The amplification of free radicals is accompanied by the principal indicators coupled with a number of progressive pathological conditions like aging, cancer, endocrine sickness and neurological disorders (Lupoli et al. 2018). N. sativa has been reported as one of the naturally occurring medicinal plants, that serves as a valuable ­antioxidant equally in vivo and in vitro (Ozdemir et  al. 2018). Components like carvacrol, t-anethole and 4-terpineol and TQ in N. sativa essential oil exhibit radical scavenging properties. In 2,2-diphenyl-1-picrylhydrazil assay (DPPH·), these compounds powerfully scavenged OH radical for non-enzymatic lipid peroxidation as compared to synthetic antioxidants. Antimicrobial Activities The medicinal plants are renowned for centuries due to their antimicrobial actions, but in the early 1900 efforts to exemplify these qualities in the research were carried out (Dorman and Deans 2000). Currently, the improvement of resistance through a pathogen to frequently used antibiotics puts forward strength to make attempts in the innovation of new microbial agents to get rid of the infection, overcome the difficulties during resistance and side effects of the presently used antimicrobial treatments (Morsi 2000). The antimicrobial activity in N. sativa is ascribed to the active constituents chiefly melanin and TQ (Bakathir and Abbas 2011), which encompasses wide-ranging actions responsible to affect the key processes of the organisms (Monika et al. 2013). Anticancer Activities One of the devastating diseases is cancer which prompts efforts to overcome it by the introduction of several natural remedies and to keep away from conventional chemotherapies. The antioxidant nature of N. sativa is studied both in vivo and in vitro techniques. The constituents, as well as oil extracted from this black cumin, are helpful against cancer of any body part like skin, liver, lungs, kidneys, breast, prostate and cervix. N. sativa exposes anti mutagenic, antimetastatic, antiproliferative, pro-apoptotic effects in malignant cell lines (Majdalweih and Fayyad 2016). There are ten traits of cancer cells that are common to the majority of the tumors. The Nigella sativa has prominent active constituents like TQ which is responsible to target all tumor cells and possess chemopreventive characteristics that are responsible to alter multiplication in cancer cell lines (Aggarwal et al. 2008; Allahgadri et al. 2010; Schneider-stock et al. 2014).

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Gastro-Protective Activities The oil and chemical constituents extracted from N. sativa have the potential to cure and prevent gastric ulcers (Khan et al. 2016). TQ owns a unique gastro defensive device by preventing proton pump, acid emission neutrophil infiltration, boost mucin exudation, and nitric oxide production (Magdy et al. 2012). In rats, experimentally induced gastric sores and basal gastric discharge were treated with N. sativa aqueous suspension to streamline this practice in herbal and Unani medicine. N. sativa considerably prohibited gastric ulcer growth triggered by necrotizing mediators by replacing the depleted stomach wall secretion content and stomach mucosal non-protein sulfhydryl concentration. Hepato-Protective Activities N. sativa eliminates the harmful properties of ischemia-reperfusion damage in rat’s liver (Yildiz et al. 2008; Zafeer et al. 2012). The administration of N. sativa showed a protective influence on the hepatic tissues from the deleterious effects of toxic metals (Kapoor 2009). Significant protection is shown by pretreatment with TQ (10 μmol/L) as indicated by the recovery of the depleted antioxidants and attenuation of protein oxidation, proving that modulatory effects have been exerted by TQ on the antioxidative defense system. Nephroprotective Activities The nephron-protective medicinal property of N. sativa oil was observed in rabbits against gentamicin-induced nephrotoxicity. N. sativa oil processed nephroprotective effects as it lowers the blood urea nitrogen (Saleem et al. 2012). The nephroprotective effects of black cumin oil revealed defensive consequences against methotrexate-induced nephrotoxicity in albino rats (Abul-Nasr et al. 2001; Yaman and Balikci 2010). Neuroprotective Activities Nowadays, depression is the most dominant ailments among neurological disorders globally. The main cause of which is the hypoactivity of neurotransmitters, especially the insufficient activity of serotonin (Perveen et al. 2014). Black cumin is the most effective medicinal herb for treating depression and many allied neurological disorders. Many studies have been conducted on neurological effects of N. sativa seeds and reported that the major active constituent of black cumin seeds i.e., TQ and its oil are potential natural therapy for the treatment of neurological complaints based on the wide range of neuropharmacological results.

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Kokoski, C. J., Kokoski, R. J., & Slama, F. J. (1958). Fluorescence of powdered vegetable drugs under ultraviolet radiation. Journal of the American Pharmaceutical Association, 47(10), 715–717. Lupoli, F., Vannocci, T., Longo, G., Niccolai, N., & Pastore, A. (2018). The role of oxidative stress in Friedreich’s ataxia. FEBS Letters, 592(5), 718–727. Magdy, M.  A., Hanan, E.  A., & Nabila, E.  M. (2012). Thymoquinone: Novel gastroprotective mechanisms. European Journal of Pharmacology, 697(1–3), 126–131. Majdalawieh, A. F., & Fayyad, M. W. (2016). Recent advances on the anti-cancer properties of Nigella sativa, a widely used food additive. Journal of Ayurveda and Integrative Medicine, 7(3), 173–180. Monika, T., Sasikala, P., & Vijaya Bhaskara Reddy, M. (2013). A investigational of antibacterial activities of Nigella sativa on mastaitis in dairy crossbred cows. International Journal of Advance Technical Research, 3, 263–272. Morsi, N. M. (2000). Antimicrobial effect of crude extracts of Nigella sativa on multiple antibiotics-­ resistant bacteria. Acta Microbiologica Polonica, 49(1), 63–74. Naz, N., Hameed, M., Ashraf, M., Ahmad, R., & Arshad, M. (2009). Eco-morphic variation for salt-tolerance in some grasses from Cholistan Desert, Pakistan. Pakistan Journal of Botany, 41(4), 1707–1714. Nikakhlagh, S., Rahim, F., Aryani, F.  H. N., Syahpoush, A., Brougerdnya, M.  G., & Saki, N. (2011). Herbal treatment of allergic rhinitis: The use of Nigella sativa. American Journal of Otolaryngology, 32(5), 402–407. Ozdemir, N., Kantekin-Erdogan, M. N., Tat, T., & Tekin, A. (2018). Effect of black cumin oil on the oxidative stability and sensory characteristics of mayonnaise. Journal of Food Science and Technology, 55(4), 1562–1568. Perveen, T., Haider, S., Zuberi, N. A., Saleem, S., Sadaf, S., & Batool, Z. (2014). Increased 5-HT levels following repeated administration of Nigella sativa L. (black seed) oil produce antidepressant effects in rats. Scientia Pharmaceutica, 82(1), 161–170. Pise, H. N., & Padwal, S. L. (2017). Evaluation of anti-inflammatory activity of Nigella sativa: An experimental study. National Journal of Physiology, Pharmacy and Pharmacology, 7(7), 707. Prasanth, D. S. N. B. K., Rao, A. S., & Prasad, Y. R. (2017). Pharmacognostic standardization of Aralia racemosa L. stem. Indian Journal of Pharmaceutical Sciences, 79, 220–226. Rachman, P. N. R., Akrom, A., & Darmawan, E. (2017). The efficacy of black cumin seed (Nigella sativa) oil and hypoglycemic drug combination to reduce HbA1c level in patients with metabolic syndrome risk. In IOP Conference Series. Materials Science and Engineering, 259, 10. Riaz, A., Younis, A., Hameed, M., & Kiran, S. (2010). Morphological and biochemical responses of turf grasses to water deficit conditions. Pakistan Journal of Botany, 42(5), 3441–3448. Saleem, U., Ahmad, B., Rehman, K., Mahmood, S., Alam, M., & Erum, A. (2012). Nephro-­ protective effect of vitamin C and Nigella sativa oil on gentamicin associated nephrotoxicity in rabbits. Pakistan Journal of Pharmaceutical Sciences, 25(4), 727–730. Schneider-Stock, R., Fakhoury, I. H., Zaki, A. M., El-Baba, C. O., & Gali-Muhtasib, H. U. (2014). Thymoquinone: Fifty years of success in the battle against cancer models. Drug Discovery Today, 19(1), 18–30. Shaheen, S., Ramzan, S., Haroon, N., & Hussain, K. (2014). Ethnopharmacological and systematic studies of selected medicinal plants of Pakistan. Pakistan Journal of Science, 66(2), 175–180. Sharma, P. C., Yelne, M. B., & Dennis, T. J. (2005). Database on medicinal plants used in Ayurveda (pp. 420–440). New Delhi: Central Council for Research in Ayurveda & Siddha. Sunita, P., Pattanayak, S.  P., & Oraon, A. (2010). Pharmacognostic studies on leaves of Dendorphthoe falcata (L.f) Ettingsh. Hamdard Medicus, 53(1), 106–112. Ullah, F., Shah, S. N., Zaman, W., Zafar, M., Ahmad, M., Ayaz, A., Sohail, A., & Saqib, S. (2019). Using palynomorphological characteristics for the identification of species of Alsinoideae (Caryophyllaceae): A systematic approach. Grana, 58, 174–184. Weiss, E. A. (2002). Spice crops. Wallingford: CABI Publishing. CABI International.

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Yaman, İ., & Balikci, E. (2010). Protective effects of Nigella sativa against gentamicin-induced nephrotoxicity in rats. Experimental and Toxicologic Pathology, 62(2), 183–190. Yarnell, E., & Abascal, K. (2011). Nigella sativa: Holy herb of the Middle East. Alternative and Complementary Therapies, 17(2), 99–105. Yildiz, F., Coban, S., Terzi, A., Ates, M., Aksoy, N., Cakir, H., et  al. (2008). Nigella sativa relieves the deleterious effects of ischemia reperfusion injury on liver. World Journal of Gastroenterology, 14(33), 5204. Zafeer, M. F., Waseem, M., Chaudhary, S., & Parvez, S. (2012). Cadmium-induced hepatotoxicity and its abrogation by thymoquinone. Journal of Biochemical and Molecular Toxicology, 26(5), 199–205. Zaman, W., Shah, S. N., Ullah, F., Ayaz, A., Ahmad, M., & Ali, A. (2018). Systematic approach to the correct identification of Asplenium dalhousiae (Aspleniaceae) with their medicinal uses. Microscopy Research and Technique, 2019(82), 459–465.

Chapter 5

Composition of Nigella sativa Seeds Hamid Mukhtar, Muhammad Waseem Mumtaz, Tooba Tauqeer, and Syed Ali Raza

Abstract  The dietary intake of Nigella sativa seeds and seed oil is associated with the cure and management of many chronic ailments and improving the general health. The medicinal properties of Nigella sativa seeds are due to the presence of various biologically functional compounds and nutritional components. The increasing interest of consumers in nutraceutical attributes of Nigella sativa seeds serves as an impetus to discuss the up to date knowledge on its compositional profile. Most of the properties of Nigella sativa seeds are due to their oil contents. The physicochemical analysis of Nigella sativa seed oil reveals that it contains substantial amounts of minerals, lipids, essential and non-essential amino acids. The essential oil from Nigella sativa is composed of thymoquinone (42.3–56.1%), p-cymene (33–38%), carvacrol (1.3–1.4%), dehydro-sabina ketone (4.4–4.5%), α-thujene (6%), camphene (11%), α-pinene (1.11%), β-pinene (7%), sabinene (1%), α-phellandrene (0.45%), β-myrcene (0.21%), γ-terpinene (5.12%), limonene (0.13%), camphor (1%), terpinolene (0.23%), thymol (10.1%), carvone (0.32%), t-anethol (1–4%), 4-terpineol (2–7%), longicyclene (0.9%), and sesquiterpene longifolene. Among alkaloids, isoquinoline derived alkaloids nigellicin, nigellimine and the two indazole derived alkaloids are present in seeds. The major phenolics of Nigella sativa seeds are thymohydroquinone, dithymoquinone, tannins, flavanol triglycosides and kaempferol 3-O-β-D-glucosylgalactoside. All these compounds contribute to the therapeutic properties of Nigella sativa seeds. Keywords  Mineral · Phenolics · Amino acids · Fatty acids · Nutrition · Alkaloids

H. Mukhtar (*) Institute of Industrial Biotechnology, Government College University, Lahore, Pakistan M. W. Mumtaz · T. Tauqeer Department of Chemistry, University of Gujrat, Gujrat, Pakistan e-mail: [email protected] S. A. Raza Department of Chemistry, Government College University, Lahore, Pakistan © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_5

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Abbreviations kg Kilogram mg Milligram PFAF Plants for a future μg Microgram

1  Nigella sativa Nigella sativa L. (black cumin), a member of family Ranunculaceae, is extensively distributed in the Middle East and Asia. The fruit of Nigella sativa is an inflated capsule with 3–7 follicles, each having many seeds (Fig.  5.1). Morphologically seeds are flat and funnel-shaped, about 0.2  cm length and 0.1  cm in width. The Nigella sativa seeds are generally known as black seeds that are widely used for culinary, medicinal purposes as well as in various traditional/folk medicinal systems for the treatment of numerous health disorders (Fig. 5.2). The medicinal potential of the seeds ought to the presence of various bioactive components. The seeds of Fig. 5.1  Nigella sativa

5  Composition of Nigella sativa Seeds

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Fig. 5.2  Seeds of Nigella sativa PFAF (2019)

Nigella sativa are consumed as a flavouring additive to prickles and bread (Al-Saleh et al. 2006; Dinagaran et al. 2016; Aisa et al. 2019; Zribi et al. 2019).

2  Composition of Black Cumin Seeds The compositional versatility in seeds of Nigella sativa has been observed. The black cumin seed when subjected to compositional estimation through proximate analysis, contained 20.8% protein, 31.9% carbohydrates, and 38.2% lipids, respectively. The moisture content of 4.64%, ash 4.37%, and crude fibre 7.94% were reported in black cumin seeds. Screening for secondary metabolites showed the presence of some functional molecules including alkaloids, flavonoids, saponins and tannins at the amount of 10.1, 3.7, 7.6 and 2.2 mg/100 g, respectively (Mamun and Absar 2018; Kazmi et al. 2019).

3  Dietary Minerals Extensive work has been done on the determination of mineral content in N. sativa seeds. Variable amounts of different minerals are present in seeds of N. sativa (Table 5.1). Among the minerals, potassium, sodium, phosphorus, magnesium and iron are the major ones in N. sativa seeds. However, zinc, copper, calcium and manganese are found at relatively low concentrations. Traces of cadmium, lead, nickel and molybdenum have also been reported in N. sativa seeds (Kabir et al. 2019).

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Table 5.1  Mineral content (mg/100 g) of N. sativa seeds Mamun and Mineral Absar (2018) Sodium 100 Calcium 579.3 Phosphorus 91.5 Iron 41.8 Magnesium 218.3 Potassium 510.3 Copper – Zinc – Manganese –

Al-Jassir (1992) 0.75 0.04 1.8 0.15 0.03 7.6 0.02 0.06 0.02

Sultan et al. (2009) 17.6 570 543 9.70 265 808 2.60 6.23 8.53

Nergiz and Ötleş (1993) 85.3 188 – 57.5 – 1180 – – –

Kabir et al. (2019) 44.8 366.7 481.5 42.6 355.2 1498.3 1.5 6.7 3.1

Cheikh-­ Rouhou et al. (2007) 18.5 564 51.9 9.4 260 708 – – –

4  Proteins The proximate analysis has revealed the presence of 20–21% total protein contents in N. sativa seeds (Babayan et al. 1978; Al-Jassir 1992). The profiling of amino acid in black cumin seeds shows the presence of various essential as well as non-­essential amino acids in variable amounts. The major amino acids in black cumin seeds are glutamic acid, aspartic acid, arginine, and glycine, which are non-essential amino acids (Table 5.2). They constitute about 50% of the total amino acids of proteins in N. sativa seeds. Among the essential amino acids, leucine has the highest percentage. Valine, lysine, threonine, phenylalanine and isoleucine are also present in N. sativa seeds. It was observed that the concentration of non-essential amino acids found in the black cumin seeds is comparatively greater than essential amino acids. The bioactivity of N. sativa seeds is mostly considered to be related to its oil and oil-soluble components of the seeds. However, the therapeutic role of natural proteins cannot be ignored and investigations are being carried out to find the bioactivity of proteins present in seeds of N. sativa (Çakir and Gülseren 2019).

5  Vitamins The vitamins are bioactive substances, essential for the proper health and normal growth of humans. Their small quantities are enough to support normal metabolic functions but their absence can create some serious health impairments. N. sativa seeds contain both fat and water-soluble vitamins. Fat-soluble vitamins are D, E, and K possess radical scavenging properties thus act as antioxidants. Black cumin seeds are the good source of these fat-soluble vitamins (Vatansev et  al. 2013). Vitamins such as B1 (thiamin), B2 (riboflavin), B6 (pyridoxine), PP (niacin) and folic acid are water-soluble vitamins and important constituents of N. sativa seeds. The list of vitamins found in N. sativa seeds and their percentage composition reported in the literature are presented in Table 5.3.

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Table 5.2  Percentage composition of amino acids present N. sativa seeds Amino acid Essential amino acid (%) Leucine Valine Lysine Threonine Phenylalanine Isoleucine Histidine Methionine Tryptophan Non-essential amino acid (%) Glutamic acid Aspartic acid Arginine Glycine Proline Serine Alanine Tyrosine Cystine

Kabir et al. (2019)

Al-Gaby (1998)

Al-Jassir (1992)

Michel et al. (2011)

6.00 4.77 3.86 3.86 3.70 3.69 2.79 1.88 1.50

6.92 5.1 3.91 3.95 4.00 3.98 2.83 1.45 0.77

5.82 4.61 4.04 3.65 3.61 3.46 3.35 1.65 –

2.04 1.34 1.10 1.21 1.16 1.06 1.05 0.53 –

23.9 9.32 8.90 6.11 4.88 4.50 4.39 3.43 2.47

22.4 10.05 9.18 6.86 6.07 3.80 4.21 3.35 1.17

24.7 8.94 9.19 5.61 4.90 4.31 3.73 3.59 1.96

7.78 3.07 2.86 1.89 1.58 1.31 1.47 – 0.75

Table 5.3  Major vitamins present in N. sativa seeds Vitamin αTocopherol (μg/g) γ-Tocopherol (μg/g) Retinol (μg/g) Vitamin D2 (μg/g) Vitamin K1 (μg/g) Vitamin K2 (μg/g) B1 (thiamin) B2 (riboflavin) B6 (pyridoxine) PP (niacin) Folic acid

Vatansev et al. (2013) 10.1

Dinagaran et al. (2016) 11.3

Nergiz and Ötleş (1993) 40

Al-Saleh et al. (2006) –

2.28

6.47

250

–

0.18 1.38

0.27 –

– –

– –

1.85

–

–

–

2.15

–

–

–

– – – – –

– – – – –

8.31 μg/100 g 0.63 μg/100 g 7.89 μg/100 g 63.1 μg/100 g 0.42 μg/100 g

14.6 mg/kg – 6.6 mg/kg 56.5 mg/kg 614 mg/kg

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6  Lipids The seeds of N. sativa are well known for their high nutritional value especially as a rich source of lipids (ca. 45.4%), and proteins (20.3%) (Al-Jassir 1992; Kabir et al. 2019). N. sativa seeds possess a relatively higher amount of neutral lipids as compared to the other classes of lipids. The major triacylglycerols (neutral lipids) mentioned are trilinoleoyl (24.6%), oleoyldilinoleoyl (19.6%), palmitoyldilinoleoyl (17.5%), palmitoyl oleoyl linoleoyl (12.9%) and dioleoyllinoleoyl (9.6%), respectively. Minor lipids included glycolipids i.e., monogalactosyl diglyceride, digalactosyl diglyceride, sterylgalactoside and acylated sterylgalactoside (Khan 1999). Phospholipids have also been reported in sufficient amounts in N. sativa seeds. The major phospholipid classes reported are phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Phosphatidylglycerol, lyso-­ phosphatidylcholine and lyso-phosphatidylethanol amine were present in minor quantities. Analysis indicated that sterols in oil of N. sativa are existed either as free sterols, steryl esters or as acylated steryl glycosides. The identified sterols were mainly cholesterol, campestanol, campesterol, stigmastanol, sitosterol, stigmasterol, ∆5-avenasterol, ∆7-avenasterol, ∆7-stigmasterol, lophenol, obtusifoliol, 24-methyl phenol, cycloeucalenol, gramisterol, 24-methylenecycloartanol, citrostadienol, taraxeroltriucollol, butyrospermol, flamyrin, cycloartenol, and 24-ethyllophenol (Takruri and Dameh 1998; Khan 1999). β-sitosterol, campesterol and stigmasterol are the prevalent sterols in seeds of N. sativa (Hamrouni-Sellami et al. 2008; Gharby et al. 2015). The fatty acids profiles of fixed oil obtained from the N. sativa seeds reveal the presence of linoleic acid, palmitic acid, oleic acid and stearic acid. Arachidic acid, linolenic acid, myristic acid, and palmitoleic acid are also present in considerable amount. Table 5.4 reveals the comparable fatty acids profiles for different landraces of N. sativa seed as reported by different researchers. It is generally observed that the unsaturated fatty acids are relatively in higher amounts than unsaturated fatty acids in N. sativa seeds. The major unsaturated fatty acids present in seed oil are oleic (C18:1) and linoleic (C18:2) acids, while among saturated fatty acids, palmitic acid (C16:0) is the major one (El-Sayed et  al. 1997; Matthaus and Özcan 2011; Kabir et al. 2019). Different classes of lipids such as triglycerides, diglycerides, monoglycerides, free fatty acids, and sterols have dissimilar ratios of fatty acid constituents. Linoleic acid is predominantly present in free acids, triglycerides and diglycerides, while sterols and monoglycerides contain a high content of myristic acid (Atta 2003). The variability in the lipid profile of N. sativa seed oil in different studies is attributed to the variety of seed grown (landrace), seed quality variables (i.e., maturity, storage conditions and damage during harvesting), oil processing procedure (i.e., cold-­ pressed extraction and solvent extraction) and the techniques used for analysis (Cheikh-Rouhou et al. 2007).

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Table 5.4  Fatty acid composition of N. sativa seed oil

Fatty acid Linoleic acid (omega-6) Palmitic acid Oleic acid Stearic acid Arachidic acid Linolenic acid Myristic acid Palmitoleic acid 11,13-Eicosadienoic acid Behenic acid Linoleic acid (omega-3) Heptadecanoic acid Pentadecanoic acid Lignoceric acid Tricosylic acid Eicosenoic acid Erucic acid

Khonche et al. (2019) (%) C18:2 48.29 C16:0 C18:1 C18:0 C20:0 C18:3 C14:0 C16:1 C20:2

20.27 5.76 5.48 0.90 – 0.63 0.94 9.22

C22:0 0.16 C18:2 1.08 C17:0 C15:0 C24:0 C23:0 C20:1 C22:1

0.28 0.18 0.10 0.04 – –

Lutterodt et al. (2010) (g/100 g) 58.90

Ramadan and Mörsel, (2002) (g/100 g) 57.3

Atta (2003) (%) 49.0

Al-Jasass and Al-Jasser (2012) (%) 68.07

13.10 24.51 2.79 0.14 0.21 – – –

13.0 24.1 3.16 – – – – 2.44

9.9 20.1 3.3 0.7 2.7 9.8 0.7 –

10.5 16.23 2.04 – 2.16 1.0 – –

– –

– –

0.8 –

– –

– – – – 0.35 –

– – – – – –

– – – – – 1.0

– – – – – –

7  Essential Oil The essential oil obtained from N. sativa seeds has well established medicinal significance due to the presence of some vital volatile components. The compounds in volatile fractions are divided into six major groups which include non-terpenoid hydrocarbons, monoterpenoid hydrocarbons, monoterpenoid ketones, monoterpenoid alcohols, sesquiterpenoid hydrocarbons and phenylpropanoid compounds (El-Sayed et al. 1997). The impact of extraction mode utilized to obtain essential oil on the compositional variation is imperative. The essential oil obtained through cryogenic grinding contains high amount of monoterpene hydrocarbons (51.2–60.2%), while ketones (46.1–59.9%) constitute the main portion in classical grinding essential oils (Nickavar et  al. 2003). Thymoquinone (42.3–56.1%), p-cymene (33–38%), carvacrol (1.3–1.4%), dehydro-sabina ketone (4.4–4.5%), α-thujene (6%), camphene (11%), α-pinene (1.11%), β-pinene (7%), sabinene (1%), α-phellandrene (0.45%), β-myrcene (0.21%), γ-terpinene (5.12%), limonene (0.13%), camphor (1%), terpinolene (0.23%), thymol (10.12%), carvone (0.32%), t-anethol (1–4%), 4-terpineol (2–7%), longicyclene (0.9%), borneol (0.43%), and sesquiterpene longifolene (1–8%) are the major bioactive constituents (Benkaci-Ali et al. 2013; Piras et al. 2013; Desai et al. 2015; Mouwakeh et al. 2019).

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Various compounds have been identified in volatile oil of N. sativa seed oil using different extraction methods including hydrodistillation, microwave-assisted distillation, supercritical fluid extraction and steam distillation. A considerable variance in components and their percentage composition have been observed in the essential oil of black cumin seeds extracted through different methods and seeds of different landraces (Burits and Bucar 2000; Kiralan 2012). The compositional variation may be reflected in the medicinal potential of N. sativa seed oil.

8  Alkaloids Two isoquinoline derived alkaloids nigellicin (1) NIGELLICIMINE N-OXIDE, (2) NIGELLICIMINE, (3) NIGELLICINE, and (4) NIGELLIDINE represent the only examples of indazole alkaloids in nature (Khan 1999; Harzallah et al. 2011).

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9  Saponins Continual interest in this field led to the isolation and characterization of saponins (5) (Merfort et al. 1997). The main saponin found in N. sativa seeds is alpha-hederin (also called melanthin). It is a triterpene saponin which releases rhamnose sugar and melanthigenin on hydrolysis and has anticancer activity (Ansari et  al. 1988; Venkatachallam et al. 2010; Adamska et al. 2019).

10  Phenolic Compounds The presence of a phenolic compound nigellone in N. sativa seeds is recognized for a long time (Kokoska et al. 2008). Other phenolic compounds present in N. sativa seeds include thymohydroquinone (6), dithymoquinone (7), tannins and some flavanol triglycosides (8a–c) and kaempferol 3-O-β-D-glucosylgalactoside (9) (Mahfouz 1960; Ali and Blunden 2003; Benkaci-Ali et al. 2007).

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11  Conclusion The composition of N. sativa seeds indicates that they are rich in important dietary components. The highly ranked evidence-based medicinal role of N. sativa seeds is due to natural metabolites and phytoconstituents of miraculous pharmacological importance. The therapeutic role of dietary ingredients of N. sativa seeds provides us nutraceutical leads to enrich the foods with medicinal functionalities which will not only reduce the disease incidence but will also reduce the socio-economic burden.

References Adamska, A., Stefanowicz-Hajduk, J., & Ochocka, J. R. (2019). Alpha-hederin, the active saponin of Nigella sativa, as an anticancer agent inducing apoptosis in the SKOV-3 cell line. Molecules, 24(16), 2958. Aisa, H., Xin, X., & Tang, D. (2019). Nigella sativa: A medicinal and edible plant that ameliorates diabetes. In Bioactive food as dietary interventions for diabetes (pp. 629–640). Cambridge, MA: Elsevier. Al-Gaby, A. (1998). Amino acid composition and biological effects of supplementing broad bean and corn proteins with Nigella sativa (black cumin) cake protein. Food/Nahrung, 42(05), 290–294. Ali, B., & Blunden, G. (2003). Pharmacological and toxicological properties of Nigella sativa. Phytotherapy Research, 17(4), 299–305. Al-Jasass, F. M., & Al-Jasser, M. S. (2012). Chemical composition and fatty acid content of some spices and herbs under Saudi Arabia conditions. The Scientific World Journal, 2012, 859892. Al-Jassir, M. S. (1992). Chemical composition and microflora of black cumin (Nigella sativa L.) seeds growing in Saudi Arabia. Food Chemistry, 45(4), 239–242. Al-Saleh, I. A., Billedo, G., & El-Doush, I. I. (2006). Levels of selenium, dl-α-tocopherol, dl-γ-­ tocopherol, all-trans-retinol, thymoquinone and thymol in different brands of Nigella sativa seeds. Journal of Food Composition and Analysis, 19(2–3), 167–175. Ansari, A. A., Hassan, S., Kenne, L., & Wehler, T. (1988). Structural studies on a saponin isolated from Nigella sativa. Phytochemistry, 27(12), 3977–3979. Atta, M. B. (2003). Some characteristics of nigella (Nigella sativa L.) seed cultivated in Egypt and its lipid profile. Food Chemistry, 83(1), 63–68. Babayan, V., Koottungal, D., & Halaby, G. (1978). Proximate analysis, fatty acid and amino acid composition of Nigella sativa L. seeds. Journal of Food Science, 43(4), 1314–1315. Benkaci-Ali, F., Baaliouamer, A., Meklati, B. Y., & Chemat, F. (2007). Chemical composition of seed essential oils from Algerian Nigella sativa extracted by microwave and hydrodistillation. Flavour and Fragrance Journal, 22(2), 148–153. Benkaci-Ali, F., Akloul, R., Boukenouche, A., & Pauw, E. D. (2013). Chemical composition of the essential oil of Nigella sativa seeds extracted by microwave steam distillation. Journal of Essential Oil-Bearing Plants, 16(6), 781–794. Burits, M., & Bucar, F. (2000). Antioxidant activity of Nigella sativa essential oil. Phytotherapy Research, 14(5), 323–328. Çakir, B., & Gülseren, İ. (2019). Identification of novel proteins from black cumin seed meals based on 2d gel electrophoresis and MALDI-TOF/TOF-MS analysis. Plant Foods for Human Nutrition, 74(3), 414–420.

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Cheikh-Rouhou, S., Besbes, S., Hentati, B., Blecker, C., Deroanne, C., & Attia, H. (2007). Nigella sativa L.: Chemical composition and physicochemical characteristics of lipid fraction. Food Chemistry, 101(2), 673–681. Desai, S., Saheb, S. H., Das, K. K., & Haseena, S. (2015). Phytochemical analysis of Nigella sativa and it’s antidiabetic effect. Journal of Pharmaceutical Sciences and Research, 7(8), 527–532. Dinagaran, S., Sridhar, S., & Eganathan, P. (2016). Chemical composition and antioxidant activities of black seed oil (Nigella sativa L.). International Journal of Pharmaceutical Sciences and Research, 7(11), 4473. El-Sayed, A. A. A., Hussiney, A., & Yassa, A. (1997). Constituents of Nigella sativa oil and evaluation of its inhibitory effect of growth and aflatoxin production by Aspergillus parasiticus. Deutsche Lebensmittel-Rundschau, 93(5), 149–152. Gharby, S., Harhar, H., Guillaume, D., Roudani, A., Boulbaroud, S., Ibrahimi, M., Ahmad, M., Sultana, S., Hadda, T. B., Chafchaouni-Moussaoui, I., & Charrouf, Z. (2015). Chemical investigation of Nigella sativa L. seed oil produced in Morocco. Journal of the Saudi Society of Agricultural Sciences, 14(2), 172–177. Hamrouni-Sellami, I., Elyes Kchouk, M., & Marzouk, B. (2008). Lipid and aroma composition of black cumin (Nigella sativa L.) seeds from Tunisia. Journal of Food Biochemistry, 32(3), 335–352. Harzallah, H. J., Kouidhi, B., Flamini, G., Bakhrouf, A., & Mahjoub, T. (2011). Chemical composition, antimicrobial potential against cariogenic bacteria and cytotoxic activity of Tunisian Nigella sativa essential oil and thymoquinone. Food Chemistry, 129(4), 1469–1474. Kabir, Y., Shirakawa, H., & Komai, M. (2019). Nutritional composition of the indigenous cultivar of black cumin seeds from Bangladesh. Progress in Nutrition, 21, 428–434. Kazmi, A., Khan, M. A., & Ali, H. (2019). Biotechnological approaches for production of bioactive secondary metabolites in Nigella sativa: An up-to-date review. International Journal of Secondary Metabolite, 6(2), 172–195. Khan, M.  A. (1999). Chemical composition and medicinal properties of Nigella sativa Linn. Inflammopharmacology, 7(1), 15–35. Khonche, A., Huseini, H. F., Gholamian, M., Mohtashami, R., Nabati, F., & Kianbakht, S. (2019). Standardized Nigella sativa seed oil ameliorates hepatic steatosis, aminotransferase and lipid levels in non-alcoholic fatty liver disease: A randomized, double-blind and placebo-controlled clinical trial. Journal of Ethnopharmacology, 234, 106–111. Kiralan, M. (2012). Volatile compounds of black cumin seeds (Nigella sativa L.) from microwave-­ heating and conventional roasting. Journal of Food Science, 77(4), C481–C484. Kokoska, L., Havlik, J., Valterova, I., Sovova, H., Sajfrtova, M., & Jankovska, I. (2008). Comparison of chemical composition and antibacterial activity of Nigella sativa seed essential oils obtained by different extraction methods. Journal of Food Protection, 71(12), 2475–2480. Lutterodt, H., Luther, M., Slavin, M., Yin, J.-J., Parry, J., Gao, J.-M., & Yu, L. L. (2010). Fatty acid profile, thymoquinone content, oxidative stability, and antioxidant properties of cold-pressed black cumin seed oils. LWT- Food Science and Technology, 43(9), 1409–1413. Mahfouz, M. (1960). The isolation of a crystalline active principle from Nigella sativa L seeds. Journal of Pharmaceutical Sciences of United Arab Republic, 1, 1–19. Mamun, M., & Absar, N. (2018). Major nutritional compositions of black cumin seeds-cultivated in Bangladesh and the physicochemical characteristics of its oil. International Food Research Journal, 25(6), 2634–2639. MatthauS, B., & ÖzCaN, M.  M. (2011). Fatty acids, tocopherol, and sterol contents of some Nigella species seed oil. Czech Journal of Food Sciences, 29(2), 145–150. Merfort, I., Wray, V., Barakat, H., Hussein, S., Nawwar, M., & Willuhn, G. (1997). Flavonol triglycosides from seeds of Nigella sativa. Phytochemistry, 46(2), 359–363. Michel, C. G., El-Sayed, N. S., Moustafa, S. F., Ezzat, S. M., Nesseem, D. I., & El-Alfy, T. S. (2011). Phytochemical and biological investigation of the extracts of Nigella sativa L. seed waste. Drug Testing and Analysis, 3(4), 245–254.

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Mouwakeh, A., Kincses, A., Nové, M., Mosolygó, T., Mohácsi-Farkas, C., Kiskó, G., & Spengler, G. (2019). Nigella sativa essential oil and its bioactive compounds as resistance modifiers against Staphylococcus aureus. Phytotherapy Research, 33(4), 1010–1018. Nergiz, C., & Ötleş, S. (1993). Chemical composition of Nigella sativa L. seeds. Food Chemistry, 48(3), 259–261. Nickavar, B., Mojab, F., Javidnia, K., & Amoli, M. A. R. (2003). Chemical composition of the fixed and volatile oils of Nigella sativa L. from Iran. Zeitschrift für Naturforschung. Section C, 58(9–10), 629–631. PFAF. (2019) https://pfaf.org/user/Plant.aspx?LatinName=Nigella+sativa Retreived on 2-12-2019. Piras, A., Rosa, A., Marongiu, B., Porcedda, S., Falconieri, D., Dessì, M.  A., Ozcelik, B., & Koca, U. (2013). Chemical composition and in vitro bioactivity of the volatile and fixed oils of Nigella sativa L. extracted by supercritical carbon dioxide. Industrial Crops and Products, 46, 317–323. Ramadan, M. F., & Mörsel, J. T. (2002). Characterization of phospholipid composition of black cumin (Nigella sativa L.) seed oil. Food/Nahrung, 46(4), 240–244. Sultan, M. T., Butt, M. S., Anjum, F. M., Jamil, A., Akhtar, S., & Nasir, M. (2009). Nutritional profile of indigenous cultivar of black cumin seeds and antioxidant potential of its fixed and essential oil. Pakistan Journal of Botany, 41(3), 1321–1330. Takruri, H.  R., & Dameh, M.  A. (1998). Study of the nutritional value of black cumin seeds (Nigella sativa L). Journal of the Science of Food and Agriculture, 76(3), 404–410. Vatansev, H., Ciftci, H., Ozkaya, A., Ozturk, B., Evliyaoglu, N., & Kiyici, A. (2013). Chemical composition of Nigella sativa L. seeds used as a medical aromatic plant from East Anatolia region, Turkey. Asian Journal of Chemistry, 25(10), 5490–5492. Venkatachallam, S. K. T., Pattekhan, H., Divakar, S., & Kadimi, U. S. (2010). Chemical composition of Nigella sativa L. seed extracts obtained by supercritical carbon dioxide. Journal of Food Science and Technology, 47(6), 598–605. Zribi, I., Ghezal, N., Sbai, H., Richard, G., Fauconnier, M. L., & Haouala, R. (2019). Biochemical composition of Tunisian Nigella sativa L. at different growth stages and assessment of the phytotoxic potential of its organic fractions. Plant Biosystems-An International Journal Dealing with all Aspects of Plant Biology, 153(2), 205–212.

Chapter 6

Nigella sativa Seed Peptides (Thionins) Ali Osman

Abstract  Black cumin (Nigella sativa) seeds are a good source of bioactive compounds with health-promoting and antimicrobial traits. Plant thionins are the first peptides with antimicrobial properties isolated from the plants, and thionins represent a family of cysteine-rich peptides with a 5 kDa molecular weight. Thionins are found in different mono- and di-cotyledonous plants. About 100 sequences of thionins have been isolated from several plant species. This chapter reviews on chemistry and biological activities of thionins isolated from Nigella sativa seeds. Keywords  Nigella sativa · Antimicrobial peptides · Cysteine-rich peptides

Abbreviations AMPs Anti-Microbial Peptides Nigella sativa N. sativa RP-HPLC Reversed-phase high-performance liquid chromatography

1  Introduction Animals, plants and humans are usually exposed to pathogens. Higher organisms naturally have immune systems that protects themselves from the chemical, physical and biological agents such as pollens, pollutants, heavy metals, pathogenic, bacteria, fungi, cold, drought, beetles, insects, and larvae (Abbas et al. 2020; Abdel-Shafi et  al. 2019a; Benko-Iseppon et  al. 2010; Stintzi et  al. 1993). The plants protect themselves by secreting the phenolic compounds, secondary metabolites and antimicrobial peptides (Abdel-Shafi et  al. 2019b; Osman et  al. 2016b; Sitohy et  al. 2012). Resistance to antimicrobial agents by bacteria has increased recently, A. Osman (*) Agricultural Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_6

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r­ epresenting several health problems (Abdel-Shafi et al. 2016). Thus, the identification of new antimicrobial agents with different mechanisms of action is highly required. Several strategies have been suggested to increase the antimicrobial potential of proteins, including chemical modifications (Osman et al. 2014), and enzymatic hydrolysis using microbial and plant proteolytic enzymes as well as proteases (Abdel-Hamid et al. 2020; Osman et al. 2016a; Saad et al. 2019). Plant produces anti-microbial peptides (AMPs) for defense against pathogenic fungi and bacteria. AMPs size ranges from 2–9 kDa consisting of 10–100 amino acid residues. These AMPs are divided into families upon the cysteine residues, their position, length of amino acids, and molecular weight (Nawrot et al. 2014). Such AMPs include families such as thionins. Thionins are the first peptides isolated from plants with antimicrobial potential (De Caleya et al. 1972), and they represent a family of cysteine-rich peptides with a molecular weight of about 5 kDa. Thionins are found in different mono- and di-­ cotyledonous plants, wherein more than 100 sequences of thionins were isolated from several species (Stec 2006). Based on the number of cysteine residues (6 or 8), thionins can be classified into two groups (Broekaert et al. 1997). Thionins are produced as proprotein with a molecular mass of approximately 18 kDa and contain a signal peptide and a C-terminal propeptide (Epple et al. 1995), wherein the removal of C-terminal propeptide is a result of post-translational modification (Ponz et al. 1983). It has been reported that thionins are localized in cell vacuoles (Romero et al. 1997). It is anticipated that the function of propeptide is to neutralize the toxic action of the thionin mature domain until the precursor could be localized into an intercellular space or vacuole (Bohlmann et  al. 1988). This chapter reviews on chemistry and biological activities of thionins isolated from Nigella sativa seeds.

2  Thionins Sources Thionins (∼5  kDa) are cysteine-rich, usually basic proteins found in monocots (grains), eudicots including dicotyledonous plants (species of mistletoe and Pyrularia pubera), and rosids (crambin). About 100 individual thionin sequences were identified in different species. The name thionins is used for two distinct groups of plant peptides (α/β-thionins, and γ-thionins). Despite the common name and very distant common origin, thionins have distinct three-dimensional architectures (Bohlmann et al. 1991). They considered separate protein families, and γ-thionins could be called plant defensins. α/β-Thionins were subdivided into 5 different classes (I-V) (Bohlmann et  al. 1991), and many researchers distinguished an additional class, γ-thionins. Type I, found in the endosperm of grains (i.e., family Poaceae), is highly basic, and contain 45 amino acids, wherein 8 of which are cysteines (Egorov et al. 2005). Type II was isolated from nuts and leaves of Pyrularia pubera (Vernon 1992) and the leaves of Hordeum vulgare (Rodríguez-Palenzuela et al. 1988). They are slightly less basic than type I and contain 46–47 amino acids. Types I and II have 4 disulfide

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bonds. Type III thionins have been extracted from stems and leaves of mistletoe species such as Phoradendron liga, Phoradendron tomentosum, Viscum album, and Dendrophthora clavata (Mellstrand and Samuelsson 1973; Samuelsson and Pettersson 1970; Thunberg 1982), and contain 45–46 amino acids. Those thionins, which have 3 disulfide bridges conserved with respect to types I and II, are as basic as type II. Type IV thionins, which contain 46 amino acids with 3 disulfide bonds, is neutral in charge, and was extracted from Abyssinian cabbage seeds (Schrader-­ Fischer and Apel 1994; VanEtten et al. 1965). Type V thionins us truncated forms of regular thionins found in some grains like wheat (Castagnaro et al. 1994). At the amino acid level, all types of α/β-thionins seems to be highly homologous. Structural work of all thionins classes exhibited the same structural motif, wherein the past classification appear less supported by three-dimensional structures. γ-Thionins are common with a large family of membrane active peptides, called defensins, found in animals and plants (Colilla et al. 1990; Mendez et al. 1990).

3  Thionins from Nigella sativa Nigella sativa seeds are rich in biologically active phytochemicals with antimicrobial potential. AMPs from black seed acidic extracts were isolated and characterized. All of them are potent antimicrobial agents that could inhibit fungal and bacterial pathogens in vitro (Rogozhin et al. 2011). Biological impacts of Nigella sativa thionins, such as bactericidal and fungicidal impacts have been reported. Isolation of thionins was carried out by combining acid extraction and fractionation with liquid chromatography techniques. N-terminal amino acid sequences were detected using Edman degradation method. In addition, the antimicrobial effects of thionins was evaluated using the microdilution broth test. By using the fluorescent spectroscopy and an atomic force microscopy it was possible to study the features of thionins mode of action of. Peptides, named NsW1 and NsW2, were isolated and possessed high affinity with heparin (an acidic polysaccharide and a member of glycosaminoglycan family). These molecules were indentified as thionins, a well-­ known family of plant antimicrobial peptides. In addition, thionins effectively inhibited the viability of Staphylococcus aureus, Bacillus subtilus, and Candida albicans that has been confirmed by using biophysical and bacteriological techniques (Vasilchenko et al. 2017).

3.1  Thionins Isolation from Nigella sativa Vasilchenko et al. (2017) isolated thionins from crushed and defatted N. sativa seeds using acidic extraction followed by precipitation using cooled acetone. The next fractionation step was performed using some liquid chromatography techniques (affinity, size-exclusion, and reversed-phase high performance). The first separation

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Fig. 6.1  Cell wall of gram-positive and gram-negative bacteria

of the acetic acid extract was to desalt it by using the reversed-phase high-­ performance liquid chromatography (RP-HPLC). The fraction bound with the column was desorbed by solvent, evaporated, and freez-dried. Instead of using peptides, the desalted extract was separated using affinity chromatography using heparin in a stepwise gradient of NaCl concentration. Three fractions were recovered: fraction A was eluted from the NaCl (100 mM) column, fraction B was eluted from the NaCl (500  mM) column, and fraction C was recovered by flushing the column with NaCl (1 M).

3.2  Thionins Mode of Action Thionins are hydrophobic molecules that interact with the hydrophobic residues and lyse the bacterial cell membranes (Fig.  6.1). Moreover, thionins interact directly with the membrane lipids as a part from interacting with membrane protein receptors as presented in Fig. 6.2 (Shai 2002).

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Fig. 6.2  Interaction between antibacterial protein (thionins) and the bacterial cell wall

References Abbas, E., Osman, A., & Sitohy, M. (2020). Biochemical control of Alternaria tenuissima infecting post-harvest fig fruit by chickpea vicillin. Journal of the Science of Food and Agriculture, 100, 2889. Abdel-Hamid, M., Romeih, E., Saporito, P., Osman, A., Mateiu, R. V., Mojsoska, B., & Jenssen, H. (2020). Camel milk whey hydrolysate inhibits growth and biofilm formation of Pseudomonas aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus. Food Control, 111, 107056. Abdel-Shafi, S., Osman, A., Enan, G., El-Nemer, M., & Sitohy, M. (2016). Antibacterial activity of methylated egg white proteins against pathogenic G+ and G− bacteria matching antibiotics. Springerplus, 5, 983. Abdel-Shafi, S., Al-Mohammadi, A.-R., Osman, A., Enan, G., Abdel-Hameid, S., & Sitohy, M. (2019a). Characterization and antibacterial activity of 7S and 11S globulins isolated from cowpea seed protein. Molecules, 24, 1082. Abdel-Shafi, S., Al-Mohammadi, A.-R., Sitohy, M., Mosa, B., Ismaiel, A., Enan, G., & Osman, A. (2019b). Antimicrobial activity and chemical constitution of the crude, phenolic-rich extracts of Hibiscus sabdariffa, Brassica oleracea and Beta vulgaris. Molecules, 24, 4280. Benko-Iseppon, A. M., Lins Galdino, S., Calsa, T., Jr., Akio Kido, E., Tossi, A., Carlos Belarmino, L., & Crovella, S. (2010). Overview on plant antimicrobial peptides. Current Protein & Peptide Science, 11, 181. Bohlmann, H., Clausen, S., Behnke, S., Giese, H., Hiller, C., Reimann-Philipp, U., Schrader, G., Barkholt, V., & Apel, K. (1988). Leaf-specific thionins of barley-a novel class of cell wall

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p­ roteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants. The EMBO Journal, 7, 1559–1565. Bohlmann, H., Apel, K., & Thionins, A. (1991). Thionins. Rev Plant Physiol Plant Mol Biol, 42, 227–240. Broekaert, W. F., Cammue, B. P., De Bolle, M. F., Thevissen, K., De Samblanx, G. W., Osborn, R. W., & Nielson, K. (1997). Antimicrobial peptides from plants. Critical Reviews in Plant Sciences, 16, 297–323. Castagnaro, A., Maraña, C., Carbonero, P., & García-Olmedo, F. (1994). cDNA cloning and nucleotide sequences of alpha 1 and alpha 2 thionins from hexaploid wheat endosperm. Plant Physiology, 106, 1221. Colilla, F. J., Rocher, A., & Mendez, E. (1990). γ-Purothionins: Amino acid sequence of two polypeptides of a new family of thionins from wheat endosperm. FEBS Letters, 270, 191–194. De Caleya, R. F., Gonzalez-Pascual, B., Garcia-Olmedo, F., & Carbonero, P. (1972). Susceptibility of phytopathogenic bacteria to wheat purothionins in  vitro. Applied and Environmental Microbiology, 23, 998–1000. Egorov, T. A., Odintsova, T. I., Pukhalsky, V. A., & Grishin, E. V. (2005). Diversity of wheat anti-­ microbial peptides. Peptides, 26, 2064–2073. Epple, P., Apel, K., & Bohlmann, H. (1995). An Arabidopsis thaliana thionin gene is inducible via a signal transduction pathway different from that for pathogenesis-related proteins. Plant Physiology, 109, 813–820. Mellstrand, S.  T., & Samuelsson, G. (1973). Phoratoxin, a toxic protein from the mistletoe Phoradendron tomentosum subsp. macrophyllum (Loranthaceae) improvements in the isolation procedure and further studies on the properties. European Journal of Biochemistry, 32, 143–147. Mendez, E., Moreno, A., Colilla, F., Pelaez, F., Limas, G. G., Mendez, R., Soriano, F., Salinas, M., & de Haro, C. (1990). Primary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, γ-hordothionin, from barley endosperm. European Journal of Biochemistry, 194, 533–539. Nawrot, R., Barylski, J., Nowicki, G., Broniarczyk, J., Buchwald, W., & Goździcka-Józefiak, A. (2014). Plant antimicrobial peptides. Folia Microbiologica, 59, 181–196. Osman, A., Mahgoub, S., El-Masry, R., Al-Gaby, A., & Sitohy, M. (2014). Extending the technological validity of raw buffalo milk at room temperature by esterified legume proteins. Journal of Food Processing and Preservation, 38, 223–231. Osman, A., Abbas, E., Mahgoub, S., & Sitohy, M. (2016a). Inhibition of Penicillium digitatum in  vitro and in postharvest orange fruit by a soy protein fraction containing mainly β-conglycinin. Journal of general plant pathology, 82(6), 293–301. Osman, A., El-Didamony, G., Sitohy, M., Khalifa, M., & Enan, G. (2016b). Soybean glycinin basic subunit inhibits methicillin resistant-vancomycin intermediate Staphylococcus aureus (MRSA-­ VISA) in vitro. International Journal of Applied Research in Natural Products, 9, 17–26. Ponz, F., Paz-Ares, J., Hernández-Lucas, C., Carbonero, P., & García-Olmedo, F. (1983). Synthesis and processing of thionin precursors in developing endosperm from barley (Hordeum vulgare L.). The EMBO Journal, 2, 1035–1040. Rodríguez-Palenzuela, P., Pintor-Toro, J.-A., Carbonero, P., & García-Olmedo, F. (1988). Nucleotide sequence and endosperm-specific expression of the structural gene for the toxin α-hordothionin in barley (Hordeum vulgare L.). Gene, 70, 271–281. Rogozhin, E. A., Oshchepkova, Y. I., Odintsova, T. I., Khadeeva, N. V., Veshkurova, O. N., Egorov, T. A., Grishin, E. V., & Salikhov, S. I. (2011). Novel antifungal defensins from Nigella sativa L. seeds. Plant Physiology and Biochemistry, 49, 131–137. Romero, A., Alamillo, J.  M., & García-Olmedo, F. (1997). Processing of thionin precursors in barley leaves by a vacuolar proteinase. European Journal of Biochemistry, 243, 202–208. Saad, A. M., Osman, A. O. M., Mohamed, A. S., & Ramadan, M. F. (2019). Enzymatic hydrolysis of Phaseolus vulgaris protein isolate: Characterization of hydrolysates and effect on the

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quality of minced beef during cold storage. International Journal of Peptide Research and Therapeutics, 26, 1–11. Samuelsson, G., & Pettersson, B. (1970). Separation of viscotoxins from the European mistletoe Viscum album L. (Loranthaceae) by chromatography on sulfoethyl Sephadex. Acta Chemica Scandinavica, 24, 2751–2756. Schrader-Fischer, G., & Apel, K. (1994). Organ-specific expression of highly divergent thionin variants that are distinct from the seed-specific crambin in the crucifer Crambe abyssinica. Molecular and General Genetics MGG, 245, 380–389. Shai, Y. (2002). Mode of action of membrane active antimicrobial peptides. Peptide Science: Original Research on Biomolecules, 66, 236–248. Sitohy, M. Z., Mahgoub, S. A., & Osman, A. O. (2012). In vitro and in situ antimicrobial action and mechanism of glycinin and its basic subunit. International Journal of Food Microbiology, 154, 19–29. Stec, B. (2006). Plant thionins-the structural perspective. Cellular and Molecular Life Sciences: CMLS, 63, 1370–1385. Stintzi, A., Heitz, T., Prasad, V., Wiedemann-Merdinoglu, S., Kauffmann, S., Geoffroy, P., Legrand, M., & Fritig, B. (1993). Plant ‘pathogenesis-related’ proteins and their role in defense against pathogens. Biochimie, 75, 687–706. Thunberg, E. (1982). Isolation and properties of ligatoxin A, a toxic protein from the mistletoe Phoradendron liga. Acta Pharmaceutica Suecica, 19(4), 285–292. VanEtten, C., Nielsen, H., & Peters, J. (1965). A crystalline polypeptide from the seed of Crambe abyssinica. Phytochemistry, 4, 467–473. Vasilchenko, A. S., Smirnov, A. N., Zavriev, S. K., Grishin, E. V., Vasilchenko, A. V., & Rogozhin, E.  A. (2017). Novel thionins from black seed (Nigella sativa L.) demonstrate antimicrobial activity. International Journal of Peptide Research and Therapeutics, 23, 171–180. Vernon, L. P. (1992). Pyrularia thionin: Physical properties, biological responses, and comparison to other thionins and cardiotoxin. Journal of Toxicology - Toxin Reviews, 11, 169–191. Osman, A., Abbas, E., Mahgoub, S., & Sitohy, M. (2016). Inhibition of Penicillium digitatum in  vitro and in postharvest orange fruit by a soy protein fraction containing mainly β-conglycinin. Journal of general plant pathology, 82(6), 293-301.

Chapter 7

Black Cumin Polysaccharides Ines Trigui, Salma Cheikh-Rouhou, Hamadi Attia, and Mohamed Ali Ayadi

Abstract  Plants have long been used as a source of food and remediation. Plant products always contain polysaccharides of different sizes, concentrations and chemical sequences. These biomolecules are stable, safe and possess various biological activities. They could represent a new natural source of additives for the food and pharmaceutical industries. The purpose of the current chapter is to characterize the polysaccharides extracted from black cumin seeds. The structural and the surface characteristics, as well as the functional and the antioxidant properties of black cumin polysaccharides (BCSP) were reported. BCSP consisted principally of galacturonic acid (30.2%), glucuronic acid (17.6%) and neutral sugar (22.9%). The structural characterization has shown that BCSP have a semi-crystalline structure. FTIR and NMR spectroscopy illustrated the typical peaks of polysaccharides. NMR data indicated that BCSP was probably a rhamnogalacturonan backbone with galactan and arabinan side chains. The foaming and emulsifying properties of BCSP were relatively high and varied with concentrations. In addition, BCSP was a promising source of natural antioxidants. Therefore, the information in this chapter could open up new opportunities for the development of effective functional ingredients for use in a wide range of food and pharmaceutical applications. Keywords  Nigella sativa · Black cumin seeds · Structural characteristics · Functional properties · Antioxidant activities

Abbreviations BCSP DPPH EAI ESI FC FS

Black cumin seeds polysaccharides 2,2-phényl-l- picrylhydrazyl Emulsifying Activity Index Emulsion Stability Index Foaming Capacity Foam Stability

I. Trigui (*) · S. Cheikh-Rouhou · H. Attia · M. A. Ayadi Valuation, Security, and Food Analysis Laboratory, National Engineering School of Sfax, Sfax, Tunisia © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_7

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FTIR HPSEC NMR OHC WHC XRD ΔH

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Fourier-Transformed Infrared spectroscopy High-Performance Size Exclusion Chromatography Nuclear Magnetic Resonance Oil Retention Capacity Water Retention Capacity X-ray Diffraction analysis Enthalpy of transition

1  Black Cumin Seeds and Their Polysaccharides Polysaccharides are among the most varied families of biopolymers and have considerable applications in the industrial and biological fields. The polyfunctional polysaccharides are widely used in food, cosmetics, and pharmaceutical industries. Polysaccharides are generally classified according to their structure, their composition, their origin and their applications. Plant products contain carbohydrates. Indeed, polysaccharides are present in plants such as starch, cellulose, hemicelluloses, gums and pectins (Ruff 2008). Furthermore, polysaccharides’ structure and composition can affect their biological activities and their functional properties (Zhao et al. 2005). Several plant polysaccharides are bioactive molecules and are notably recognized for their anticarcinogens (Chen et al. 2010), immunomodulators (Angone et  al. 2010), antibacterial and anti-inflammatory properties and also for their antioxidant activities (Slima et  al. 2018; Wu et  al. 2014) and their antiviral activities (Song et al. 2013a, b). Some oilseeds, such as seeds of black cumin (Nigella sativa L), are a source of polysaccharides with a high nutritional, industrial and pharmaceutical interest. Black cumin is among the most used medicinal plants that have aroused great interest in the Mediterranean countries (Gharib-zahedi et al. 2010), and Asian countries (Kooti et  al. 2016). Black cumin is an annual herbaceous plant belonging to the Renonculaceae family (Srinivasan 2018) and cultivated in different parts of the world (Ramadan and Mörsel 2002). It is especially remarkable for its oilseeds, whose flavor is pungent and bitter (Ahmad et al. 2013). Black cumin seeds are traditionally used in nutrition and as a medicament (Kiralan et al. 2014). Indeed, seeds are used as flavoring and spice (Cheikh-Rouhou et al. 2007). The seeds of black cumin are also used thanks to its therapeutic and pharmacological properties including anti-inflammatory, antitumoral, antidiabetic, antimicrobial and antioxidant (Kooti et al. 2016; Tavakkoli et al. 2017; Trigui et al. 2019). The composition of black cumin seeds is very rich and diverse (Al-Saleh et al. 2006). It depends on the studied cultivars, the stage maturation, the geographical and climatic conditions, as well as the methods of extraction (Karadağoğlu et al. 2019). Black cumin seeds are a relevant source of dietary protein (19.8%), lipids (37.0%) and carbohydrate (30.0%) (Ali et  al. 2012). Thus, seeds of black cumin may serve as a potential source of bioactive and functional proteins and

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polysaccharides for application in the pharmaceutical and food industries. In recent years, many studies have reported the physicochemical composition of black cumin seeds (Kabir et al. 2019) and have explored the scope of its therapeutic possibilities (Koshak et al. 2017; Yimer et al. 2019) or analyzed the lipid fraction of these seeds (Cheikh-Rouhou et al. 2007; Gharby et al. 2015; Hosseini et al. 2018). However, studies about the extraction and characterization of black cumin seeds polysaccharides are very limited. The study of black cumin seeds carbohydrate composition shows a wealth of glucose (Ramadan 2007), maltose and sucrose (Mehta et al. 2008). A subsequent study of black cumin seeds metabolism shows that at the beginning of development, the rate of arabinose, lyxose, and xylose begins to increase, but then decreases and other sugars such as galactinol and raffinose whose presence is linked to desiccation stage appear (Song et al. 2013a, b). Another study of the composition of polysaccharides of black cumin seeds of Indian origin has revealed the presence of rhamnose, arabinose, xylose, galactose, glucose and uronic acid content of 30  mg/g (Manjegowda et al. 2017). Moreover, structural studies reported that this polysaccharide is a type of rhamnogalacturonan-I having the skeleton of rhamnogalacturonan with side chains galactan and arabinan at a varied degree of polymerization (Manjegowda et al. 2017). This study suggested the use of black cumin seeds polysaccharides as nutraceuticals and as remedial tools against chronic diseases such as gastric ulcers (Manjegowda et al. 2017). Other results approve the use of these biopolymers in food applications as functional ingredients for use in a wide range of food formulations as well as bioactive compounds and a natural source of phytochemicals and antioxidants (Trigui et al. 2018). It was also reported that the structure and the composition of black cumin seeds polysaccharides have affected the antioxidant activities and the functional properties (Trigui et al. 2018).

2  Extraction of Polysaccharides The plant cell wall is characterized by the chemical richness and structural diversity of macromolecules, mainly polysaccharides. Protocols must, therefore, be sufficiently selective to allow the extraction of a well-defined category of macromolecules with a high yield of product/raw material. The polysaccharides extracted must be undegraded, pure, homogeneous and contain very few proteins, lipids and nucleic acids. Studies of the literature in the field of polysaccharide extraction have reported a large number of extraction protocols. Indeed, the choice of polysaccharide extraction method depends generally on the plant material, the envisaged properties, and applications, as well as on the solubility of the polysaccharide in the water at different pH. In addition, the use of sufficiently high extraction conditions (low pH, high temperature, and long extraction time) often allows obtaining high extraction yields. However, such conditions can lead to degradation, which negatively influences the extraction yield and the functional properties of the polysaccharides and also they

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promote the extraction of harmful and undesirable compounds. Generally, the protocols for extracting polysaccharides are carried out at relatively high temperatures between 80 °C and 90 °C using different chemical agents. New polysaccharide extraction methods have recently been applied, such as hot water extraction, microwave-assisted extraction, and supercritical fluid extraction. These methods are characterized by their efficiency and rentability and also allow the preservation of the environment and the protection of the polysaccharide structure (Le Normand et al. 2014; Song et al. 2013a, b). A study was carried out on the extraction of bioactive polysaccharides by hot water, enzymatic, ultrasonic and microwave (Cheng et  al. 2013). The antioxidant activity of the polysaccharides obtained by extraction with hot water is greater than for those obtained with the other methods. However, hot water extraction combined with microwave extraction, ultrasound and enzymatic pretreatment methods, often allows for increasing the efficiency of extraction yield and also the preservation of biological activities of polysaccharides. For black cumin seeds polysaccharides, according to Manjegowda et al. (2017), starch and protein of black cumin seeds defatted powder were removed by enzymatic procedure. The powder was then extracted by boiling with an ammonium oxalate solution in order to obtain pectic polysaccharide. Moreover, black cumin seeds polysaccharides (BCSP) were extracted (Fig. 7.1) as described by Trigui et al. (2018). The polysaccharide yield (%, w/w) was determined as follows:

Fig. 7.1  Extraction diagram of black cumin seeds polysaccharides (Trigui et al. 2018)

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Y ( % ) = ( W 1 / W 0 ) × 100



(7.1)

W1: Extracted polysaccharide weight (g) W0: Dried sample weight (g)

3  Physicochemical Characteristics Previous studies highlighted a general deficiency of fundamental knowledge linked with the characterization of polysaccharides extracted from black cumin seeds. BCSP were obtained by hot water extraction as well as by ethanol precipitation, deproteinization and dialysis (Trigui et al. 2018). As reported in Table 7.1, the polysaccharides have the largest portion (71.5%) of the extract. The yield of BCSP was 5.18% (Table 7.1). The same yield was obtained for polysaccharides extracted from Pharbitis nil seeds (Wang et al. 2014). The protein content recorded for BCSP was 8.83%. Similar protein levels have been reported for polysaccharides extracted from Table 7.1 Physicochemical composition of black cumin seeds polysaccharides (Trigui et al. 2018)

Dry matter (%) 95.8 Polysaccharides (%) 71.5 Proteins (%) 8.83 Yield (%) 5.18 Colour L* 58.3 a* 3.22 b* 4.28 Thermal properties T0 (°C) 46.9 Td (°C) 84.7 ΔH (J/g) 441 Carbohydrate composition (%) Galacturonic acid 30.2 Glucuronic acid 17.6 Total neutral sugars 22.9 Rhamnose 2.74 Arabinose 5.83 Xylose 3.19 Mannose 2.28 Glucose 3.18 Galactose 5.76 %: basis on the dry matter T0, onset denaturation temperature; Td, peak denaturation temperature; ΔH, enthalpy of transition

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the flaxseed kernel (Ding et  al. 2014), sorghum sheaths (Slima et  al. 2018), and fenugreek (Ktari et al. 2017). However, other studies have reported lower protein content for polysaccharides extracted from olive leaves (Khemakhem et al. 2018). This difference can be explained in particular by the difference in the extraction and deproteinization method. Color is one of the properties that determine the suitability of the BCSP for food application for which it is intended. The color characteristics (L*, a*, b*) of BCSP are given in Table 7.1. Results show a light yellow color for BCSP. This feature enhances their relevance in food formulations. Table 7.1 showed also the thermal properties of BCSP. The onset denaturation temperature (T0), peak denaturation temperature (Td) and enthalpy of transition (ΔH) values were, respectively, 46.99 °C, 84.72 °C and 441.00 J/g (Trigui et al. 2018). The study of thermal properties is very useful in the food industry, particularly in cooking and food storage processes. The evaluation of the monosaccharide composition of BCSP is very important in order to understand the structure-function relationship of the polysaccharides. Table 7.1 showed the composition of monosaccharides of BCSP. Galacturonic acid (30.2%) and glucuronic acid (17.6%) are the dominant sugars in polysaccharides (Trigui et al. 2018). BCSP contains also 22.9% of neutral sugars consisting mainly of arabinose (5.83%) and galactose (5.76%). The sugar content of BCSP is comparable to that obtained for other plant-based polysaccharides (Romdhane et al. 2017). Manjegowda et al. (2017) have also shown the presence of rhamnose, arabinose, xylose, galactose, glucose as major sugar residues with a uronic acid content of 30 mg/g in the dietary pectic polysaccharide from black cumin seeds. Another important parameter in the characterization of polysaccharides is the molecular weight distribution. The molecular weight distribution of black cumin seeds polysaccharides was determined by size exclusion chromatography (HPSEC) (Trigui et al. 2018). The study of the elution profile indicates that the average molecular weight of BCSP was about 800 kDa (Trigui et al. 2018). However, Manjegowda et  al. (2017) reported that the molecular weight of dietary pectic polysaccharide from black cumin seeds was determined as about 95  kDa by size exclusion chromatography.

4  Structural Properties The bioactivity of polysaccharides is strongly related to their structural properties. Therefore, the determination of the structure-function relationships of polysaccharides is necessary. In order to determine the partial or total structure of polysaccharides, analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy (1H and 13C NMR) analysis, X-ray diffraction analysis (XRD) and fourier-­ transformed infrared spectroscopy (FTIR) analysis were used to obtain indications concerning the nature of the main chain, the crystallinity, and the functional groups of the polysaccharides. In addition, high-performance liquid chromatography (HPLC) and mass spectrometry (MS) were used for structural analysis of

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polysaccharides (Garozzo et al. 1999). However, to have a complete and detailed structural analysis of polysaccharides, the combination of different methods of analysis is preferable. Previous research has focused on the structural analysis of plant polysaccharides such as those on polysaccharides extracted from watermelon rinds (Romdhane et al. 2017), and chickpea (Mokni Ghribi et al. 2015). Structural analyses were carried out to characterize the polysaccharides of black cumin seeds (Manjegowda et  al. 2017; Trigui et  al. 2018). The XRD diagram recorded to study the crystallography of polysaccharides revealed that BCSP has a typical XRD profile for semi-crystalline polymers (Trigui et al. 2018). The result was similar to that found for other plant polysaccharides (Jeddou et al. 2016; Mokni Ghribi et  al. 2015). The FTIR spectrum of BCSP showed typical characteristic peaks of polysaccharides (Trigui et al. 2018). In fact, the spectrum showed the presence of uronic acids in polysaccharides and the pyranose form of sugars (Manjegowda et al. 2017; Trigui et al. 2018). Moreover, according to Manjegowda et al. (2017), FTIR spectral analysis has indicated that BCSP was a polymer of pectic polysaccharide type. FTIR spectroscopy was supported by NMR spectroscopy, to determine the structural profile of the BCSP. The 1H and 13C NMR spectra confirm the presence of α and β anomers in BCSP (Huang et al. 2011; Trigui et al. 2018; Xu et al. 2016). In addition, NMR spectra revealed that BCSP is composed of rhamnogalacturonan backbone (Habibi et al. 2005). The 13C NMR spectrum showed also signals indicating the presence of an arabinan-like structure as a side chain (Trigui et al. 2018). However, 1H NMR spectrum shows the presence of a side chain of galactan (Manjegowda et al. 2017). Overall, structural studies suggest that BCSP is a pectic polysaccharide having a rhamnogalacturonan backbone with galactan and arabinan side chains (Manjegowda et al. 2017; Trigui et al. 2018).

5  Functional Properties Polysaccharides of plant origin have functional properties that are relevant for many applications, particularly in the food and pharmaceutical industry, such as solubility, emulsifying and foaming capacities and the absorption and retention of fats and water. Functional properties are a set of physicochemical and organoleptic properties that influence the structure, technological quality, nutritional quality and acceptability of a product (Kinsella 1981). Generally, protein, as well as polysaccharides functionalities, are closely related to amino acid composition or monosaccharide composition and sequence, molecular weight, electrostatic charge distribution and structure (Deng et al. 2019). Measurements of electrical mobility are generally used to characterize the functional properties of macromolecules as well as variations in composition and structural changes. Several factors, such as chemical composition, concentration, pH, and temperature, can affect Zeta (ζ) potential values. The ζ-potential indicates the charge distribution at the interface of molecules. The surface charge, as indicated by

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the ζ potential of BCSP was reported by Trigui et al. (2018). Results showed that the surface charge of BCSP was affected by pH and that BCSP are negatively charged throughout the pH range (3–9). These results suggest that BCSP are potential water-­ soluble compounds capable of donating electrons and ensuring good stability of the solution. Knowledge of the phenomena involved in the construction of food products allows the control of their quality as well as their stability. Surface properties are of great importance. A substance that is capable of lowering the interfacial tension by adsorbing and forming a film at the interfaces is called surface-active compounds. The formulation of many foods requires the use of surface-active compounds that condition the formation and stability of systems dispersed in the air or in the fat. The surface activity of BCSP was evaluated by Trigui et al. (2018) in order to evaluate the potential of BCSP to reduce the surface tension at the air-water interface. As reported by Trigui et al. (2018), BCSP are able to decrease interfacial tension compared to that of water (72  mN/m), which indicates that BCSP are surface-active compounds. Surface tension was affected by BCSP concentrations. The interfacial behavior of BCSP can probably be explained by the increase in the net charges of the polysaccharides. The functional properties are generally related to the improvement of the texture. The interaction between components, including water and lipids, is water retention capacity (WHC) and oil retention capacity (OHC). The evolution of the WHC of BCSP at different temperatures (25, 50 and 75 °C) was determined (Trigui et al. 2018). WHC of BCSP increase with increasing temperature, this can be explained by the increase of the solubility of polysaccharides at high temperatures (Trigui et  al. 2018). BCSP had higher WHC than those obtained for polysaccharides extracted from watermelon rinds (Romdhane et  al. 2017), and sorghum sheaths (Slima et al. 2018). The water retention capacity recorded in this study suggests that BCSP can be used as a functional ingredient in food products. OHC is an important feature of polysaccharides. It is a technological property related to the chemical structure of polysaccharides. The evolution of OHC of BCSP at different temperatures (25, 50 and 75  °C) was reported by Trigui et  al. (2018). OHC of BCSP decreased with increasing temperature. Results are superior to those obtained for pea polysaccharides and okara (Mateos-Aparicio et al. 2010). As a result, BCSP can be used as functional ingredients in industrial products, because they improve the flavor retention and the mouthfeel. Proteins are generally used as emulsifiers because of their hydrophilic and hydrophobic chains. Indeed, the presence of small protein fractions in some polysaccharides has often been reported as responsible for the observed emulsifying properties (Huang et al. 2001). Emulsifying activity index (EAI) and emulsion stability index (ESI) of BCSP were studied by Trigui et al. (2018). EAI and ESI of BCSP decreased with increasing concentrations. In fact, the small protein fractions in BCSP are probably responsible for the emulsifying properties of polysaccharides. Therefore, it can be deduced that the different interactions between proteins and polysaccharides in BCSP may affect the properties of the emulsion. Because of their ability to

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form solid interfacial films, BCSP can be used as ingredients to stabilize food emulsions. Foaming capacity (FC) and foam stability (FS) of BCSP are also investigated by Trigui et  al. (2018). The results revealed that FC and FS of BCSP increase with increasing concentration. The foaming properties of BCSP can be attributed to the ability of the polysaccharides to improve the viscosity of the aqueous phase. Adequate concentrations of BCSP are needed to cover the surface of the air bubble, create a rigid film around it and, therefore, produce more stable foam. These results suggest that BCSP can be used as a foaming agent in food formulations.

6  Antioxidant Activities The phenomenon of oxidation can damage macromolecules in the body such as proteins and DNA, resulting in various diseases, including cardiovascular disease, cancer and aging-related disorders. It is widely accepted that an antioxidant is capable of slowing or inhibiting the oxidation of a naturally oxidizable product in the air and protecting biological systems (Park et al. 2001). Studies have shown that natural polysaccharides allow the prevention of stress-induced oxidative damage in free radical scavengers in living organisms (Gao et al. 2015). The antioxidant activities of polysaccharides are strongly related to the structural properties of polysaccharides and also depend on the unit of use, degree of branching, glycoside bonds, polysaccharide conformation and molecular weight (Li et al. 2014). However, hot water-soluble extraction of polysaccharides is the most effective method for preserving antioxidant activity (Liu et al. 2015). Antioxidants have often been used in industrial treatment systems to reduce damage to the human body and prolong stability during food storage. In general, many plant polysaccharides have shown strong antioxidant properties and can be explored as promising new antioxidants. Methods for measuring the antioxidant capacity of polysaccharides extract differ due to the diversity of antioxidant action and the oxidation process. Therefore, combining more than one method to assess antioxidant capacity is necessary (Pérez-Jiménez et al. 2008). DPPH· is a stable free radical widely used to determine the antioxidant capacity of the components. The mechanism of the antiradical activity is based on the reduction of DPPH· (violet color) in DPPH·-H (yellow color) by a proton donor substrate (antioxidant) thus leading to the reduction of the propagation of the oxidative reaction (Li et al. 2007). The DPPH· radical scavenging capacity of BCSP was studied by Trigui et al. (2018). BCSP showed a concentration-dependent DPPH· radical scavenging ability. The highest DPPH· radical scavenging activity (63.2%) was obtained at a concentration of 1 mg/mL (Trigui et al. 2018). Results were superior to those previously reported by Chen and Huang (2019), for pumpkin polysaccharides. Previous studies have shown that the radical scavenging activity of polysaccharides is influenced by the composition of monosaccharides and the molecular weight (Feng et  al. 2014). Indeed, the antiradical activity of BCSP can be attributed to the existence of

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hydroxyl groups and carboxyl groups of uronic acids in polysaccharides, which behave as hydrogen donors (Nara et al. 2009). The reducing power assay allows evaluating the potential of a natural antioxidant to give an electron to a free radical in order to make it more stable (Fan et al. 2012). The reducing power is determined using the potassium ferricyanide reduction method in which the iron (Fe3+) oxidation form is converted to ferrous form (Fe2+) by antioxidant compounds. The Fe2+ form can be monitored by measuring Prussian blue formation at 700 nm (Xing et al. 2008). The ability of BCSP to reduce Fe3+ to Fe2+ has been studied by Trigui et  al. (2018). The results show that the reducing power of BCSP depends on the concentration. In addition, the reducing power of BCSP increases with increasing concentration of polysaccharides. The IC50 value recorded for BCSP was 0.68 mg/mL (Trigui et al. 2018). Therefore, BCSP contains substances that are electron donors that can react with free radicals to convert them into more stable products. Our results are superior to those previously reported by Chen and Huang (2019) for pumpkin polysaccharides. Carbohydrates are also sensitive to oxidation, but they are less sensitive than lipids and proteins (Zhuang et al. 2013). The β-carotene bleaching test was used to study the ability of BCSP to inhibit oxidation of linoleic acid existing in an emulsified system. In fact, in this system, linoleic acid acts as a generator of free radicals. The presence of antioxidants can neutralize the generated free radicals and therefore maintain the β-carotene color for a longer period in the sample. The ability of BCSP to prevent or minimize the reduction of β-carotene is investigated by Trigui et al. (2018). The antioxidant activity of BCSP increases with the increase in polysaccharide concentration. The antioxidant activity was 53.9% at a concentration of 1 mg/mL. Moreover, the chelating activity of metals is based on ferrozine chelation of ferrous Fe2+ ions, which can quantitatively form a complex with them. In the presence of a chelating agent, the formation of the Ferrozine-Fe2+ complex is inhibited, which leads to a decrease in its red color. Measuring the decrease in the color of the complex at 562 nm therefore makes it possible to estimate the chelating capacity of the present chelator (Li et al. 2013). The chelating power is considered one of the mechanisms of the antioxidant activity since it can trap the transition metals, which can initiate lipid oxidation reactions. Among the transition metals, Fe2+ iron is known as the most important pro-oxidant, which promotes lipid oxidation because of its high reactivity (Li et al. 2013). The chelating power of BCSP has been reported by Trigui et al. (2018). The chelating activity of BCSP showed a concentration-dependent response. The chelating power of polysaccharides increased with increasing concentration. The IC50 value of BCSP obtained was 0.78 mg/mL. Moreover, data obtained by Manjegowda et al. (2017) provide evidence for the defining roles of antioxidants and pectic polysaccharide extracted from black cumin seeds.

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Chapter 8

Thymoquinone: Chemistry and Functionality Amr E. Edris

Abstract  Thymoquinone (TQ) is found in nature as one of the constituents of the volatile oil fraction of black seeds (Nigella sativa L.). Nowadays, more research interest is directed toward TQ due to its versatile biological activities especially its anticancer potentials. This chapter will present an overview that covers different aspects of TQ including its origin in N. sativa seeds and the different factors that can affect its content in the crude and the volatile oil of the seeds. The analytical chromatographic tools that are usually used for the detection and quantification of TQ in the crude oil of N. sativa and its volatile fraction will be reviewed. The author will discuss the biological activities of TQ with especial emphasis on its anticancer potentials. Strategies to overcome the drawbacks that can limit the practical applications of TQ as a promising anticancer agent such as its potential toxicity and low stability will be also discussed. Finally, the author will refer to the production of TQ either by isolation from its natural sources like N. sativa oil or by organic synthesis from petrochemicals. That segment of the chapter will also refer to the aspects of enhancing the anticancer activity of TQ by synthesizing new TQ-analogs. Keywords  Origin · Content · Analysis · Anticancer · Toxicity · Nanoencapsulation · Isolation · Synthesis

Abbreviations GC Gas chromatography HPLC High-performance liquid chromatography TQ Thymoquinone

A. E. Edris (*) Aroma & Flavor Chemistry Department, Food Industries & Nutrition Division, National Research Center, Dokki, Cairo, Egypt © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_8

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1  Origin of Thymoquinone and Factors Affecting Its Content Thymoquinone (TQ) is a natural phytochemical, which present in the volatile oil fraction of the seeds of Nigella sativa (family Ranunculaceae). El-Dakhakhny (1963) was the first to identify and report the presence of TQ as a major constituent among the composition of volatile oil. He determined the chemical structure of that compound as 2-isopropyl-5-methyl-benzoquinone. TQ is biosynthesized in the seeds of N. sativa from γ-terpinene, which is considered to be the precursor of TQ. γ-Terpinene undergoes dehydrogenation and aromatization, which leads to the formation of another cyclic monoterpene namely p-cymene. The later is converted by hydroxylation to thymol, which is finally oxidized to TQ (Poulose and Croteau 1978). Genus Nigella comprises around 20 spices; however, among these the “sativa” species is the one, which is characterized by the presence of TQ. The other famous species of Nigella such as N. damascena and N. orientalis do not contain TQ among their volatile oil composition (Moretti et al. 2004; Kokoska et al. 2005). Because TQ is a plant-derived secondary metabolite, its amount can vary radically in the seeds of N. sativa depending on many factors including the agricultural treatment, geographical location, and genetic diversity. These parameters control the biochemical pathways that lead ultimately to the biosynthesis of this compound. In addition, the method of oil extraction from N. sativa seeds can also be considered an important factor that determines the content of TQ in the extracted crude and volatile oil. The following passages will discuss these factors with more details and explanations. Effect of Agricultural Treatments on TQ Content Agriculture treatments can affect the yield of the volatile oil of N. sativa, which in turn can affect the content of TQ in that volatile fraction (Seyyedi et  al. 2015). Fertilization during plant growth, especially nitrogen and phosphorous levels plays an important role in the chemical composition of the volatile oil of N. sativa and its content of TQ (Ashraf et al. 2006; El-Ghawwas 2002). The levels of these two elements were optimized in order to give the best volatile oil and TQ yields (Özgüven and Sekeroglu 2007). Effect of Geographical Location on TQ Content The geographical location of growing any medicinal plant determines the quantity and composition of its secondary metabolite. That is because each geographical location has its characteristic environmental conditions depending on its latitude and altitude. Both elements can control the temperature, periods of sun shin, humidity, and rainfall levels. The effect of geographical location factor on TQ content in the volatile oil of N. sativa was evaluated by many investigators using gas chromatography (GC) analysis. For instance, the TQ percentage can range from 27% to 57% of the volatile oil composition depending on the seeds’ origin (Burits and Bucar 2000). On the other hand, it was found that TQ in the volatile oil of N. sativa seeds cultivated in Poland was in trace amounts, while p-cymene (60%) and γ-terpinene (12%) were the major constituents of the volatile oil (Wajs et al. 2008).

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Similar results were obtained for TQ in the volatile oil of N. sativa cultivated in Iran, which was found to be only 0.6% of the volatile oil fraction, while trans-anethole (38.3%), and p-cymene (14.8%) were the major volatile compounds (Nickavar et al. 2003). The volatile oil of N. sativa cultivated in Tunisia showed only 1.65% TQ content, while p-cymene represented the major compound (43.5%) followed by α-pinene (13.7%) (Toma et al. 2010). Similarly, TQ content in the volatile oil of Italian N. sativa was only 3.8%, while p-cymene (33.8%) and thymol (26.8%) represented the major constituents (Moretti et al. 2004). On the other hand, TQ in the volatile oil of some varieties of Egyptian N. sativa can vary from 30 to 63% (Edris 2010). More about the effect of geographical location on the TQ content can be detected by the reader himself in other relevant studies. One should also consider the fact that seeds obtained from the local market at a certain geographical location do not necessarily mean that they were grown in that location. It could be imported by traders to that region from another geographical location that differs climatically from the region of marketing. Therefore, any investigation that tends to correlate TQ content to certain geographical locations should be performed using seeds sown and grown under the climatic conditions of the same location of the study. The fluctuation of the amounts of TQ as a function of geographical location can also be evaluated directly in the crude oil of N. sativa  by HPLC (not just in the volatile oil fraction by using GC). For instance, TQ content was found to vary from 0.29  mg to 13.3  mg per 100  g of crude oil for seeds collected from 30 different geographical locations (Gad and El-Ahmady 2018). In a similar study, N. sativa seeds collected from 5 geographical locations contain an amount of TQ that varied from 1270 mg to 3089 mg per Kg seeds (Al-Saleh et al. 2006).  It is clear that there is a radical variation of content for TQ in the N. sativa volatile oil and crude oil depending on the geographical location. It is also obvious to consider that this variation will ultimately affect the biological activities of the crude and the volatile oil (Nguyen et al. 2019).  Effect of Genetic Factor on TQ Content Even within the same geographical location, the content of TQ can vary from a certain type of seeds to another depending on the genetic diversity of the mother herb. For instance, it was found that the volatile oil of N. sativa seeds cultivated in Egypt contains 30% TQ and 11% γ-terpinene (Edris 2010). On the other hand, TQ content reached 56%–63% and γ-terpinene 0.4% in a volatile oil extracted from other seeds grown also in Egypt. This variation was partially justified based on the role of genetic diversity in the regulation of biochemical pathways that lead to the formation of TQ in the seeds. From the early beginning of this chapter, it was indicated that TQ is biosynthesized from γ-terpinene, which is converted through different intermediate compounds into TQ (Poulose and Croteau 1978). Thus, due to some genetic factors, these biochemical pathways may not precede to the end where TQ is supposed to be accumulated. Therefore, the volatile oil in such cases accumulates high amounts of precursor compounds at the expense of TQ. These compounds

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could include γ-terpinene and p-cymene (Wajs et al. 2008), α-pinene and p-cymene (Toma et al. 2010) or even thymol and p-cymene (Moretti et al. 2004). Effect of Oil Extraction Method on TQ Content The extraction methods of the crude oil or the volatile oil from the N. sativa seeds also play a crucial role in the content of TQ in both oils. It is well known that the crude oil (fixed plus volatile) can be extracted from the seeds of N. sativa by pressing (Hamed et  al. 2017) or by using organic solvents like n-hexane (Edris et  al. 2016). Among different organic solvents, benzene was found to be the best extraction solvent that was able to extract the maximum amount of TQ from the seeds of N. sativa (Iqbal et  al. 2018). However, benzene should be totally avoided in the extraction process of any oilseed due to its proven carcinogenic effect as one of the hazards organic aromatic hydrocarbons that should be abandoned (Loomis et al. 2017). When the volatile oil of N. sativa (which bears TQ) becomes the fraction of interest, so it should be extracted from the seeds using a modified distillation method that depend on two successive stages (Edris 2010; Kokoska et al. 2005). The first stage is initiated by the extraction of the whole crude oil (fixed plus volatile) from the seeds via pressing or via the appropriate organic solvents. Then the extracted crude oil is subjected to the second stage of extraction using hydro- or steam-distillation to isolate the volatile oil fraction. This double-stage extraction protocol was reported to keeps TQ at its maximum level in the isolated volatile oil. Unfortunately, most of the newcomers to the field of N. sativa do not realize that fact and just subject the crushed seeds to one-stage distillation. This practice leads to diminishing the amount of TQ in the isolated volatile oil to very low limits, most probably due to degradation of TQ. As an illustration, to prove that statement, Edris (2010) found via GC analysis that the percentage of TQ in the volatile oil isolated directly from the crushed seeds of N. sativa using a one-stage hydrodistillation process was 2.5% (relative to the total volatiles). Interestingly, that percentage was significantly increased to 30% using the two-stage extraction method that involved the extraction of the whole crude oil from the seeds by n-hexane followed by hydrodistillation of the crude oil to isolate the desired volatile oil fraction with a maximum yield of TQ (30%). The trend of using a two-stage extraction method to maximize the yield of TQ in the volatile oil of N. sativa seeds was also confirmed in a second successive study (Edris et al. 2018). Environmental friendly techniques such as supercritical fluid extraction (SFE) can also be used for extraction of the whole crude oil from the seeds of N. sativa. The conditions of extraction including the pressure and temperature could affect the content of TQ in the oil. For instance, high extraction pressure (up to 400 bar) and temperature (50 °C) leads to crude oil rich in the fixed oil fraction at the expense of volatile oil and TQ (Venkatachallam et al. 2010; Rao et al. 2007). On the other hand, by adjusting the extraction conditions of SFE to 600 bar and 40 °C a TQ-rich fraction could be obtained (Norsharina et al. 2011).

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Recently, a method was developed to obtain a volatile oil-rich fraction from the seeds of N. sativa using CO2 under subcritical conditions instead of supercritical conditions (Edris et al. 2018). The pressure (70 bar) and temperature (30 °C) which were applied to the seeds in this method led to the extraction of the volatile oil-rich fraction that accounts for 66.6% of the total obtained extract. Consequently, much TQ content was found in the volatile oil fraction reaching 60.5% of the total volatiles. This percentage was found to be higher than that obtained using the one-stage traditional extraction methods like hydrodistillation (TQ, 10.2%) or steam distillation (TQ, 23.7%), taking into consideration that the same fresh seeds were used in the three extraction methods (Edris et al. 2018).

2  Chromatographic Analyses for Detection and Quantification of TQ Simple techniques such as thin-layer chromatography (TLC) can be used for the detection of TQ in the oil of N. sativa (Abou-Basha et al. 1995). In addition, a more advanced form of that technique like the high performance thin layer chromatography (HPTLC) can also be used for the detection and quantification of TQ (Taleuzzaman et al. 2017). On the other hand, TQ can also be detected using other chromatographic techniques like the high performance liquid chromatography (HPLC) equipped with a UV-vis detector, where TQ can be detected at 256 nm. This technique is considered to be one of the most important and straight forward analytical tools which are frequently used to detect and quantify TQ directly in the crude oil of N. sativa (Ghosheh et al. 1999; Gad and El-Ahmady, 2018; Isik et al. 2017; Iqbal et al. 2018; Al-Saleh et al. 2006). A more sophisticated form of HPLC like ultra-HPLC can also be used for the same purpose (Ahmad et al. 2018). The author would like to draw the attention of the reader to the fact that HPLC can detect TQ by direct analysis of the whole crude oil of N. sativa seeds. On the other hand,  for the detection of TQ in the pure volatile oil fraction, that will require the use of another basic chromatographic tool such as the gas chromatography (GC). In such a kind of chromatographic analysis, the volatile oil must be first separated from the fixed oil (e.g. hydro-distillation), then subjected to GC analysis for the assessment of TQ. A huge number of studies have used GC analysis as an effective analytical tool for that purpose and the reader can check that using the appropriate keywords like “chemical analysis and volatile oil of N. sativa”.

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3  B  iological Activities of TQ with Emphasis of Its Anticancer Properties TQ is a versatile phytochemical which has diverse biological activities and medicinal properties that were addressed in myriads of publications. These activities include antioxidant, anti-inflammatory, hepatoprotective, neuroprotective and immunomodulatory properties. In this passage of the chapter, the author will extensively focus on the anticancer potentials of TQ. That is due to the evidence-based and promising potentials of TQ in the therapy and protection against various types of cancer (Mahmoud and Abdelrazek 2019; Imran et al. 2018; Goyal et al. 2017; Khan et al. 2017; Schneider-Stock et al. 2014). This disease is so far the most elusive and life-threatening ailment with ~14.1 million cancer cases around the world (Sultana et al. 2014). TQ can also be used as an adjuvant to combine with classical anticancer drugs. For instance, TQ was evaluated as an adjuvant to cabazitaxel and showed a synergistic effect with the drug against breast cancer (Kommineni et al. 2018). The same trend was also adopted in mice by a combination of TQ with the traditional anticancer drug resveratrol (Alobaedi et al. 2017). That combination therapy caused a significant decrease in tumor size, induced geographic necrosis and enhanced apoptosis,  as claimed by the reference. The anticancer activity of doxorubicin against adult T-cell leukemia in vitro and in vivo was enhanced by combination with TQ (Fatfat et al. 2019). The successful trend of combining TQ with anticancer drugs was also extended to the modern field of irradiation which applies the Gamma-­ knife. It was found that the combination of Gamma-knife irradiation with TQ led to enhancing the cytotoxicity, genotoxicity, and apoptosis in B16-F10 melanoma cells (Hatiboglu et al. 2019). It is worth indicating that the maximum tolerated a dose of TQ was found to be lower in females than in males (AbuKhader 2012). That deficiency can be compensated in female-related cancers like breast cancer by combined therapy using TQ combined with an anticancer drug like doxorubicin (Effenberger-Neidnicht and Schobert 2011). Another advantage of TQ combination with anticancer drugs is the reduction of the toxic effect of the later on somebody organs. For instance, doxorubicin toxicity on different body organs was reduced and its activity was enhanced by combination with TQ (El-Ashmawy et al. 2017; Nagi and Mansour 2000). In the same trend, oral administration of TQ was found to ameliorate the toxic effect of cisplatin on different body organs including the kidney and the brush border membrane (Farooqui et al. 2017). In addition, cisplatin-induced neurotoxicity which usually accompanies treatment of cancer patients with that drug can be protected by combination with TQ (Üstün et al. 2018). The suggested mechanism includes the promotion of the neuronal cell viability and neurite outgrowth against cisplatin toxicity under the effect of TQ.  In another relevant investigation, the neuroprotective effect of TQ against cisplatin-induced neurotoxicity was correlated to the down-regulation of the

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apoptotic markers p38 mitogen-activated protein kinase and also to the role of TQ in the prevention of oxidative stress (Kandeil et al. 2019). More details about the mechanism of action of TQ against different types of cancers were reported in different studies (Fatfat et  al. 2019; Mahmoud and Abdelrazek 2019; Shanmugama et  al. 2018; Schneider-Stock et  al. 2014, Dergarabetian et  al. 2013). Generally, TQ can induce its pharmacologic effects through the modulation of the physiological and biochemical events responsible for the generation of reactive oxygen species (ROS). In normal tissues, TQ behaves as a powerful antioxidant but in cancer tissue, it works as pro-oxidant which induced the generation of ROS and decreased GSH levels in a dose- and time-dependent manner. On the molecular levels, TQ was reported to have a modulating effect on different oncogenic transcription factors. The above-mentioned literature clearly illustrates the anticancer activity of TQ and its promising role in cancer-fighting in different ways. Therefore, it was expected that legislative authorities would respond to the calls of investigators by allowing TQ for the final level of clinical evaluation on cancer patients (Khan et al. 2017; Islam et  al. 2016). However, that cannot be granted before comprehensive investigations of the toxicity of TQ in order to assess its maximum tolerated dose that could induce a therapeutic effect. That subject will be discussed in detail in the next passage.

4  Toxicological Aspects of TQ TQ is metabolized in the body by the reductases enzymes into two main metabolites depending on the number of reduction cycles. These metabolites include semiquinone, which has pro-oxidant activity or thymohydroquinone which has antioxidant activity (El-Najjar et al. 2010). TQ can also be reduced to dihydro-thymoquinone, which in turn leads to the formation of ROS that cause various adverse effects on the cells due to the induction of oxidative stress (Mashayekhi-Sardoo et al. 2018; Huq and Mazumder 2010). Due to these potentially toxic metabolites, a study was initiated to investigate the cytotoxicity and genotoxicity of TQ in vitro (Khader et al. 2009). Their results indicated that TQ concentrations from 50 μM down to 10 μM showed cytotoxic effects by inducing high levels of necroses compared to the control. These results indicate that TQ can cause glutathione depletion and liver damage at high concentrations. TQ also showed concentration-dependent genotoxicity as evidenced by a significant increase in the frequency of chromosomal aberrations at 2.5 μM. In another investigation, the acute and sub-acute oral toxicity of TQ in mice after encapsulation in nanostructured lipid carriers was studied (Ong et al. 2016). The results showed that the encapsulation process can decrease the toxicity of TQ compared to the un-encapsulated TQ. No observed adverse effect level was found at an oral dose of 10  mg/kg mice/day for long-term oral consumption, indicating safe consumption at that dose. The same research team in a previous relevant i­ nvestigation

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indicated that a long-term oral consumption TQ or TQ-encapsulated in nanostructured lipid carrier is safe for human consumption at the dose of 0.813 mg/kg human body weight/day (Ng et al. 2015). The LD50 studies of TQ administered via different routes were investigated by various researchers. For instance, Badary et al. (1998) indicated that the LD50 of TQ in mice was 2.4 g/kg after acute oral administration. At higher doses (2–3 g/kg) TQ induced signs of toxicity manifested by hypoactivity, difficulty in respiration, a significant reduction in tissue (liver, kidneys, and heart) in addition to reduced glutathione (GSH) content. On the other hand, intraperitoneal (i.p.) LD50 of TQ in mice was found to be 90.3 mg/kg (Mansour et al. 2001). They concluded that i.p. route of administration is more toxic than an oral route. Administration of TQ into mice via i.p. route up to 20 mg/kg had no adverse effect on body or liver or kidney weights (Harzallah et al. 2012). However, at 40 mg/ kg TQ showed an increase in malondialdehyde and catalase activities indicating toxic effects. Another investigation showed that the LD50 of TQ in mice via i.p. the route was 104.7 mg/kg while oral route showed LD50 870.9 mg/kg (Al-Ali et al. 2008). They also indicated that in the case of rats the LD50 of TQ via i.p. the route was 57.5 mg/kg and via oral route was 794.3 mg/kg. According to the claims of Al-Ali et al. (2008), LD50 values in mice are 10-times (for i.p. administration) to 15-times (for oral administration) greater than the doses of TQ which is reported for achieving its anticancer effects. The same LD50 values reported by Al-Ali et  al. (2008) in rate were also greater 100-times (for i.p.) to 150-times (for oral administration) than the doses of TQ to produce the same anticancer effects. For more data about the LD50, acute, subacute and subchronic toxicity of TQ and its adverse effects we refer to the comprehensive review of Mashayekhi-Sardoo et al. (2018).

5  S  trategies to Overcome the Toxicity and Other Physical Properties-Related Issues of TQ From the above mentioned, it is clear that TQ could be toxic at certain levels of administration. Moreover, TQ suffers from some other physical properties-related issues that can hamper its full utilization as a promising anticancer agent. These physical property issues include low stability in aqueous solutions, low water solubility and consequently low bioavailability (Salmani et al. 2014). Investigators indicated that TQ has low stability in aqueous solutions especially at pH > 7 (alkaline solutions) with rabid degradation. They also found that the water-solubility of TQ was 549–669 μg/mL, which is considered to be low and justify the low bioavailability of this compound. Exposure to light can also induce the degradation of TQ, so that even a short period of exposure can cause severe degradation of this compound. TQ is also found to be sensitive toward temperature even after formulation in water-based emulsion (Tubesha et al. 2013a, b). The best condition that leads to

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minimizing degradation of TQ in the emulsion is by storage at 4 °C–25 °C at which only 10% of TQ degradation can take place. Based on these issues and drawbacks, researchers try to develop some strategies to overcome the toxicity of TQ and to enhance its physical stability. That will allow getting the maximum therapeutic potential of this compound as a promising anticancer agent. A strategy that is based on nanoencapsulation technology seems to be appropriate for the fulfillment of the target of reducing toxicity (Ballout, et al. 2018; El-Far et al. 2018). In addition, the problem of low water solubility and its consequent low bioavailability can also be overcome by nanoencapsulation. That is because TQ nanocapsules are formulated in the water-based stable colloidal system, which allows TQ nanoparticles to become water-born at any concentrations. The carrier materials that are used as nanocapsules for enveloping TQ can encompass: (1) Synthetic polymeric nanoparticles: including polyethylene glycol (Bhattacharya et al. 2015) and poly-lactide-co-glycolide, known as PLGA (Dinarvand et al. 2011; Ravindran et al. 2010). (2) Polymeric micelle: including the nonionic triblock copolymers like Pluronic F127 and Pluronic F68 (Shaarani et al. 2017). (3) Natural polymeric nanoparticles: including β-cyclodextine (Al-Qubaisi et  al. 2019) and chitosan (Fakhria et al. 2019). (4) Nano-lipid carriers: including nanoliposomes (Mohammadabadi and Mozafari 2018), niosomes (Rajput et  al. 2015), and solid lipid nanoparticles (Ramachandran and Thangarajan 2016). (5) Nano-structured lipid carrier: including a combination of liquid oil and crystallized oil (Ong et al. 2016; Ng et al. 2015). (6) Nanoemulsions (Ahmed et al. 2016; Tubesha et al. 2013a, b). (7) Microemulsions (Velho-Pereira et al. 2017) (8) Silica nanoparticles: upon which TQ can be impregnated instead of encapsulated (Fahmy et al. 2019).

6  Synthesis of TQ and Its Analogs From a future perspective, TQ could potentially be approved one day as part of cancer treatment protocols. In this case, large quantities of this compound must be secured for the pharmaceutical industry to satisfy the demands. The natural source of providing pure TQ is the volatile oil fraction of N. sativa seeds, where this compound can reach up to 67% of the volatile oil composition of high-quality seeds (Hamed et al. 2017). Some studies that quantified TQ in N. sativa seeds by HPLC indicated that this compound can reach up to ~3 g/kg seeds in certain varieties of seeds (Al-Saleh et al. 2006). An attempt has been made for the isolation of natural TQ from the whole crude oil of N. sativa by mixing the later with n-hexane followed by deep freezing at −20 °C (El-Tahir et al. 2003). Another technique for isolation of

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TQ from the volatile oil of N. sativa was developed based on one of the nanotechnology approaches (Edris 2017). According to the author, the technique involves designing a solubilization system made of water, surfactant(s) and other excipients. When the volatile oil of N. sativa is mixed with that solubilization system at room temperature, a microemulsion is formed spontaneously transferring all the components of the volatile oil into nanoparticles except for TQ which precipitate in the form of pure crystals. However, from the practical point of view, there are not enough natural resources to fulfill the expected large demand for TQ on a global-wide level. Therefore, organic synthesis becomes an unavoidable route for the manufacture of TQ on a large scale with a reasonable cost compared with the isolation of TQ from natural sources. In this regard, a simple method for the synthesis of TQ was reported (Dockal et al. 1985) that depend on catalyzed oxidation of thymol or carvacrol using Co(II). The maximum yield of TQ can vary from 84% to 93% depending on the starting materials (thymol or carvacrol). There is always an endeavor for enhancing the anticancer activity of TQ by studying the structure-activity relationship of this compound. Therefore, different TQ analogs were synthesized for that purpose aiming to find more anticancer potent derivatives of that compound. One of these studies revealed that some of TQ-analogs were more anticancer active than TQ against ovarian cancer (Johnson-Ajinwo et al. 2018). Similarly, the synthesis of TQ-artemisinin hybrids showed high activity against colon cancer (Fröhlich et  al. 2018) and leukemia (Fröhlich et  al. 2017). TQ-analogs containing gallate and fluorogallate showed superior anti-proliferative and chemo-sensitizing activity against pancreatic cancer in vitro, whether tested alone or in combination with a traditional anticancer drug like Gemcitabine (Yusufi et  al. 2013). The authors also indicated that TQ alone from the volatile oil of N. sativa has moderate activity against pancreatic cancer compared to the synthesized TQ-analogs.

7  Conclusion The author wishes that this chapter be comprehensive enough for the inquiring scholars who are seeking collective topics relevant to TQ as a versatile phytochemical and promising anticancer agent.

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Chapter 9

Novel Prospective of N. sativa Essential Oil Analysis, Culinary and Medicinal Uses Doaa M. Abo-Atya, Mohamed F. El-Mallah, Hesham R. El-Seedi, and Mohamed A. Farag

Abstract  Nigella is a genus belonging to family Ranunculaceae. Its essential oil components have long been used in traditional medicine as well as in culinary food and more recently cosmetics. Its major volatile component thymoquinone is an emerging natural drug to exhibit hepatoprotective, anti-inflammatory, antioxidant and cytotoxic effects. In Nigella sativa, thymoquinone is the pharmacologically active component in its essential oil that is the most-frequently examined for its quality control analysis. In contrast, much less is reported on other volatiles composition and in response to the different extraction methods. Current review focuses on Nigella genus volatiles composition in the context of its different genotypes, geographical origin as well as processing or extraction methods. A review of Nigella volatiles application in the field of cosmetics, food flavoring and phyto-medicine and underlying chemistry is presented. Keywords  Nigella · Essential oil · GC-MS · Food flavor · Analysis

D. M. Abo-Atya · M. F. El-Mallah Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Koom, Egypt H. R. El-Seedi Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Koom, Egypt Division of Pharmacognosy, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden M. A. Farag (*) Pharmacognosy Department, College of Pharmacy, Cairo University, Cairo, Egypt Department of Chemistry, School of Sciences & Engineering, The American University in Cairo, New Cairo, Egypt e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_9

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Abbreviations ASE Accelerated Solvent Extraction CVDs Cardiovascular Diseases DM Diabetes Mellitus DN Diabetic Nephropathy DNA Deoxyribonucleic Acid DTQ Dithymoquinone EDE Experimental Dry Eye GC Gas Chromatography GC-FID Gas Chromatography-Flame Ionization Detector GC-MS Gas Chromatography-Mass Spectrometry GC-MS-O Gas Chromatography-Mass Spectrometry-Olfactometry GSH Gastric Mucosal Glutathione HD Hydro Distillation Extraction HDL High Denisty Lipoprotein HLB Hydrophilic-Lipophilic Balance HPLC High Performance Liquid Chromatography HPTLC High Performance Thin-Layer Chromatography HS-SPME Headspace Solid Phase Micro Extraction IC50 Inhibitory Concentration LDL Low Denisty Lipoprotein LPS Lipopolysaccharide MAE Microwave Assisted Extraction MCF-7 One Of Breast Cancer Cell Types MD Microwave Distillation MDA Malondialdehyde NaF Sodium Fluoride NMR Nuclear Magnetic Resonance NO Nitric Oxide NSEO-NE N. Sativa Essential Oil Nanoemulsion PGU Purity Gum Ultra RNA Ribonucleic Acid ROS Reactive Oxygen Species RP-HPLC Reversed Phase High Performance Liquid Chromatography SC-CO2 Supercritical CO2 Extraction SD Steam Distillation Extraction SE Solvent Extraction SE-SD Steam Distillation Of Crude Oil Obtained By Solvent Extraction SFE Supercritical Fluid Extraction SFE-SD Steam Distillation Of Crude Oil Obtained By SFE SOD Superoxide Dismutase SPME Solid-Phase Micro-Extraction THQ Thymohydroquinone

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THY Thymol TLC Thin Layer Chromatography TQ Thymoquinone γ-PARP Gamma Poly [ADP-Ribose] Polymerase 1

1  Introduction Nigella sativa is one of the 25 species within Nigella genus belonging to family Ranunculaceae (buttercup or crowfoot) (Edris 2009; Farag et al. 2017). The term “Nigella” originates from Niger meaning black in Latin as referred to the color of its seeds (Kalidasuet al. 2017). It is widely distributed in Middle Eastern Mediterranean region, Southern Europe, Northern India, Pakistan, Syria, Turkey, Iran, and Saudi Arabia (Srinivasan 2018). The most valued and medicinally used part of Nigella is the seeds, known as Panacea (in old Latin means cure all), Kalonji (India), Hak Jung Chou (China), Habbatu Sawda, Habatul Baraka (in Arabic means the blessed seed), black cumin, black seed, black caraway (English), chernushka (Russian), çörek out (Turkish), and Cyah-daneh in Persian (Aggarwal et al. 2008; Amin and Hosseinzadeh 2016). Its traditional uses are recognized by different religions (Islamic books and Holy Bible), and throughout history by the ancient Egyptians, Romans and Greeks. Figure 9.1 exhibits the traditional uses of N. sativa in different cultures.

Fig. 9.1  Traditional uses of N. sativa in different cultures

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The medicinal value of the seeds is approved by Arab/Unani system of medicine and other ethno-medicinal systems Ayurveda and Siddha (Gali-Muhtasib et  al. 2006; Paarakh 2010). N. sativa volatile oil is still used nowadays as a flavor for bread and cheese and as a spice in various kinds of meals (Wajs et al. 2008). The seed extract plays a vital role in cosmetic applications for skin, tooth and hair caring and wound healing (Aljabre et  al. 2015; Eid et  al. 2017; Sudhir et  al. 2016; Yaman et al. 2010). With regards to seed and its volatile oil medicinal uses, it exhibits a myriad of pharmacological activities that can help in the treatment of blood, skin, lung, cervix, kidney, prostate, liver and breast cancers (Khan et al. 2011; Swamy and Tan 2000). The seeds are proven to be effective against inflammation (inhibition effects on Carrageenan induced paw oedema) (Al-Ghamdi 2001), spasms (Gilani et al. 2001), bacteria (Hanafy and Hatem 1991), gastric ulcers (Rajkapoor et  al. 2002), and hyperglycemia (Al-Hader et al. 1993). The seeds exhibit diuretic and antihypertensive activity (El Tahir et  al. 1993a; Zaoui et  al. 2000), anti-nociceptive (Abdel-­ Fattah et al. 2000), analgesic (Al-Naggar et al. 2003), hepato-protective (Daba and Abdel-Rahman 1998), anti-oxytocic (Aqel and Shaheen 1996) and for respiratory stimulation (El Tahir et al. 1993b). Although the seed is rich in several metabolite classes, it is the essential oil (Gali-Muhtasib et al. 2006) and quinone content mainly mediate for most of the pharmacological activities mentioned above. Thymoquinone (TQ) is the main active constituent of black seed essential oil that amounts for ca. 70–90% of its volatiles composition. Seeds fixed and volatile oil amount for 35–40% and 0.05%, respectively, in addition to alkaloids, tannins, vitamins, mucilages, lipases, organic acids, phytosterols, resins, sugars and minerals (Cheikh-Rouhou et al. 2007; Farag et al. 2014). Till now, several extraction and isolation techniques have been used traditionally to identify N. sativa volatile oil composition to include hydro distillation extraction (HD) using Clevenger or Likens Nickerson apparatus, steam distillation extraction (SD), solvent extraction using maceration or Soxhlet and ultrasound assisted extraction (Edris 2010; Gerige et al. 2009; Kiralan and Kiralan 2015; Velho-Pereira et al. 2011). More advanced volatiles extraction methods include headspace solid phase micro extraction (HS-SPME), microwave assisted extraction (MAE), supercritical fluid extraction (SFE), steam distillation of crude oil obtained by SFE (SFE-SD), microwave assisted solid-phase micro extraction and accelerated solvent extraction (ASE) (Benkaci-Ali et al. 2006; Benkaci–Ali et al. 2007; Edris et al. 2018; Farag et al. 2017; Ghahramanloo et al. 2017; Kiralan 2012; Kokoska et al. 2008; Liu et al. 2012, 2013). A further step post isolation of volatile oil involves its chemical characterization using mostly hyphenated chromatographic methods such as thin layer chromatography (TLC) (Liu et  al. 2011), gas chromatography (GC) (Wajs et  al. 2008), gas chromatography-mass spectrometry (GC-MS) (Farag et al. 2017), gas chromatography-flame ionization Detector (GC-FID) (Edris 2010), gas chromatography-­mass spectrometry-olfactometry (GC-MS-O) (Kesen et al. 2018), high performance liquid chromatography (HPLC) (Piras et al. 2013), high performance thin-layer chromatography (HPTLC) (Velho-Pereira et  al. 2011) and/or nuclear magnetic resonance (NMR) (Venkatachallam et al. 2010).

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The main focus of this chapter is to review N. sativa essential oil role as food flavor, condiment, herbal medicine and in cosmetics. Further, the different methods employed for characterizing volatiles compositions and attempts to improve its physicochemical properties, stability, pharmacokinetic parameters in different preparations are also outlined.

2  Volatiles of Nigella Seeds N. sativa seeds comprise several metabolites but mostly fixed oil that amount for its major composition at 30% w/w, with 85% of total unsaturated fatty acids (TFAs) (Gali-Muhtasib et al. 2006). Linoleic acid (C18:2) is the main unsaturated fatty acid with 65.1% followed by oleic acid (C18:1) with 12.7 percentage (Botnick et  al. 2012), though fixed oil is not the focus of this chapter. Volatile oil was found to be abundant in the mature seed coat (Botnick et al. 2012) that accounted for its distinct aroma and/or biological effects. Variation in volatile oil yield ranging from 0.4% to 2.5% and/or qualitative composition has been observed in relation to seeds origin (Nickavar et al. 2003), season and time of cultivation (D’Antuono et al. 2002), geographical and storage conditions (El–Dakhakhny 1963; Piras et  al. 2013). Agricultural practice similarly influenced the oil composition in accordance to the grade of sunlight (Farid Benkaci-Ali et al. 2010), nutrients (El-Sayed et al. 2000), seeds cultivar (Edris 2010) and irrigation system (Kalidasuet al. 2017). Volatile oil composition was also found to be influenced by other processing factors. Roasting decreased the amount of TQ (2-isopropyl-5-methyl-1,4-­benzoquinone) concurrent with an increase in thymol, furans and sesquiterpenes (Farag et al. 2017). Pyrazine derivatives dominated in case of microwave roasting, while 3-­methylbutanal and furfural dominated in case of conventional roasting (Kiralan 2012). Other than this variation, common components of N. sativa essential oil reports were found to be TQ (27.8–57.0%), p-cymene (7.1–15.5%), carvacrol (5.8–11.6%), t-anethole (0.25–2.3%), 4-terpineol (2–6.6%), and longifolene (1.0–8.0%) (Hosseinzadeh and Parvardeh 2004). Quinones exemplified by (TQ) appeared to be the major volatile subclass in seeds and the one mediating for its pharmacological properties (Padhye et al. 2008). Quinones exhibit a major oxidative stability against thermal oxidation (60 and 100 °C), also further considered as potential chemotaxonomic marker for distinguishing N. sativa from other closely related drugs (Farag et  al. 2017). Reviewing literature reports of the volatile oil composition in N. sativa so far confirmed the significant existence of quinone monoterpenoids from samples collected from different regions and/or analyzed by several methods (Table 9.1). TQ is a photosensitive compound which dimerizes upon exposure to sunlight to produce dithymoquinone (DTQ) (Venkatachallam et al. 2010). This sensitivity to sunlight warrants proper storage to avoid its dimerization. Equally important, the volatile constituents of N. sativa ground seeds showed more sensitivity to eco-­ factors of air, heat, and light than that of whole seeds (Ahamad Bustamam et al. 2017), suggestive that ground seeds exhibit less shelf life compared to the whole seed.

SD −

+ − − − −

−

− − −

− + − − − − − −

HD −

− − − − −

−

− − −

− + − − − − − −

Egypt

Extraction method 1,4-Dimethyl-3-cyclohexenyl methyl ketone 1,8-cineole 1-Ethyl-2,3-dimethyl benzene 1-Methyl-3-propyl benzene 2(1H)-Naphthalenone 2-(3,5-Dimethylphenyl)-2-­ propanol 2-Methyl-6-methylene-octa-1,7-­ dien-­3-ol 2-Tridecanone 3-methyl Nonane 4-Isopropyl-6-methoxy-1-methyl1-cyclohexene 4-Methoxy-2,3,6-trimethylphenol 4-Methoxythujane Anethole Anisaldehyde Apiole Aromandendrene Bergamotene Biformene

Origin

Iran

− − − − − − − −

− − −

−

− − − − −

SFE −

− + − − − + + −

− − −

+

− − − − +

− + − − − + + −

− − −

+

− − − − +

SPME − −

− − − − − − − −

− − −

−

− − − − −

−

Farag et al. (2017)

− + − − − + + −

− − −

+

− − − − +

−

+ − − − − − − +

+ − +

−

− − − − −

MAE +

Liu et al. (2013)

India Turkey Egypt Syria India Bangladesh

− − − − − − − −

− − −

−

− − − − −

−

Ghahramanloo et al. (2017)

Edris et al. (2018)

Reference

India Turkey Egypt Syria India Bangladesh

Iran

Egypt

Origin

India

− − +++ + + − − −

− + −

−

− + + + −

HD −

India Gerige et al. (2009)

− − − − − − − −

− − −

−

− − − − −

Turkey

− − − − − − − −

− − −

−

− − − − −

− − − − − − − −

− − −

−

− − − − −

− − − − − − − −

− − −

−

− − − − −

Kokoska et al. (2008) SE-­ HD SD SD SFE-SD − − − −

Turkey

Table 9.1  The chemical composition of Nigella sativa essential oils, collected from different regions, extracted by different methods and analyzed by GC-MS

102 D. M. Abo-Atya et al.

HD − − + − − + − − − − − +++ − − − − − − − − − − − − −

Reference

Extraction method Bisabolene Borneol acetate Bornyl acetate Cadinene Camphor Carvacrol Carvone Caryophyllene Copaene Cyclocitral Cymen-8-ol Cymene Cymenene Dehydrothymoquinone Dihydrocarvone Diisooctyl phthalate Dimethyl-benzyl alcohol Diphenyl-2-pyridylmethane Elemene Eremophilene Estragole Farnesene Farnesol Fenchone Isoterpinolene

SD − − + − − + − − − − − +++ − + − − − − − − − − − − −

Edris et al. (2018) SFE − − − − − + − − − − − + − − − − − − − − − − − − − − − − − + − − − − − − + − − − − − − − − − − − − −

Ghahramanloo et al. (2017) SPME − − − − − − − − − − − +++ − − − − + − + − − + − − − − − − − − − − − − − − +++ − − − − + − + − − + − − −

− − − − − − − − − − − − − − − − − − − − − − − − −

Farag et al. (2017)

− − − − − − − − − − − +++ − − − − + − + − − + − − −

MAE + + − − − + + − + + − +++ − − − + − + − + − − − − −

Liu et al. (2013) HD − − − − − + + − − − + + − − + − − − − − + − − + −

Gerige et al. (2009)

(continued)

Kokoska et al. (2008) SE-­ HD SD SD SFE-SD + + − + − − − − + − + + + − + + − + − − + + + + + + + + + + − + − − − − − − + − − − + − +++ +++ +++ + − + + + − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − + − − − − − − − + 9  Novel Prospective of N. sativa Essential Oil Analysis, Culinary and Medicinal Uses 103

Egypt

Iran

SPME + − − − − − + − + − − − + − + − − + − − + + − − − − − + − + − − − +++ − + − − + − − +

− − − − − − − − − − − − − − − − − + − − −

Farag et al. (2017)

+ − − − − − + − − − − − +++ − + − − + − − +

MAE − + − − − − + + − − − − − − − + + − − + +

Liu et al. (2013)

India Turkey Egypt Syria India Bangladesh

− − − − − +++ − − − − + − − +++ − − − − + − −

Origin

SFE − − − − − +++ − − − − + − − + − − − − − + −

HD − + − − − − + + − − − − − − − + + − − + +

Extraction method Lilac aldehyde C Limonene Limonene oxide Linalool Linalool acetate Linoleic acid Longifolene Longipinene Muurolene Myrcene Myristic acid Myristicin Nerol Oleic acid Phellandrene Pinene Sabinene Santalene t-Butylhydroquinone Terpinen-4-ol Terpinene

SD − + − − − − + + − − − − − − − + + − − + +

Ghahramanloo et al. (2017)

Edris et al. (2018)

Reference

India Turkey Egypt Syria India Bangladesh

Iran

Egypt

Origin

Table 9.1 (continued)

India

HD − + − − − − + + − + − + + − + + + − − + +

India Gerige et al. (2009)

Turkey

Kokoska et al. (2008) SE-­ HD SD SD SFE-SD − − − − + + + − − − + − + + + + + + + + − − − − + + + + + + + + − − − − − − − − − − − − − − − − − − − − − − − − + + − − + + + + + + + + − − − − − − − − + + + + + + + +

Turkey

104 D. M. Abo-Atya et al.

HD +++ − − − +++ −

Reference

Extraction method Thujene Thymohydroquinone Thymol Thymol methyl ether Thymoquinone Uvidine

SFE + − − − + − − − − − + −

Ghahramanloo et al. (2017) SPME + − − + +++ − + − − + +++ −

− − + − +++ −

Farag et al. (2017)

+ − +++ + + −

MAE + − − − +++ −

Liu et al. (2013)

− − − +

HD +

Gerige et al. (2009) Kokoska et al. (2008) SE-­ HD SD SD SFE-SD +++ +++ + + + − + − − + − + − − − − + + +++ +++ − − − −

HD Hydro distillation, SD Steam distillation, SFE Supercritical fluid extraction, MAE Microwave assisted extraction, MD Microwave distillation, SE-SD steam distillation of crude oil obtained by solvent extraction, SFE-SD steam distillation of crude oil obtained by SFE

SD + + − − +++ −

Edris et al. (2018)

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Traditional extraction methods as hydro- and steam distillation (HD, SD), produce low TQ levels compared to other advanced methods such as supercritical fluid extraction (SFE), solid-phase micro extraction (SPME), and microwave assisted extraction (MAE) (Edris et al. 2016; Kokoska et al. 2008; Wajs et al. 2008). Such differences in TQ yield are attributed to either the poor extraction efficiency and or degradation of thermolabile constituents (Abedi et al. 2017; Kubátová et al. 2001; Liu et al. 2012). The chemical composition of essential oils isolated by HD and SD were closely similar, chiefly containing p-cymene (56.2% for HD and 52.0% for SD). Whereas, thujene (15.1% for HD and 17.5% for SD) and TQ was the third major detected compound isolated by SD (4.3%), and only at trace levels (0.5%) in case of hydro-­ distilled oil. p-Cymene (42.4%) and TQ (30.7%) were detected from SE-SD treated oil. In contrast, TQ (76.7%) was the major compound in oil isolated by SFE-SD (Kokoska et al. 2008). A study conducted by Ghahramanloo et  al. (2017), revealed that supercritical fluid extraction (SFE) showed significant extraction efficiency than other traditional methods when using N. sativa essential oil from two different regions (Iran and India) (Ghahramanloo et al. 2017). Farag et al. (2017) investigated N. sativa samples from Syria, Turkey, India and Egypt, employing the SPME-GC-MS. This study revealed that aromatics relative percentiles are at 99.8%, 80.8%, 79% and 67%, respectively in all N. sativa accessions. TQ was the pharmacologically active constituent, though showing larger variation from 99.7% in samples from Syria to only 1.8% in samples from India (Farag et al. 2017). Analysis of N. sativa essential oil using MAE coupled to GC-MS displayed high yield of TQ and p-cymene (Xue Liu et al. 2013). The results point to that N. sativa seeds grown in different regions i.e. Egypt, Turkey, Bangladesh, India and Syria, and extracted using different methods encompass several volatile classes mostly terpenoids. Only in samples extracted using supercritical fluid extraction (SFE), that higher levels of unsaturated fatty acids (linoleic and oleic acids) were reported likely due to the lyophilic nature of the extracting solvent in such case (Khaw et al. 2017). Figure 9.2 displays chemical structures of N. sativa volatile oil components, grouped based on their class type.

3  A  nalytical Techniques Reported Towards Nigella Volatiles Characterization This section shall cover reports made towards qualitative and quantitative assays for volatiles in Nigella using different extraction or detection methods and highlighting advantages and limitation for each if any.

9  Novel Prospective of N. sativa Essential Oil Analysis, Culinary and Medicinal Uses

A

C

107

B

D

E

F

Fig. 9.2  Major volatile classes detected in N. sativa volatile oil: (a) Monoterpene hydrocarbons; (b) Monoterpene alcohols; (c) Terpenic phenol; (d) Monoterpene ethers; (e) Monoterpene ketones; and (f) Unsaturated fatty acids

3.1  Extraction Methods 3.1.1  Traditional Volatile Oil Extraction Methods Traditional methods used for volatile oil extraction on a large scale include mostly heat or solvent treatment being easy, simple and inexpensive methods. However, there are some limitations such as the high energy consumption, solvents exhausted extremely, poor extraction efficiency, long time consuming and toxic solvents

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(Abedi et al. 2017; Xue Liu et al. 2012). Also, volatile degradation owing to extra purification of low quality essential oils produced by conventional methods (Ghahramanloo et al. 2017), leading to low TQ percentage (10.1 and 23.7%) when extracted by HD and SD, respectively, compared to other methods such as n-hexane extraction (37.6 and 41.6%) or subcritical CO2 extraction (60.5%) (Edris et al. 2018). Solvent extraction (SE) applied for volatiles extraction is mostly done using methanol, ethanol, acetone, chloroform and n-hexane in Soxhlet apparatus (Ramadan and Mörsel 2002; Rao et al. 2007; Singh et al. 2005; Velho-Pereira et al. 2011). Using a Soxhlet extractor at 70 °C or other hot solvent extraction methods at 40–60 °C can affect the oil properties and characteristics due to change in volatile components at the high temperature of distillation (Cheikh-Rouhou et al. 2007). 3.1.2  Advanced Volatiles Extraction Methods Considering limitations in the above methods and to overcome disadvantages of traditional extraction methods, novel techniques are continuously developed. These advanced techniques offer superior yield, less extraction time, high accuracy, less solvent consumption, and the most important feature is prevention of thermolabile components degradation. 3.1.2.1  Solid-Phase Micro-Extraction (SPME) Solid-phase micro-extraction (SPME) is considered as one of the relatively modern methods, invented by Pawliszyn in 1989, and used for volatiles extraction being simple, rapid, accurate, efficient, eliminate solvent costs and toxicity and requiring no heat (Farag et al. 2017; Vas and Vekey 2004). In addition, a characteristic feature of SPME is maintaining the volatile aroma without any modifications as in case of hydro- and steam distillation (Marsili 2001). It allows extraction of less abundant volatiles from liquid or gas samples through desorption from fused-silica fiber, depending on many factors including fiber choice, adsorption time and adsorption temperature (Prosen and Zupančič-Kralj 1999; United Nations Industrial Development et al. 2008). A major limitation of SPME is that it does not allow oil recovery to be further tested but only the characterization of aroma compounds. HS-SPME was employed to confirm that isomeric methyl ethers are natural components of N. sativa and are not produced as artifacts during fractional distillation or hydro-distillation (Wajs et al. 2008). 3.1.2.2  Microwave-Assisted Extraction (MAE) Microwave-assisted extraction (MAE) is a novel technique reported for the first time by Ganzler et al. (1986) (Ganzler et al. 1986). It is an efficient extraction tool exhibiting many advantages including superior yield, effective and rapid heat transfer,

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elimination of organic solvents, low experimental cost and less extraction time (Gavahian et al. 2015). The less the extraction time during microwave extraction, the less the oxidation and hydrolysis rate of the compounds (Qi et al. 2014). The MAE mode of action can be elucidated by transfer of heat from inside to outside of the sample, leading to increase in internal moisture temperature and hence rupture of the external cell walls and egress of essential oil. In contrast, HD leads to thermal degradation of thermolabile substances due to transfer of heat from outside to the inside of samples (Lucchesi et al. 2007). We suggest that the variation in extraction efficiency between the two techniques is based on the different mode of heating. Benkaci-Ali et al. (Benkaci-Ali et al. 2006, 2007), Liu et al. (2013) and Abedi et al. (2017) conducted several studies on MAE of N. sativa volatile oil including a comparison between MAE techniques and other traditional methods (Abedi et al. 2017; Benkaci-Ali et al. 2006, 2007; Liu et al. 2013). MAE was found superior to HD extraction method by providing a superior TQ extraction efficiency where MAE after 30  min yielded (0.316%, w/w), whereas HD after 3 h yielded (0.23%, w/w) (Abedi et al. 2017). These results are in agreement with Benkaci–Ali et  al. (2007) revealing that microwave extraction of N. sativa seeds yielded higher oxygenated monoterpenes yields than by HD (Benkaci–Ali et al. 2007). Benkaci-Ali et al. (2006) results suggested that 90% of N. sativa volatile oil can be extracted within 6 min and to significantly save both heat energy and extraction time, concurrent with a reduction of compounds produced from thermal degradation such as DTQ (F. Benkaci-Ali et al. 2006). In contrast, Liu et al. (2013) reported that the optimum MAE time is 10 min. TQ and p-cymene were the predominant components in N. sativa (Liu et al. 2013). 3.1.2.3  Supercritical Fluid Extraction (SFE) Supercritical fluid extraction (SFE) is an excellent method for extraction of volatiles from aromatic plants particularly N. sativa despite its relatively high equipment cost (Abedi et al. 2017; Piras et al. 2013). It exhibits many advantages to include being rapid, selective, avoid oxygen in extraction medium, less solvent consumption, avoiding degradation of bioactive compounds, and with extraction solvent can be separated easily leaving no solvent memory effect (Machmudah et  al. 2005; Venkatachallam et al. 2010). Ghahramanloo et al. (2017) reported the extraction efficiency of N. sativa oil using SFE technique is higher than solvent extraction methods. Nevertheless, SFE provided low TQ level compared to TQ recorded in other samples (Ghahramanloo et al. 2017). As SFE utilizes safe, non-flammable, cheap and pure solvents such as liquefied CO2, it can surpass conventional extraction methods such as SD, HD and SE by getting rid of hydrolysis and oxidation related to volatile components (Lang and Wai 2001; Piras et al. 2009; Piras et al. 2012; Porcedda et al. 2010). Solati et al. (2012)

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reported that supercritical CO2 extraction (SC-CO2) displayed a superior oil yield and potent antioxidant activity at elevated pressure. Optimum extraction conditions for TQ production were 150 bar, 40 °C, 120 min, and for oil yield were 350 bar, 60 °C and 120 min concurrent with the best antioxidant effect reported at extraction conditions of 350 bar, 50 °C, 60 min (Solati et al. 2012). Edris et al. (2018) results confirmed the work of Ghahramanloo et al. (2017) suggesting that extraction using solvent i.e. n-hexane, hydro- and steam distillation methods produced lower TQ content compared to that produced by SC-CO2. Nevertheless, it is the amount of monoterpene hydrocarbons extracted by SC-CO2 that appeared to be less than that extracted using other techniques (Edris et al. 2018; Ghahramanloo et al. 2017). 3.1.2.4  Accelerated Solvent Extraction (ASE) Accelerated solvent extraction (ASE) exceeds traditional extraction methods by offering higher yield, better reproducibility and with less solvent consumption (Kaufmann and Christen 2002). In addition, it also requires less time being carried out at elevated temperature and/or pressure. The utilization of elevated temperature aims to improve extraction efficiency asides it is the elevated pressure that maintains solvent in a liquid status ensuring rapid and safe extraction (Wang and Weller 2006). Disadvantages for ASE include its relatively higher cost and degradation of thermolabile compounds at elevated temperature (GiergielewiczMożajska et al. 2001). A study conducted on N. sativa essential oil extraction using ASE revealed that carrying it out at optimal selected conditions can eliminate volatiles degradation. The existence of some classes such as monoterpene hydrocarbons (p-cymene, α-thujene) and oxygenated compounds (TQ, carvacrol) post extraction using ASE suggested that no obvious degradation occurred in oil (Liu et al. 2012).

3.2  C  hromatographic and Identification Techniques of Nigella Volatiles 3.2.1  Thin Layer Chromatography (TLC) Quantitative TLC technique provides a simple, fast, reproducible analytical method (Y.-Z.  Liang et  al. 2004) for analysis of natural products routinely done in most laboratories. TLC was used for the determination of TQ, thymol and DTQ in N. Sativa seed oil (Basha et al. 1995).

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3.2.2  High-Performance Thin-Layer Chromatography (HPTLC) HPTLC surpasses regular TLC by offering higher sensitivity and improved separation efficiency (Ram et al. 2011). HPTLC was reported for the quantification of TQ in N. sativa extract, commercial oil and formulations (Velho-Pereira et al. 2011), posing it as an optimum technique to be applied for QC analysis of Nigella oil and determining its authenticity. 3.2.3  High Performance Liquid Chromatography (HPLC) Compared to TLC, HPLC has many advantages including its sensitivity, rapidness (owing to smaller analytical time) and precision (Anjum et al. 2012). HPLC was employed to quantify for quinone and associated components in N. sativa seed oil (Ghosheh et  al. 1999). The HPLC was used for the identification of N. sativa seed oil bioactive ingredients including TQ, DTQ, thymohydroquinone (THQ), and thymol (THY) (Ghosheh et al. 1999). The quantification of TQ was carried out using reversed phase high performance liquid chromatography (RPHPLC) (Iqbal et al. 2018). 3.2.4  Gas Chromatography-Mass Spectroscopy (GC-MS) GC-MS is a robust identification technique, allowing for the identification of a large number of compounds. About 112 compounds were identified from N. sativa seed oil using GC-MS (Benkaci–Ali et al. 2007). GC-MS revealed that the highest TQ level was observed in N. sativa oil samples from Turkey (Kazemi 2015; Piras et al. 2013). Two-step extraction methods (HD & SE) of N. sativa seeds showed higher TQ level than HD alone (Edris 2010). 3.2.5  G  as Chromatography-Mass Spectrometry-Olfactometry (GC-MS-O) GC-MS-O was recently applied for the determination of N. sativa aroma-active compounds characteristic odors (Kesen et al. 2018) by offering aroma type linked to each components and providing better insight on the sensory characters of Nigella aroma compounds. The technique allows for the classification of volatile compounds into aroma and non-aroma-active compounds in the investigated samples. The investigation of N. sativa seed extracts characteristic odors was dominated by buttery, cheesy, balsamic, citrusy, and spicy odors (Kesen et al. 2013).

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3.2.6  Nuclear Magnetic Resonance (NMR) NMR is one of the spectroscopic techniques that is increasingly applied for analyzing various food products i.e. beverages, oils, lipids, plants and milk goods. Though is less sensitive than MS, NMR tools are most powerful in the structural elucidation of compounds especially if new and not reported in literature. Application for analysis of N. sativa essential oil include the identification of the 2 monoterponoid compounds (cis- and trans-4-methoxythujane) (Wajs et al. 2008).

4  Nano-Formulation of N. sativa Volatile Oil Recently, nanoemulsion formulation is increasingly applied to improve the physicochemical characters i.e. solubility and stability of pharmaceuticals including essential oil. Nanoemulsions are ultrafine dispersions of oil in water at a size range of 10–600  nm (Solans et  al. 2003). Although, N. sativa essential oil exhibit strong therapeutic effect, it is less applied clinically due to its poor solubility and absorption (Odeh et al. 2012). Nanoemulsion-based formulation is a commonly used system for improving lipophilic material solubility as in case of N. sativa essential oil Liu et al. 2007). Several nanoemulsion techniques are used, such as high-pressure homogenisation, microfluidisation, phase inversion, spontaneous emulsification, solvent evaporation and ultrasonication (Date et  al. 2010). Among these protocols, ultrasonic emulsification is a simple, cost-effective, high-energy, smooth, rapid and aseptic (Mahdi Jafari et al. 2006). A N. sativa essential oil nanoemulsion (NSEO-NE) was prepared and assessed for its anti-cancer effect on MCF-7 human breast cancer cells. The preparation consisted of N. sativa essential oil (3%), polysorbate80 as a non ionic surfactant and water. Such nanoemulsion of N. sativa oil led to droplet size decrease dependant on both surfactant and oil composition (Ziani et  al. 2011). Nanoemulsions with 1:2 (v/v) and 1:3 (v/v) ratio exhibited a droplet diameter of 44.6 and 37.4 nm, respectively. These results are in agreement with that reporting on increase of surfactant ratio parallel to a decrease in oil droplet size (Gutiérrez et al. 2008). The inhibitory effect of NSEO-NE on the viability of human breast cancer cells was evaluated at 24 and 48 h at a dose level of 10–100 μL/mL and revealing that NSEO-NE markedly inhibited breast cancer cell viability (Periasamy et al. 2016). Another approach for preparing NSEO-NE included the use of purity gum ultra (PGU) as a surfactant to react with N. sativa essential oil (NSEO) as active ingredient. The PGU-stabilized NE exhibited high negative Zeta potential (> −30), leading to longer shelf-life (Bonilla et al. 2012) in addition to smaller mean droplet diameter (MDD). Though it should be noted that MDD of nanoemulsion changed by 20–30 nm during 1 month of storage at room temperature (Abbas et al. 2015; Liang et al. 2012; Majeed et al. 2016). PGU-based NSEO-NE was compared with pure NSEO regarding its antimicrobial activity against Bacillus cereus and Listeria

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monocytogenes growth, with stronger inhibition observed in the encapsulated NSEO in comparison to pure NSEO.  The anitmicrobial mechanism of PGU-­ stabilized NSEO-NE against Gram-positive bacteria was found to be mediated via the disruption of cytoplasmic membrane (Majeed et al. 2016).

5  Nigella Volatiles in Food Flavor and Culinary Uses The unique aroma characteristics of N. sativa volatile oil contributes to its taste and hence its culinary uses as food flavor (Botnick et al. 2012), described as being peppery, slightly bitter, smoky and spice (Edris 2010). Several aroma-active compounds were identified in N. sativa with its characteristic odor to include: eugenol (spicy-­ smoky), phenethyl alcohol (honey, floral), guaiacol (smoky), isobutanoic acid (cheesy), hexanoic acid (cheesy), acetoin (buttery) and limonene (citrus) (Kesen et al. 2018), suggestive that the characteristic odor of N. sativa seeds is mediated by a blend of volatile compounds and not just a single one. Edris et al. (2016) reported that formulated N. sativa L. oleoresin could be used in the fortification of nutraceuticals, processed food and dairy products (Edris et al. 2016). Cakir et al. (2016) reported that addition of N. sativa enhances cheese aroma and affects positively the consumers score values resulting in better palatability. Such flavor enhancement was attributed to increase in total levels of active volatiles i.e. alcohols, aldehydes, ketones, sulfur compounds, esters, terpenes and miscellaneous compounds (Cakir et  al. 2016) as secondary proteolysis products of the cheese. Another application of N. sativa oil is in the production of low fat non-dairy creamer by using fluidized bed coating technology. Sensory evaluation revealed an increase in consuming the developed coffee creamer, with less moisture content, high water solubility and strong antioxidant activity (Mohammed et al. 2019). Aside from N. sativa seeds culinary uses, its seeds are known as functional therapeutics, attributed partially to its volatile oil composition (Ali and Blunden 2003) to be discussed in details over the next section.

6  Beneficial Effect of N. sativa Essential Oil N. sativa essential oil exhibit a myriad of pharmacological effects to include anti-­ inflammatory (Bordoni et  al. 2019), anti-Alzheimer (Alhebshi et  al. 2013), anti-­ cancer (Peng et al. 2013), anti-microbial (Bita et al. 2012), anti-oxidant (Abbasnezhad et al. 2015), anti-schistosomiasis (Mohamed et al. 2005), anti-diabetic (Althnaian et  al. 2019), anti-stress (Roshan et  al. 2010), cardiovascular protective (Nemmar et al. 2011), and nephron-protective action (Saleem et al. 2012). The majority of these activities are attributed mostly to its TQ, thymol, DTQ, α-pinene, β-pinene, p-cymene and thymohydroquinone (Al-Saleh et al. 2006) (Fig. 9.3).

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Fig. 9.3  Major component of N. sativa volatile oil and its pharmacological activities

Although researchers have conducted several preclinical studies on Nigella oil, further research on in vivo and clinical assessment of its medicinal uses are still needed (Mukhtar et al. 2019).

6.1  Anti-inflammatory Effect Inflammatory responses are endogenous mediators that perform defensive reaction to remove damaging substances (Majdalawieh and Ro 2010). These harmful materials trigger diseases such as arthritis, asthma and eczema, chronic rhinitis and ulcerative colitis (Yousefi et al. 2013). Volatile oil of N. sativa exhibited anti-inflammatory efficacy mediated via the inhibition of eicosanoid and lipid peroxidation (Mutabagani and El-Mahdy 1997). In addition to reduction of NO production in LPS-challenged primary glial cells (Alemi et al. 2013). Recent research explored N. sativa oil’s anti-­ inflammatory activity in allergic rhinitis (Nikakhlagh et al. 2011), in which patients were administered 0.5 mL of N. sativa oil for 30 days and showing reduction of nasal mucosal congestion, nasal itching, and sneezing attacks (Nikakhlagh et  al. 2011). Intraperitoneal injection of N. sativa oil (1.55 mL/kg) resulted in the suppression of hind paw edema by 96.3% (Mutabagani and El-Mahdy 1997) superior to that of indomethacin (3 mg/kg) showing 46.9% inhibition of edema (Mutabagani and El-Mahdy 1997).

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6.2  Anti-diabetic Effect Diabetes mellitus (DM) is a metabolic disorder worldwide, and results in chronic glucotoxicity (Lenzen 2008). Raise of glucotoxicity in blood arise from the production of reactive oxygen species (ROS), which promote cellular damage and contribute to the development of several of diabetes known complications (Kaatabi et al. 2015). N. sativa volatile oil showed efficacy for treatment of diabetes or glucose intolerance acting via the secretion of glucose-induced insulin without adversely affecting the absorption of glucose (Kapoor 2009). Volatile oil may also reduce diabetes-induced production of malondialdehyde (MDA) as well as enhanced superoxide dismutase (SOD), and level of insulin concentrations (Pari and Sankaranarayanan 2009). In a randomized controlled trial, the anti-hyperglycemic impact of adjuvant therapy of N. sativa volatile oil was assessed in comparison to anti-diabetic drug, in type 2 diabetes. All observed parameters were reduced relative to base line with placebo groups, and with no side affected observed in N. sativa treated patients (Fallah Huseini et al. 2013). TQ alleviated diabetic nephropathy (DN) by reducing kidney injury markers that increased in diabetic rats compared to control i.e. creatinine, urea protein and oxidative stress markers, including (TBARS) (Al-Majed et  al. 2006). Hyperglycemia leads to oxidative and osmotic stress, resulting in diabetic vascular complications (Dunlop 2000; Sugimoto et al. 2001). TQ functions also as a γ-PARP antagonist, γ-PARP plays a key role in DN and kidney disease progression (Drel et al. 2006; Obrosova et al. 2005) in addition to inhibition of lipolysis and proteolysis in rats fed ethanol and a high-fat diet (Suguna et al. 2013). TQ stimulates the uptake and utilization of glucose by peripheral tissues, with an additional pancreatic impact and a mild toxicity effect (Fararh et al. 2005; Gianfrate and Ferraris 1998).

6.3  Cardio-protective Effects Cardiovascular diseases (CVDs) are one of the world’s top causes of mortality (Ding and Mozaffarian 2006). World Health Organization reports that the most significant risk factor for creating CVDs are unhealthy diet. One of the main efficient strategies in protecting against CVDs is N. sativa volatile oil (Ahmad and Beg 2013). A quantity of N. sativa volatile oil (4 mL/kg) exhibited a cardio-protective effect against cardiotoxicity caused by lead acetate in (Ahmed and Hassanein 2013). Treatment of rats with N. Sativa oil (2 mL/kg injection, po) reduced heart injury and lipid peroxidation caused by cyclosporine (25 mg/kg po for 3w) (Ebru et al. 2008). N. sativa volatile oil (4–32  μL/kg, intravenous) decreased dose-dependent blood pressure and heart rate in guinea pigs (El Tahir and Ageel 1994) suggestive for a potential hypotensive effect. A research assessed TQ’s cardio-protective effect on cyclophosphamide-induced cardiotoxicity in albino rats revealing that TQ mitigated against all biochemical

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alterations induced by cyclophosphamide concurrent with an enhanced antioxidant enzymes, mitochondrial activity and heart tissue energy production (Nagi et  al. 2011). The therapeutic effect of TQ (8–10 mg/kg in drinking water) on doxorubicin-­ induced cardiotoxicity (15–20  mg/kg single IP injection) was also reported (Al-Shabanah et al. 1998; Nagi and Mansour 2000). Endothelial dysfunction with aging is ascribed to a discharge of vasoconstrictor prostaglandin and the imbalance between NO manufacturing and hyperpolarizing force. Daily administration of TQ (10  mg/kg/day) enhanced endothelial activity (Idris-Khodja and Schini-Kerth 2012), whereas a dose of (20 mg/kg) showed hypercholesterolemic effect in rabbit via reduction of LDL-C, MDA concurrent with an increase in HDL-C (Ragheb et al. 2008).

6.4  Anti-cancer Activity Anti-cancer efficiency of N. sativa volatile oil is not only limited to a tumor inhibition effect (Randhawa and Alghamdi 2011; Salem 2005), but rather a cheompreventive effect on different neurons and cells from radiation (Iddamaldeniya et al. 2003). The first reported safe anticancer compound in Nigella oil was TQ (Al-Ali et al. 2008), that showed effects against lung cancer (Huat and Swamy 2003), cervical cancer (Shafi et  al. 2009), breast cancer (El-Aziz et  al. 2005), blood cancer (El-Mahdy et  al. 2005), pancreatic cancer (Chehl et  al. 2009) and other various cancers. TQ also showed anti-neoplastic impact against colon cancer (Gali-Muhtasib et al. 2004) found to inhibit colon carcinogenesis in the post-intonation stage (Salim and Fukushima 2003). Pancreatic cancer is the lowest survival rate of all prevalent cancers for the previous 5  years (Allemani et  al. 2018). TQ caused apoptosis of pancreatic ductal adenocarcinoma cells and inhibited its proliferation (Chehl et al. 2009). Oral administration of TQ increased the activity of quinone reductase and glutathione transferase posing it as an efficient prophylactic agent against chemical induces cancer (Nagi and Almakki 2009; Randhawa and Alghamdi 2011). Another major component of N. sativa volatile oil is α-hederin to exhibit antitumor effect (Huat and Swamy 2003). TQ was also found to augment gemcitabine and oxaliplatin antitumor activity against pancreatic cancer (Banerjee et al. 2009).

6.5  Anti-oxidant Activity In our body, natural antioxidant protection systems balance the production of reactive oxygen species (ROS). The excess ROS alters the defence mechanism and supports disease pathogenesis and affect biomacromolecules i.e. DNA, RNA and lipid.

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The antioxidant action of N. sativa essential oil using TLC screening technique identified radical scavenging properties for TQ, caravacrole, (E)-anethole and 4-­terpineol (Burits and Bucar 2000). N. Sativa volatile oil major component (TQ) played a major role in protecting organs from oxidative damage caused by ROS (Baynes 1991) mediated via increasing serum enzymatic activities of SOD and GSH-Px concurrent with a decrease in total oxidant status, malondialdehyde and NO levels (Nagi and Mansour 2000). TQ has a dual antioxidant and anti-inflammatory effect to prevent the negative impacts of (ROS), and change the inflammatory processes occuring in diseases such as bowel disease and colorectal cancer (El-Abhar et al. 2003). In a rat-based study, TQ mitigated against changes in gastric mucosal glutathione (GSH) and superoxide dismutase (SOD) caused by ischemia/reperfusion (El-Abhar et  al. 2003). Supplementation with TQ counteracted the induced hepatotoxicity of sodium fluoride (NaF) owing to TQ powerful antioxidant activity (Abdel-Wahab 2013). Generation of superoxide anion (O2−) by the xanthine/xanthine oxidase system could be inhibited by TQ in a dose-dependent manner (IC50  =  3.4  M) (Badary et al. 2003).

6.6  Treatment of Dry Eyes Dry eye is a chronic disease that accounts for a vast majority of diseases (Al-Ghamdi 2001) being classified as chronic illness due to multiple factors, including age, defects of hormones, medicines, surgery and systemic autoimmune diseases. TQ is one of biologically effective major component of N. sativa volatile seed oil, that was found active to interfere with the pathogenesis of dry eye disease through its anti-­ inflammatory effects (Pflugfelder 2003). A dose of 0.4% of TQ exhibited significant therapeutic efficacy for experimental dry eye (EDE) in comparison with 0.2% benzalkonium chloride, and with no side effects (Erdurmus et al. 2007).

7  Nigella Volatiles and Cosmetics The potent applications of N. sativa seed or essential oil in cosmetics are closely related to the pharmacological properties of its bioactive compounds. N. sativa can be used as an antiaging component in cosmetics due to its antioxidant activity (S. Amin et al. 2010). N. sativa seed oil is considered a vital ingredient in the production of toothpaste and mouthwash due to its effect against pathogens such as Streptococcus mutans, Candida albicans and Streptococcus mitis (Hanafy and Hatem 1991). Figure 9.4 displays general applications of N. sativa seeds oil in food flavor, culinary uses and cosmetics.

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Fig. 9.4  General applications of N. sativa in food flavor, culinary uses and cosmetics

7.1  Hair Loss A clinical study conducted on a mixture of hair oil containing N. sativa oil besides other different oils was found to combat hair fall gradually and to last for 90 days (Dulal et al. 2014).

7.2  Skin Infections In the Prophetic Medicine documented by Imam Ibn Qayyim Al-Jauziyah, it was reported that N. sativa seed can be used for the treatment of skin ulcers after roasting and mixing with waxes and henna or its oil, also it can be used for dandruff and ailments such as black pigmentation and leprosy (Al-Bukhari and Sahi 1976). N. sativa oil can be mixed with bee wax and applied for skin infections, burns, also as anti-aging agent moisturizer and joint pain reliever (Mohamed Fawzy Ramadan 2007).

7.3  Acne Vulgaris N. sativa seed powder can be used for the treatment of acne when mixed with honey (Lebling and Pepperdine 2006). A clinical study conducted using N. sativa oil lotion 10% to evaluate its effect on Acne vulgaris revealed that N. sativa oil lotion led to a decrease in the mean number of papules and pustules (Al-Harchan 2010). Antimicrobial, anti-inflammatory and immunomodulatory activities of N. sativa were chiefly responsible for the previous results.

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7.4  Skin Pigmentation A clinical study revealed the effect of N. sativa seed and fish oil on Vitiligo (the autoimmune skin disease manifested with skin white patches secondary to damaging the skin melanocytes). After application of N. sativa seed and fish oil on people suffering from vitiligo, results revealed that N. sativa was more effective then fish oil. The upper extremities, head and neck showed greater improvement (Ghorbanibirgani et al. 2014). TQ displayed an evidential melanin dispersion effect after exposure of pigment cells to extract or TQ (S. A. Ali and Meitei 2011).

7.5  Sun Protection An in vitro study conducted using 5% N. sativa oil showed an actual sunscreen activity, as SPF value is 1.05 with ultra-boot star rating of 2 (Shantanu et al. 2010).

7.6  Wound Healing TQ the powerful antioxidant agent in N. sativa oil, exhibited potential effect in prevention lipid peroxidation of tissues membrane accordingly, accelerate wound healing (Yaman et al. 2010). A clinical study on rats wound model proved the effect of N. sativa oil on wound healing was higher than that of silver sulfadiazine. Owing to the anti-inflammatory and immunomodulatory effects (Yaman et al. 2010). Another study confirmed the potential effect of N. sativa oil regarding the enhancement of collagen formation and epithelization rate where it has a superior effect as moisturizer and wound healing (Sarkhail et al. 2011).

7.7  Tooth Caring N. sativa seed oil is considered a potential tooth caring plant. It exhibits a potent activity against several cariogenic bacteria including Streptococcus mutans, Streptococcus mitis, and Candida albicans. The enrichment of N. sativa with phenolics i.e. thymoquinone makes it ideal in the treatment of oral infections (Sudhir et al. 2016) to be applied in preparations such as toothpaste and mouthwash.

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8  Conclusion Nigella Sativa and its volatile oil exhibit distinctive characteristics among the natural herbaceous plants. N. sativa volatile oil varies in yield and qualitative composition based on ecological factors as well as extraction methods. Such variation is controlled by many factors include seed origin, time of cultivation, geographical and storage conditions. Agricultural practice found to influence oil composition included amount of sunlight, nutrients, seed cultivars or variety type and irrigation system. Such variation in volatile composition warrants for detailed studies reporting on practices that can help maximize certain targeted molecules in N. sativa oil i.e. thymoquinone for improvement of its drug effect. Previous studies indicated that most of N. sativa volatile oil pharmacological activities have been associated with its major constituents, particularly TQ. Based on TQ strong biological effects, researchers are encouraged to explore how it interacts with body enzymes/receptors to produce these effects. Moreover, structure activity relationship of TQ analogues can help identify more active agents with fewer side effects. The biosynthesis and genetic annotation for pathway leading to the production of TQ, of rare occurrence in planta has also yet to be annotated in N. sativa. Acknowledgement  We would like to thank Dr. Shaden A.M. Khalifa, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden for her contributions during the writing and revision of the manuscript, as well as for her financial support.

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Srinivasan, K. (2018). Cumin (Cuminum cyminum) and black cumin (Nigella sativa) seeds: Traditional uses, chemical constituents, and nutraceutical effects. Food Quality and Safety, 2(1), 1–16. Sudhir, S. P., Deshmukh, V. O., & Verma, H. N. (2016). Nigella sativa seed, a novel beauty care ingredient: A review. International Journal of Pharmaceutical Sciences and Research, 7(8), 3185. Sugimoto, K. i., Tsuruoka, S., & Fujimura, A. (2001). Effect of enalapril on diabetic nephropathy in oletf rats: The role of an anti-oxidative action in its protective properties. Clinical and Experimental Pharmacology and Physiology, 28(10), 826–830. Suguna, P., Geetha, A., Aruna, R., & Siva, G. V. (2013). Effect of thymoquinone on ethanol and high fat diet induced chronic pancreatitis – A dose response study in rats. Indian Journal of Experimental Biology, 51(4), 292–302. Swamy, S. M. K., & Tan, B. K. H. (2000). Cytotoxic and immunopotentiating effects of ethanolic extract of Nigella sativa L. seeds. Journal of Ethnopharmacology, 70(1), 1–7. United Nations Industrial Development Organization, Handa, S. S., Khanuja, S. P. S., Longo, G., & Rakesh, D. D. (2008). Extraction technologies for medicinal and aromatic plants. Trieste: Earth, Environmental and Marine Sciences and Technologies. Vas, G., & Vekey, K. (2004). Solid-phase microextraction: A powerful sample preparation tool prior to mass spectrometric analysis. Journal of Mass Spectrometry, 39(3), 233–254. Velho-Pereira, R.  M., Barhate, C.  R., Kulkarni, S.  R., & Jagtap, A.  G. (2011). Validated high-­ performance thin-layer chromatographic method for the quantification of thymoquinone in Nigella Sativa extracts and formulations. Phytochemical Analysis, 22(4), 367–373. Venkatachallam, S. K. T., Pattekhan, H., Divakar, S., & Kadimi, U. S. (2010). Chemical composition of Nigella sativa L. seed extracts obtained by supercritical carbon dioxide. Journal of Food Science and Technology, 47(6), 598–605. Wajs, A., Bonikowski, R., & Kalemba, D. (2008). Composition of essential oil from seeds of Nigella sativa L. cultivated in Poland. Flavour and Fragrance Journal, 23(2), 126–132. Wang, L., & Weller, C.  L. (2006). Recent advances in extraction of nutraceuticals from plants. Trends in Food Science & Technology, 17(6), 300–312. Yaman, I., Durmus, A. S., Ceribasi, S., & Yaman, M. (2010). Effects of Nigella sativa and silver sulfadiazine on burn wound healing in rats. Veterinární Medicína, 55(12), 619–624. Yousefi, M., Barikbin, B., Kamalinejad, M., Abolhasani, E., Ebadi, A., Younespour, S., et  al. (2013). Comparison of therapeutic effect of topical Nigella with betamethasone and Eucerin in hand eczema. Journal of the European Academy of Dermatology and Venereology, 27(12), 1498–1504. Zaoui, A., Cherrah, Y., Lacaille-Dubois, M. A., Settaf, A., Amarouch, H., & Hassar, M. (2000). Diuretic and hypotensive effects of Nigella sativa in the spontaneously hypertensive rat. Thérapie, 55(3), 379–382. Ziani, K., Chang, Y., McLandsborough, L., & McClements, D. J. (2011). Influence of surfactant charge on antimicrobial efficacy of surfactant-stabilized thyme oil nanoemulsions. Journal of Agricultural and Food Chemistry, 59(11), 6247–6255.

Chapter 10

Rediscovering Nigella Seeds Bioactives Chemical Composition Using Metabolomics Technologies Mohamed A. Farag, Hamada H. Saad, and Nesrine M. Hegazi

Abstract  Nigella sativa commonly known as black cumin is a herbal supplement used in traditional Middle Eastern and European medicine mainly for chronic respiratory disorders. Its seed is well recognized for a myriad of biological effects and is one of the top-selling and most well-studied herbs in the Middle East. Nevertheless, there is no definite profile for its quality control (QC) analysis to ensure uniformity in bioactivity or to recognise adulteration among samples. Its complete metabolic profile has yet to be elucidated, an essential step to explain its medicinal uses. Innovative analytical tools and multivariate analysis are competent approaches for studying plant samples chemical composition and hence allowing their authentication, and is increasingly applied for QC of herbal drugs. The current review focuses on the application of large-scale metabolomics analyses for exploring Nigella seeds and its allied drugs. Applications to include numerous spectral techniques (IR, NMR and MS) as well as chromatographic analysis to characterize Nigella fingerprint that aids in its authentication as well as for the comparative chemical composition of its different species or seeds origin. Further, metabolomics applications

M. A. Farag (*) Pharmacognosy Department, College of Pharmacy Cairo University, Cairo, Egypt Department of Chemistry, School of Sciences & Engineering, The American University in Cairo, New Cairo, Egypt e-mail: [email protected] H. H. Saad Phytochemistry and Plant Systematics Department, Division of Pharmaceutical Industries, National Research Centre, Cairo, Egypt Department of Pharmaceutical Biology, Pharmaceutical Institute, Eberhard Karls University of Tübingen, Tübingen, Germany N. M. Hegazi (*) Phytochemistry and Plant Systematics Department, Division of Pharmaceutical Industries, National Research Centre, Cairo, Egypt e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_10

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either to improve the yield of its bioactive chemicals or to probe involved biosynthetic pathways are also presented. Keywords  Chemometrics · Metabolomics · Nigella sativa · LCMS · NMR Secondary metabolites · Quality control

1  Introduction Nigella sativa is an annual angiosperm belonging to the Ranunculaceae. Its commonly known as black seed, black cumin (English), Habbatul Barakah (Arabic), and Tikur azmud (Amharic). It is widely cultivated in the Middle Eastern Mediterranean region, Southern Europe, Northern India, Pakistan, Syria, Turkey, Iran, and Saudi Arabia. The seeds and oil have been extensively used in Indian and Arabian culture. The seed has a pungent bitter taste and is recognised as a spice besides a flavouring additive in food. It is traditionally utilized for the treatment of asthma, diabetes, hypertension, fever, inflammation, bronchitis, dizziness, eczema, and gastrointestinal disturbances. Historically, ancient Egyptians used its seeds as a preservative in mummification (Srinivasan 2018). N. sativa seeds encompass various beneficial vitamins and minerals like Cu, P, Zn, and Fe. Its primary metabolites nutritional composition include 38.2% fat, 20–85% protein, 31.9% carbohydrates and 7–94% crude fibres (Srinivasan 2018). Phytochemical investigation of N. sativa secondary metabolites revealed the existence of over 100 metabolites comprising alkaloids, saponins, sterols, and essential oil. Thymoquinone (TQ) was recognised as the major volatile constituent that exhibited broad biological activities (Haseena et al. 2015). N. sativa fixed oil is rich in saturated (i.e. palmitic and stearic acids) and unsaturated (i.e. linoleic and oleic acids) fatty acids (Mehta et al. 2008). With regards to N. sativa pharmacological activities, it exhibits in vivo and in vitro antioxidant (Mostafa et al. 2013; Sultan et al. 2015), antidiabetic (Daryabeygi-­ Khotbehsara et al. 2017; El Rabey et al. 2017), anti-hypertensive (Rizka et al. 2018; Jaarin et al. 2015), anti-inflammatory, analgesic (Al-Ghamdi 2001), neuroprotective (Abulfadl et  al. 2018; Sharaf et  al. 2014), anti-bacterial (Maryam et  al. 2016; Abdallah 2017), anti-fungal (Abdallah 2017), anti-viral (Onifade et al. 2013), anti-­ parasitic (El-Hack et al. 2016) activities (Kolahdooz et al. 2014). N. sativa is largely consumed for its presumed wide health promoting or disease-­ preventing effects (Edris 2007; Nazrul Islam et al. 2004). Thus, in the latest years many efforts have been made towards addressing the complex chemical compositions of Nigella to meet the increasing legal demands for the herbal supplements safety and uniformity of active compounds content ensuring consistency for such popular herbal supplement.

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2  Quality Control (QC) Approaches in Nigella The mounting interest in herbal supplements and their efficacy drove the development of analytical approaches for the QC of complex mixtures as found in valuable plant extracts for the pharmaceutical industry (Farag et al. 2012; Farag 2014). However, plant extracts encompass a complex mixture of metabolites often derived from multiple geographical origins and/or cultivated and prepared under different conditions. These raise variations regarding their QC, which is under increasing surveillance by the commercial sector and health authorities. Although, N. sativa is highly consumed and among the most investigated nutraceuticals in Middle Eastern region, there is no defined monograph for its QC analysis. Therefore, driven by the well-known beneficial health effects and economic importance of N. sativa that might be subjected to mislabeling or even adulteration, establishing a reliable analytical method for its QC seems warranted. Thymoquinone, the most significant metabolite in N. sativa, is well recognised as marker for prove of drug efficacy. The QC of N. sativa seeds and oil has long relied on its quantification. Numerous analytical techniques including thin-layer chromatography (Abou Basha et al. 1995), gas chromatography (Houghton et al. 1995), high performance liquid chromatography coupled to UV sperctrophotometry (HPLC- UV) (Aboul-Enein and Abou-Basha 1995, Ghosheh et  al. 1999), HPLC fluorescence (Iqbal et  al. 2013) and differential pulse polarography (Michelitsch and Rittmannsberger 2003) were employed for the quantitative determination of TQ. In the 2000’s, high performance thin layer chromatography (HPTLC) was introduced for quantitative analysis of chemicals in herbal drugs. It was successfully employed for accurate and robust assay of TQ in Nigella extracts and oils (Velho-Pereira et al. 2011; Belete and Dagne 2014; Ahmad et al. 2014) aside from being relatively affordable in routine labs. Nevertheless, it is well acknowledged that biological activities of herbal drugs as exemplified in case of Nigella are mostly attributed to the synergistic effect of its phytochemical composition. This species contains a myriad of metabolites such as proteins, flavonoid glycosides, alkaloids, saponins, fixed and volatile oils (Fig. 10.1). The complete metabolic profile has yet to be enlightened to clarify its medicinal use and to guarantee its clinical efficiency and or safety. Consequently, QC evaluation of targeting the assay of few chemicals or even a single marker might be deficient for its QC analysis, identification from its closely allied drugs and/or adulteration detection especially when present in extracts, in which no plant material is found to aid in its morphological or microscopical identification. Fingerprinting techniques, which are well known with a multi-component and comprehensive QC pattern, are generally adopted as a robust analytical method to reliably evaluate the bioactive content for the standardization of herbal supplements. Even though N. sativa was extensively investigated in numerous studies, not much information is found regarding the comparative metabolic profile of other Nigella species. Much more focus has been devoted towards the analysis of the official N. sativa with less known on other species within that genus. Only recently,

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Monoterpenes

Sesquiterpenes

Thymoquinone

Thymol

b-elemene

p-cymene

O O

HO

Flavonoid glycosides

OH

Kaempferol 3-O-sophorotrioside 7-O-rhamnoside OH O

O

HO

OH OH

O OH O

OH

O

HO

O O

O OH

O OH

O

OH

Saponins

OH

HO

O

HO

Magnoflorine

HO

O OH

O

OH O N+

O

HO O HO

O

HO O O O

tauroside G2

OH

O Alkaloids

HO

O OH

O OH OH

OH

O OH

HO OH

Fig. 10.1  Secondary metabolite classes present in N. sativa seeds

secondary metabolomics analysis was adopted for the large scale profiling of Nigella in the context of its different genotype resulting in a successful discrimination of the commonly confused species based on their chromatographic fingerprints (Farag et al. 2014, 2017; Yun et al. 2014).

3  Plant Metabolomics Metabolomics is a unique tool in modern system biology that has been employed for fingerprinting and/or profiling of plant-derived foods (Pinu 2015). Detecting food components on a molecular level offers valuable insights into the complex relationships bioactive phytochemicals and its nutritive and therapeutic effects (Farag et al. 2019a, b). Metabolomics mostly employ hyphenated techniques such as gas (GC) or liquid chromatography (LC) coupled to mass analyser (MS) for the analysis of volatile or non-voltile polar metabolites. Considering the relatively polar nature in most natural product classes i.e. saponins and phenolics, LC-MS is employed as the technique of choice in most plant profiling projects. Compared to

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classical LC-MS, ultra-performance liquid chromatography UPLC/MS offers several advantages including its high efficiency, excellent resolution with relatively short analysis times in addition to higher mass accuracy when coupled to high-­ resolution mass spectrometry, a key step in metabolite identification (Castro-Puyana et al. 2017). Hence, high throughput UPLC/MS based metabolomics has been used extensively to investigate phytochemical variations across different plant genera as well as similarly related species and taxa (Farag et al. 2015a, b, 2016; Rocchetti et al. 2019). Since assessing the potential aspects of the widely used nutraceuticals e.g. N. sativa to ensure safety and efficacy necessitates a complete knowledge of its phytochemical composition. Thus, the need for the application of such multimodalities analytical procedures is likely to provide maximal metabotyping for its different constituents. Nevertheless, a common drawback upon utilizing mass spectroscopy for metabolites detection lies in its non-universality in terms of detection type aside from the impact of the ionization potential of certain metabolites that is likely to suppress other less ionisable compounds offering a biased profile of the metabolome. In contrast, nuclear magnetic resonance spectroscopy (NMR) provides a universal detection of metabolites niche in an extract besides its stronger structural elucidation power (especially by 2D NMR experiments) which is another valuable tool in case of unknown compounds elucidation. Though this technique presents the highest level of structural detail and is applicable to all compounds by providing similar response factor for the different classes of constituents, it is less sensitive in comparison to mass spectrometry (Farag 2014). Consequently, a synergism of such informative and complementary analytical tools as a multi-technique approach facilitates untargeted, high-throughput and global analysis for nutraceutical extracts charting a complete image of its metabolites composition in a single step as exemplified in several comparative metabolomics approach comprising a multiplex approach of both MS and NMR techniques (Rasheed et al. 2018; Tugizimana et al. 2014). Over the next sections, we provide a review on the application of these techniques viz. MS, UV, IR, NMR for analysis of N. sativa highlighting both advantages and limitations from each study as summarised in Table 10.1.

4  Mass Spectrometry Based Application for Nigella Analysis 4.1  LC-MS Liquid chromatography is the most routinely used platform for plant extracts profiling commonly integrated with UV spectrophotometry and electrospray ionisation (ESI) mass spectrometry generating useful information for the structural elucidation of the detected metabolites. This tool is broadly applied for the determination of different classes of natural products including flavonoids, alkaloids and saponins (Farag 2014).

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Table 10.1  Application of the different metabolomics platforms for the analysis of Nigella seeds Technique Sample type HPLC-UV- MS/ N. sativa seed MS GC-MS N. sativa seed

UPLC-­ qTOF-­MS/MS and GC-MS combined with chemometrics

6 Nigella types

UPLC-Q-TOF/ MS/MS, HPLC fingerprinting combined with multivariate analysis NMR

N. glandulifera and N. sativa seeds

Targeted UPLC-FTMS 1 H NMR

N. sativa seed

N. sativa seed

N. sativa seed from different geographic origins

UPLC-PDA-MS N. sativa seed and callus and and callus seedling chemometrics sprout treated with methyl jasmonate (MeJA) Five Nigella SPME-GCMS analysed using species from multivariate data different geographic analysis origins N. sativa fresh SPME-GCMS analysed using and roasted seeds multivariate data and fixed oil formulation analysis GC-MS N. sativa seed

Detected metabolite Saponins

Remarks, application Quantitative analysis of saponins Volatiles Monitoring volatile constituents during seed development and maturation Saponins, flavonoids, A combined phenolics, alkaloids, metabolic approach for the and fatty acids chemotaxonomic classification of different Nigella species and for the QC of N.sativa seeds Discrimination and Flavonoids, QC of alkaloids, N. glandulifera and hederagenin N. sativa seeds glycosides

Reference Avula et al. (2010). Xue et al. (2013)

Sugars, amino acids, organic acids Benzylisoquinoline alkaloids Phenolics, sugars, terpenoid and fatty acid

Hagel et al. (2015)

Flavonoids, hydroxycinnamates, fatty acids.

Aromatics, sesquiterpenes, monoterpenes

Aromatics, sesquiterpenes, monoterpenes Volatiles

Assessing the correlation between N. sativa seeds from different geographical origins Studying the metabolic profile of whole N. sativa seeds as opposed to callus and upon addition of MeJA

Farag et al. (2014)

Yun et al. (2014)

Maulidiani et al. (2015)

Farag et al. (2015b)

Assess nigella aroma Farag et al. (2017) composition in relation to its genetic or geographical origin Impact on processing on volatiles composition Studied volatiles stability under different storage conditions

Ahamad Bustamam et al. (2017)

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Avula et al., developed an analytical method compromising HPLC-UV coupled to ESI-TOF- MS/MS for the quantitative and qualitatitive analysis of saponins (i.e. tauroside H2, tauroside G3, decaisoside D, sapindoside B, tauroside E) in N. sativa seeds using the common observed positive full scan ion mode along with the extracted ion chromatogram and the observed fragment ions (Avula et  al. 2010). Additionally, the authors applied the validated analytical method for QC to ascertain a commercial dietary supplement containing N. sativa (Avula et al. 2010) In 2014, Hu et  al., developed and further optimized a reliable liquid chromatography-­ multiple reaction monitoring (LC-MRM) method to quantify Nigella A in rat plasma in negative ESI mode, which was thoroughly approved in terms of selectivity, sensitivity and accuracy. In response to the official indexing of N. glandulifera in Chinese Pharmacopoeia since 1995 as a local herbal medicine and its poor commercial formulation quality by only two bioactive constituents hederagenin and thymoquinone, Yun et al. (2014) designed a pattern recognition method to sort out two morphologically and microscopically similar Nigella species, Chinese N. glandulifera and Indian N. sativa, based on their chromatographic fingerprints. HPLC fingerprint analysis of 20 samples comprising N. glandulifera, and N. sativa, revealed the presence of 18 shared peaks in the chromatographic profiles representing the major components of both N. glandulifera and N. sativa with a broad polarity range from lipophilic constituents (e.g., TQ) to hydrophilic constituents (e.g., L-tryptophan). The method was approved to meet the technical fingerprint standards regarding its specificity, precision, reproducibility and stability using both nigellanoside and TQ as reference standards (Yun et al. 2014). In 2017, Kadam and Lele (2017) optimized an extraction process of N. sativa seeds after removing the oil and further analysed it via LC-ESI-Q-TOF-MS/MS. 34 Major peaks were detected in such phenolics-enriched extract including phenolic acids, flavonoids, esters, triterpene saponins, alkaloids, vitamins and fatty acids. Precursors of the major bioactive phenolic ‘‘thymoquinone” were identified as thermoquinol glucoside well as thymol-O-sophoroside, suggestive that in nigella seeds, TQ is released upon hydrolysis of its stored glyocisidic conjugate.

4.2  UPLC-MS Currently, most metabolites profiling studies are executed with state-of-the-art high-resolution LC-MS tools employing ultra-high performance liquid chromatography (UPLC) that allows for expedite metabolite analysis alongside better peak resolution than traditional HPLC method to be ionized by high-resolution MS for molecular formula assignment. Such highly sensitive hyphenated analytical technique represents a promising new technology for the QC analysis of herbal medicines (Farag et al. 2013a). To highlight for environmental diversity and taxonomic relevance within genus Nigella, Farag et al. (2014) performed the first comparative secondary metabolomics study of 6 different Nigella species seeds (N. arvensis,

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N. damascena, N. hispanica, N. nigellastrum, N. orientalis, and N. sativa) collected from different geographical origin (Egypt, Syria, Saudi Arabia, Turkey, and Ethiopia) using UPLC/PDA/(-)ESI-qTOF-MS. The authors were the first to pursue an integrated approach to unveil the metabolic differences among Nigella species and identifying several chemotaxonomic markers to distinguish N. sativa from its intimately related species. N. sativa was rich in the alkaloid ‘‘norargemonine” in comparison to other species, while kaempferol 3-O-[glucopyranosyl-(1–2)galactopyranosyl-(1–2)-glucopyranoside was recognised as a potential taxonomic marker for discriminating black cumin from the studied close species. Other annotated peaks belonged to various metabolite families including phenolic glycosides (i.e., thymoquinol), flavonoid glycosides, triterpene saponins (i.e., tauroside), alkaloids (i.e., magnoflorine) and fatty acids (i.e., linolenic acid), with fatty acids being the most ample class in all of the studied extracts (Farag et al. 2014). Unlike the work of Kadam and Lele (2017). this study involved no prior removal of seed lipoidal matter prior to MS dectection. The biotechnological production of secondary metabolites in Nigella cell or organ culture represents an attractive alternative to that of whole seed material. Consequently, the need for studying the metabolites profile of plant callus cells in comparison with that of differentiated native ones is a priori. A comparative metabolomics study between the non-differentiated cells of N. sativa versus its official seeds was reported via UPLC-MS by Farag et al. (2015b). N. sativa callus sustained the ability to accumulate N. sativa charecteristic hydroxycinnamates i.e., p-­coumaroylquinic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid and 5-O-feruloyl quinic acid and presented the major secondary metabolites in callus. Albeit, TQ and even its precursor glycosides, the recognised cytotoxic agent in N. sativa, were undetectable in callus, suggestive for the need of cell differentiation for its production. Further, impact of the phytohormone methyl jasmonate (MeJA) was experimented for enhanced production of secondary metabolites. Compared to control callus, MeJA was found to upregulate O-methylation of O-caffeoylquinic acid forming O-feruloylquinic acid as revealed via OPLS analysis (Farag et  al. 2015b) and probably to be implicated in a defence response in N. sativa i.e. insect damage. The exclusive detection of methylated sucrose in MeJA callus was an additional evidence for the activation of such methylation reaction as a defence response (Farag et al. 2015b). The identification and description of OMT enzyme responsible for the production of O-feruloyl quinic acid from O-caffeoylquinic has still to be cloned from N. sativa. Untargeted UPLC-FTMS profiling was performed by Hagel et al. (2015) in positive ion mode generating an wide-ranging mass lists representing a broad range of metabolites, with a focus on benzylisoquinoline alkaloids (BIAs). Alkaloids are more suited to be detected in positive ion mode considering their positive charge bearing nitrogen. Since the putative assignments of alkaloids was mostly based on its empirical formulae, retention time elution, and as structural isomerism is quite common among BIAs, twenty one BIAs representing 32 detected m/z features were annotated as masses sharing the same formula assigned to groups of alkaloids, not

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a single BIA (Hagel et  al. 2015). Since such compounds family exhibits many medicinal values such as narcotic analgesics, antimicrobial agents, and anticancer properties, further selective MS/MS analysis of BIAs is warranted for their proper annotation, which could be achieved using a series of standards covering such differences in structural isomerism via multiple neutral loss scanning combined with substructure recognition.

4.3  GC-MS Gas chromatography mass spectrometry (GC-MS) represents another platform that is more suitable for the analysis of volatiles metabolites or low molecular weight non-volatiles post derivatization i.e. silylation pending metabolites thermostability at running temperatures. GC surpasses LC in terms of its high separation efficiency and sensitivity, being suited for the analysis of 100 metabolites in plant extracts both qualitatively and quantitatively. Such technique has long been applied for plants metabolite profiling with a wide-ranging coverage of various metabolites classes such as organic/amino acids, sugars, sugar alcohols, terpenes, phosphorylated intermediates and lipophilic compounds. Nevertheless, its lower metabolome coverage capturing only volatile compounds that are stable inside GC-MS limits its application for profiling of polar high molecular weight compounds (Beale et al. 2018). Quantitative and qualitative analysis of N. sativa aroma and its fixed oil utilising GC-MS has long been documented in numerous studies. The occurrence of fatty acids, phenyl propanoids, monoterpenes, sesquiterpenes together with nonterpenoid hydrocarbons was commonly reported (Nickavar et  al. 2003, Benkaci-Ali et  al. 2007, Venkatachallam et  al. 2010, Piras et  al. 2013, Mahmoudvand et  al. 2014, Gharby et al. 2015, Nadaf et al. 2015, Hadi et al. 2016 and Nivetha and Prasanna 2016). Thymoquinone and thymol belonging to phenylpropanoids represented the most abundant metabolites in N. sativa seeds and to more account for its chief biological effect posing the value of using GC-MS for N. sativa QC analysis. In contrast, sesquiterpenes viz. β-elemene, δ-cadinene and α-selinene appeared to predominate Polish N. orientalis seeds (Wajs and Kalemba 2010), and suggestive that TQ serves well as a chemotaxonomic marker for N. sativa genotype. GC-MS analysis was employed for the discrimination of N. sativa from its closely related species that is N. damascena where the two species exhibited different volatile profiles. With N. damascena found enriched in sesquiterpenes mainly β-elemene and methyl 3-methoxy-N-methyl anthranilate, whereas monoterpenes (p-cymene) and TQ were more abundant in N. sativa (Rchid et al. 2004 and Moretti et al. 2004). However, it should be noted that abundance of p-cymene in N. sativa could reflect the possible degradation of TQ being less stable especially at the high operating temperature of the GC-MS and/or using steam distillation (Farag et al. 2017). Xue et  al. studied N. sativa seeds as an exemplary structure to elucidate the undergoing changes during the production of volatile constituents and the build-up

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of the storage reserves that are distinctive of seed development and maturation. The variation of primary and secondary metabolites during N. sativa seeds development was studied via GC-MS network-based analysis. More than 70 metabolites from seeds through 14 different time points from initial, through mid to late development were quantified (Xue et al. 2013). The authors proposed that through the development and maturation stages of Nigella seeds, fatty and amino acids serve as the building units for the biosynthesis of volatiles. These findings concur the biosynthetic pathway of volatiles (Schwab et al. 2008) identifying aromatic and branched chain amino acids along with methionine as precursors for many N. sativa aroma constituents (Gonda et al. 2010, 2013). Farag et al. reported a cold extraction method of Nigella volatiles using a headspace solid phase micro-extraction SPME coupled to GC-MS in which five Nigella species from different geographical sources including N. arvensis (Germany), N. damascene (Italy, Germany, Romania), N. nigallastrum (Czech Republic), N. orientalis (Turkey, Germany), N. sativa (unknown origin, Egypt, Syria, India) were investigated (Farag et al. 2017). SPME led to the identification of 34 volatiles including aromatics and terpenes, and with N. sativa found enriched in aromatics and in agreement with former studies (Rchid et al. 2004 and Moretti et al. 2004). Amongst major aromatics, TQ, p-cymene, and (E)-anethole found most enriched in Syrian N. sativa seed specimen at 99.7% in contrast to 2% in Indian seeds. Though morphologically similar, N. damascena exhibited a distinct volatiles profile comprised principally of β-elemene, from all its accessions collected from Italy, Romania and Germany. Conclusively, these results suggest for biosynthetic pathways activation leading to phenylpropanoids in N. sativa versus sesquiterpenes in N. damascena. Such results are nevertheless hypothesis and need to be confirmed by monitoring other cellular products i.e., enzyme activity or gene transcripts to provide a better insight of volatiles regulation and or biosynthesis in that genus. The stability of N. sativa seeds aroma constituents under different storage conditions was studied by Ahamad Bustamam et al. to assess its shelf life and metabolites consistency. The whole and ground seeds were analyzed using headspace-gas chromatography-­mass spectrometry (HS-GC-MS) and further determined the effect of air, heat, and light on seeds aroma compounds. The whole and ground seeds showed similar profiles, but were different in terms of concentration levels, with ground seeds showing higher levels for its major constituents compared to whole seeds. The highest decrease in volatile level was observed in exposed whole and ground seeds post 28 days of storage (Ahamad Bustamam et al. 2017). One study employing GC-MS for primary metabolites profiling in Nigella seeds was reported in 2014 by Farag et al. The study comprised 14 samples representing 6 distinct species (N. arvensis, N. damascena, N. hispanica, N. nigellastrum, N. orientalis and in comparison to the official drug N. sativa). As expectedly, fatty acids were the most abundant metabolites followed by sugars and amino acids. Linoleic and oleic acids were the major fatty acids suggestive that N. sativa oil is not of high omega 3 versus 6 ratio as observed in case of linseed oil. These findings are in agreement with results reported by Atta (2003) and Matthaus and Özcan (2011). Nevertheless, it should be noted that these results were based on solvent extraction

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of seeds with methanol and has yet to be replicated for isolated fixed oil from seeds. For detailed referral to volatiles distribution in Nigella, authors are encouraged to read other chapter in this book.

5  N  uclear Magnetic Resonance (NMR) Based Application for Nigella Analysis Despite being less sensitive compared to MS, NMR serves as an integral tool for the fingerprinting of plant extracts (Ward et al. 2007). Such platform rely on high field NMR of crude and/or fractionated extracts to provide NMR metabolomics fingerprints which can be statistically compared to evaluate peak similarities. More advanced processing includes assignment of specific chemical shifts to specific metabolite identities. NMR spectroscopy is also well suited for metabolites quantification comparable to HPLC though with no need of standards to be used for each targeted compound (Farag et al. 2012).

5.1  1 D-NMR Based Metabolites Fingerprinting Proton nuclear magnetic resonance (1H-NMR) spectrometric data represents a straightforward and robust tool for most NMR based plant metabolomics studies. Maulidiani et al. classified N. sativa samples from 4 different regions (Saudi Arabia, Yemen, Syria and E3thiopia) based on its NMR fingerprint. 1H NMR spectra were attained using three different solvents [chloroform, methanol and aqueous methanol (1:1)] revealing the predominance fatty acids, terpenoids, carbohydrates and phenolic compounds. 9 Compounds were identified in comparison with previously reported data providing an NMR fingerprint to sort out the origin of unknown samples (Maulidiani et al. 2015). However, this method relied on only 9 variables in the classification compared to the data driven from the UPLC-MS analysis employed by Farag et al. (2014) for the classification of Nigella samples from different geographical origins. Further, Hagel et al. (2015) was able to identify larger number of primary metabolites totalling 29 in N. sativa seeds via 1H-NMR, among a survey study covering 20 benzylisoquinoline alkaloid-producing plant lines. The abundance of primary metabolites in Nigella extract via NMR in both studies and the less detection of secondary metabolites i.e. flavonoids, alkaloids and saponins can be explained via samples preparation methods targeting enrichment of these metabolite classes prior to detection. It is well recognized that 1D-NMR technology on its own is not enough to give a complete overview of primary and secondary metabolites present in herbal extracts and to experience considerable signal overlap, which limit metabolites identification. Innovative NMR methodologies have expanded for better signal resolution to include 2D-NMR experiments, which would aid in the unambiguous identification of more metabolites in plant extracts.

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5.2  2 D-NMR Based Metabolites Fingerprinting Although requires more time acquisition, 2D-NMR experiments helps further confirm metabolites identification especially when present in crude extracts without purification as typical in most plant metabolomics studies. Complex 2D-NMR experiments include 2D 1H TOCSY, 2D HSQC and 2D HMBC in which metabolites are annotated according to their 1H and 13C chemical shifts, J-coupling patterns, along with 1H-1H and 1H-13C covalent linkage patterns from the TOCSY and HSQC experimentation (Kim et al. 2010; Mahrous & Farag, 2015). Venkatachallam et al. utilised 2D-HSQC, 1H and 13C-NMR spectroscopy to analyse phenolic constituents in Indian N. sativa oil and proved the occurrence of TQ, thymohydroquinone and thymol, though without any quantification data. The authors additionally highlighted the effect of extraction protocol on phenolic metabolites recovery from the oil and recommended the use of super critical CO2 for extraction of quinones in Nigella oil (Venkatachallam et al. 2010). Moreover, Maulidiani and co-authors further confirmed the presence of thymol, thymoquinone, fatty acids, p-cymene, thymol-β-glucopyranoside, and sucrose in N. sativa via 2D J-resolved and HMBC experiments (Maulidiani et al. 2015). We propose that the extension of other 2D-NMR experiments including HSQC and or HMBC to provide the relevant metabolic fingerprint of N. sativa complex extracts has yet to be fully exploited. Distinct HMBC correlation 1H and 13C NMR signals patterns would indeed allow for the unequivocal assignment of more metabolites in such complex extract (Farag et al. 2014). From our past experience in similar projects, 1D-NMR technology solely cannot always provide definite metabolites identification and suffers from signal overlap. HMBC metabolites cross peak patterns is certainly an auspicious approach that has ultimately to be examined for Nigella extracts classification especially if multivariate data analysis is engaged as detailed over the next section.

6  IR Spectroscopy Infrared spectroscopy has been increasingly applied in herbal drugs QC analysis via acquiring IR fingerprints of the analysed samples (Vlachos et al. 2006) aside from its ability to provide rich information on drug excipients. It represents a speedy and simple analytical tool for the qualitative analysis of organic compounds that is readily available in most laboratories compared to MS or NMR. Two studies reported the successful employment of FTIR spectroscopy with multivariate analysis for detecting Nigella seed oil adulteration with grape seed oil (Nurrulhidayah et  al. 2011), corn oil and soya bean oil (Rohman and Ariani 2013). FTIR proved to be a rapid and simple analytical tool for routine monitoring of oil adulteration as minimal sample preparation is required without the use of reagents and chemicals.

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7  M  ultivariate Data Analyses Application in Nigella QC Analysis Considering the complexity of spectral MS and NMR info of intricate plant samples as in Nigella, multivariate data analysis are frequently employed to identify compositional differences among species and possible chemical markers for QC measures. Multivariate data analysis methods often involve a number of processing steps, many of which are optional prior to the final classification. Multivariate data analyses are broadly classified into two categories: unsupervised i.e. principal component analysis (PCA), hierarchical cluster analysis (HCA) versus supervised tools i.e. partial least square (PLS) to be employed for data visualization, biomarkers identification, samples classification and or to correlate biological outcome of metabolite composition as typical in case of PLS. They are commonly engaged in phytomedicines QC research to pinpoint the inter-relationship among variables and to detect chemotype relation among varieties or species (Okada et al. 2010). PCA analysis of the GC-MS derived data during different phases of Nigella seed development revealed that samples from early, mid, and late maturation stages belonged to distinct clusters in which the seeds displayed remarkable changes in metabolism, as observed by two changes in C-N metabolism. The first change was characterised by compelling variation in sugars levels, sugar alcohols and precursors of triacyglycerols metabolism supporting the production of fatty acids. In the second shift, hexoses, sugar alcohols and fatty acids together with shikimic acid, 3,4-dihydroxyphenyl-acetate and dopamine decreased which supported the inception of volatiles biosynthesis (Xue et al. 2013). Farag et  al. further compared PCA models derived from UPLC-MS targeting secondary metabolites versus GC-MS datasets targeting primary metabolites in the context of Nigella accessions classification power. Examined Nigella species included N. arvensis, N.damascena, N. hispanica, N. nigellastrum, N. orientalis, and N. sativa from different geographical origins. The differentiation capability of both UPLC-MS and GC-MS were compared from their respective PCA results. The PCA score plot obtained from a reduced UPLC-MS dataset (rt 100 till 550 sec) and excluding the fatty acid elution region was found the most relevant in specimens classification compared to the full UPLC-MS and GC-MS datasets. PCA plot revealed that the discrimination of Nigella species was mostly determined by the type and level of flavonoids i.e. kaempferol conjugates along with a still unknown alkaloid with the mass m/z 698.328. N. hispanica was the most remote among all examined species and also from N. sativa owing to is richness in kaempferol-­3-O-sophorotrioside-7-O-rhamnoside (Farag et al. 2014). Both N. sativa and N. hispanica appeared as the most remote ones of the studied species, however, it failed to differentiate N. sativa derived from diverse geographical regions except when modelled individually. In contrast, PCA plots derived from GC-MS dataset steadily identified N. hispanica and N. nigellastrum as the most secluded from other species conforming with UPLC-MS findings. The fact that N. hispanica displayed different phytochemistry warrants to be verified from other origins as it is usually

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considered as an analogue relative to N. sativa and N. arvensis (Comes et al. 2008). Though, GC-MS based PCA failed to differentiate between other Nigella species and with lower model power in classification as evident from covered variance. Results derived from both GC-MS and UPLC-MS points for the relevance of secondary metabolites in specimens classification compared to primary metabolites, later being more affected by growing habitat and as previously reported in other plant research (Farag et al. 2013b). It is worth to mention that impact of weather conditions on secondary metabolites production in Nigella was clearly depicted from PCA that showed segregation of seeds collected from similar climatic areas i.e. Egypt, Syria and Turkey distant from those grown in relatively hot climate i.e. Ethiopia and Saudi Arabia (Farag et al. 2014). The advantage of applying comparative modelling based on more than one analytical platform i.e. GCMS versus LCMS provides a stronger evidence for the classification built hypothesis. Yun et  al., in 2014 adopted pattern recognition methods such as PCA and PLS-DA of the UPLC-MS chromatographic fingerprints belonging to the Chinese N. glandulifera, and Indian N. sativa. PCA successfully separated specimens into two distinct clusters corresponding to N. glandulifera and N. sativa whereas PLS-DA, as a supervised multivariate analysis method, was used to specify the specific compounds responsible for such distinction and revealing comparable segregation as observed in PCA.  The variable importance plot (VIP) categorised (L-tryptophan, salfredin B11, nigelflavonoside B, methyl nigellidine, p-­ hydroxybenzoicacid, fuzitine chloride, thymoquinone, 3-O-[β-D-­ xylopyranosyl-(1  →  3)-α-L-rhamnopyranosyl-(1  →  4)-β-D-glucopyranosyl]-11methoxy-16- hydroxy-17-acetoxy hederagenin, nigeglanoside, nigelloside and hydrate salfredin B11) as significant classification markers owing to the significant difference in their average areas in the two genotypes (Yun et al. 2014). A multivariate PCA data modelling was also employed by Farag et al. to analyze phytohormone elicitation impact on N. sativa callus secondary metabolome as analysed via UPLC-MS, with clear separation of N. sativa seeds from its callus samples, later being more fortified with lipids (i.e. saturated and unsaturated fatty acids) (Farag et al. 2015b). Maulidiani et al., in 2015 implemented PCA for classifying 1 H-NMR spectra (δH 0.52–10.00 ppm) for 12 N. sativa samples from four different origins (Qasemi, Syrian, Yemeni and Ethiopian), and to further prioritize solvent type used for extraction. Correlation built model was employed by authors to correlate metabolite profile and bioactivities (α-glucosidase inhibition, nitric oxide (NO) inhibition and DPPH radical scavenging activity assay) utilizing the variable importance in the projection (VIP) analysis and the PLS derived regression coefficient. The VIP analysis confirmed the contribution of the major metabolites (TQ, thymol and fatty acids) as major contributor to monitored effects. Thymoquinone and thymol exhibited positive effect on all three bioactivities, whereas fatty acid showed positive influence to α-glucosidase inhibition, but negative to nitric oxide inhibition and DPPH radical scavenging activity (Maulidiani et al. 2015). Likewise, PLS scores reflected the relationship between the detected metabolites and seeds origin, with Ethiopian specimens found enriched in TQ and thymol, while in the Qasemi and Syrian samples, higher fatty acid levels was observed. Such results are not fully in agreement with that of Farag et al. (2017) showing Syrian N. sativa as

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the richest in TQ. Such inconsistency opt for analysis of more specimens from these origins to prove or disprove results. This discrepancy could be attributed to intrinsic (i.e. genetic characteristics) or extrinsic (i.e. environmental) influences (Maulidiani et al. 2015). To prove classification model based on PCA, authors evaluated the reliability of other supervised tools i.e. PLSDA for the discrimination of samples from different origins using contingency matrix and the Cohen’s Kappa analysis, with an overall accuracy of 79.2% and kappa coefficient of 0.71 in samples origin prediction (Maulidiani et al. 2015). Such built model can indeed be employed in the future for prediction of samples from unknown origin as typical in most QC research. To determine the guidelines for seeds optimum storage conditions, Ahamad Bustamam et  al. employed a targeted multivariate analysis of PCA to assess the effects of air, heat, and light, on N. sativa whole and ground seeds aroma under six storage conditions. The absolute peak areas of the dominant constituents, particularly, 2- thujene, p-cymene, carvone, TQ, carvacrol, and longifolene were used as variables in the PCA. All of the tested storage conditions showed volatiles variation as influenced by the 4 ecofactors of air, heat, and light. Additionally, the nature of the stored seeds either whole or ground strongly influenced seeds aroma composition. As concluded by authors, the best storage conditions proved to be the whole seeds in a transparent air-free container in dark environment (Ahamad Bustamam et al. 2017). The impact of nigella seeds processing and oil extraction was assessed using HS-SPME by comparing analysis of volatiles in nigella oil versus roasted seeds as analysed using chemometrics (Farag et al. 2017) viz. supervised orthogonal projection to latent structures-discriminate analysis (OPLS). Thymoquinone was the chief component in unroasted seeds, whereas p-cymene was the main constituent in the fixed oil, suggestive that fixed oil might not exhibit the same biological effect in N. sativa seed being deprived of such valuable chemical. Moreover, food safety is evolving as a major concern globally especially detecting the presence of toxins, or residues of chemical substances released from food processing viz. seeds roasting or thermal effect accompanying oil extraction as typical in case of N. sativa seed. Future coupling of these discrepancy metabolites profile data with gene transcript levels can additionally help in the investigation of the involved genes and eventually the hidden biosynthetic pathways. Nothing is known on the underlying biosynthetic machineries or genes regulation involved in natural products production in N. sativa. Undeniably, the correlative analysis of differential metabolic profiling and gene expression profiling in several medicinal plants has confirmed to be a strong methodology for the recognition of candidate genes and enzymes, principally for those of defence-related metabolism (Goossens et al. 2003).

8  Conclusion and Future Developments Nigella is widely distributed in the Middle Eastern Mediterranean region, Southern Europe and Northern India. It is one of the oldest herbs used in folk medicine all over the world for its wide range of beneficial effects. Several pharmacological activities were documented for Nigella seeds among which antimicrobial, antioxi-

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dant, immuno-stimulatory, analgesic and antitumor (Farag et al. 2017). These benefits of nigella seeds enormously favour the coverage of its metabolomics with focus on the major classes of its secondary metabolites. This review provides insights into the applications and state-of-the-art progress in the metabolomics studies focused on nigella seeds analysis. It highlights the employment of different metabolomics approaches in the QC of nigella seeds, chemotaxonomic classification of different Nigella species, assessing the impact of processing and different storage conditions on its aroma constituents, and in elucidating the biosynthetic mechanisms of its significant metabolites (Table 10.1). As concluded, no single approach can offer full overview of nigella metabolic profile. Results reported using LCMS showed higher sensitivity and broader coverage of secondary metabolites while NMR analysis offered reliable quantification of the detected major metabolites. Hence, combining different analytical tools offers a broader view of various classes of primary and secondary metabolites. Coupling of UPLC with MS and NMR represents an emerging approach for the detection of less abundant metabolites. Yet to come, the use of 2D-NMR with chemometrics has to be more employed for nigella seeds classification and quality control applications. Acknowledgements  Dr. Mohamed Farag acknowledges the funding of the Alexander von Humboldt foundation, Germany.

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Onifade, A. A., Jewell, A. P., & Adedeji, W. A. (2013). Nigella sativa concoction induced sustained seroreversion in HIV patient. African Journal of Traditional, Complementary and Alternative Medicines, 10(5), 332–335. Pinu, F.  R. (2015). Metabolomics-The new frontier in food safety and quality research. Food Research International, 72, 80–81. Piras, A., Rosa, A., Marongiu, B., Porcedda, S., Falconieri, D., Dessì, M. A., et al. (2013). Chemical composition and in vitro bioactivity of the volatile and fixed oils of Nigella sativa L. extracted by supercritical carbon dioxide. Industrial Crops and Products, 46, 317–323. Rasheed, D. M., Porzel, A., Frolov, A., Wessjohann, L. W., & Farag, M. A. (2018). Comparative analysis of Hibiscus sabdariffa (roselle) hot and cold extracts in respect to their potential for alpha-glucosidase inhibition. Food Chemistry, 250, 236–244. Rchid, H., Nmila, R., Bessiere, J. M., Sauvaire, Y., & Chokairi, M. (2004). Volatile components of Nigella damascena L. and Nigella sativa L. seeds. Journal of Essential Oil Research, 16(6), 585–587. Rizka, A., Setiati, S., Lydia, A., & Dewiasty, E. (2018). Effect of Nigella sativa seed extract for hypertension in elderly: A double-blind, randomized controlled trial. Acta Medica Indonesiana, 49(4), 307. Rocchetti, G., Bhumireddy, S. R., Giuberti, G., Mandal, R., Lucini, L., & Wishart, D. S. (2019). Edible nuts deliver polyphenols and their transformation products to the large intestine: An in vitro fermentation model combining targeted/untargeted metabolomics. Food Research International, 116, 786–794. Rohman, A., & Ariani, R. (2013). Authentication of Nigella sativa seed oil in binary and ternary mixtures with corn oil and soybean oil using FTIR spectroscopy coupled with partial least square. The Scientific World Journal, 2013. Schwab, W., Davidovich-Rikanati, R., & Lewinsohn, E. (2008). Biosynthesis of plant-derived flavor compounds. The Plant Journal, 54(4), 712–732. Sharaf, N., Mansour, R., Elsayed, N., & Mahran, L. (2014). P078: Neuroprotective effect of thymoquinone against lipopolysaccharide-induced Alzheimer’s disease in an animal model. European Geriatric Medicine, 5, S106–S107. Srinivasan, K. (2018). Cumin (Cuminum cyminum) and black cumin (Nigella sativa) seeds: Traditional uses, chemical constituents, and nutraceutical effects. Food Quality and Safety, 2(1), 1–16. Sultan, M. T., Butt, M. S., Karim, R., Ahmed, W., Kaka, U., Ahmad, S., et al. (2015). Nigella sativa fixed and essential oil modulates glutathione redox enzymes in potassium bromate induced oxidative stress. BMC Complementary and Alternative Medicine, 15(1), 330. Tugizimana, F., Steenkamp, P. A., Piater, L. A., & Dubery, I. A. (2014). Multi-platform metabolomic analyses of ergosterol-induced dynamic changes in Nicotiana tabacum cells. PLoS One, 9(1), e87846. Velho-Pereira, R.  M., Barhate, C.  R., Kulkarni, S.  R., & Jagtap, A.  G. (2011). Validated high-­ performance thin-layer chromatographic method for the quantification of thymoquinone in Nigella sativa extracts and formulations. Phytochemical Analysis, 22(4), 367–373. Venkatachallam, S. K. T., Pattekhan, H., Divakar, S., & Kadimi, U. S. (2010). Chemical composition of Nigella sativa L. seed extracts obtained by supercritical carbon dioxide. Journal of Food Science and Technology, 47(6), 598–605. Vlachos, N., Skopelitis, Y., Psaroudaki, M., Konstantinidou, V., Chatzilazarou, A., & Tegou, E. (2006). Applications of Fourier transform-infrared spectroscopy to edible oils. Analytica Chimica Acta, 573, 459–465.

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Ward, J. L., Baker, J. M., & Beale, M. H. (2007). Recent applications of NMR spectroscopy in plant metabolomics. The FEBS Journal, 274(5), 1126–1131. Wajs, A., & Kalemba, D. (2010). Volatile constituents of seeds of Nigella orientalis cultivated in Poland. Journal of Essential Oil Research, 22(3), 232–234. Xue, W., Batushansky, A., Toubiana, D., Botnick, I., Szymanski, J., Khozin-Goldberg, I., & Fait, A. (2013). The investment in scent: time-resolved metabolic processes in developing volatileproducing Nigella sativa L. seeds. PloS one, 8(9), e73061. Yun, Q., Liu, Q., He, C., Ma, X., Gao, X., Talbi, A., & Zhou, J. (2014). UPLC-Q-TOF/MS characterization, HPLC fingerprint analysis and species differentiation for quality control of Nigella glandulifera Freyn et Sint seeds and Nigella sativa L. seeds. Analytical Methods, 6(13), 4845–4852.

Chapter 11

Health Promoting Activities of Nigella sativa Seeds Ebru Aydin and Arzu Kart

Abstract  Nigella sativa L. (black cumin, family Ranunculaceae) is an annual flowering plant. Its black trigonal seeds are the source of the active ingredients of the plant. It is native to South Asian and East Mediterranean countries, where it has been used traditionally and clinically in different parts of the world to treat many diseases. The use of N. sativa seed showed wide range worldwide in foods (preservative, spice, and flavoring agent), painkiller (headache, and back pain), some ailment treatments (diabetes, infection, inflammation, hypertension), pharmacology (antioxidant, antidiabetic, antihypertensive, neuroprotective, anti-inflammatory, analgesic effects, antimicrobial, antibacterial, antiviral, antiparasitic, antifungal, anticancer and male infertility). Black cumin seeds contain a substantial amount of vegetable protein, fiber, lipids, carbohydrates, minerals, and vitamins. The major bioactive component of black cumin seed is thymoquinone which found in the essential oil and provides wide range of therapeutic benefits (i.e., gastrointestinal protective effects). Fatty acid composition of the seed oil is reported mainly to contain linoleic, linolenic, oleic, palmitoleic and palmitic acids. The major sterols of seeds are β-sitosterol, campesterol, stigmasterol and 5-avenasterol which known to lower cholesterol levels. The purpose of this chapter is to provide a comprehensive review based on the scientific reports about the health-promoting activities of N. sativa seed and to discuss the therapeutic properties and functionality of the seed that lead the appliactions of seeds in the pharmaceuticals and food industry. Keywords  Thymoquinone · Medicinal plants · Health · Anti-virus · H9N2 avian influenza virus · Hepatitis C · Diabetes · Cardiovascular diseases · Gastric ulcers · Fertility

E. Aydin (*) · A. Kart Faculty of Engineering, Department of Food Engineering, Suleyman Demirel University, Isparta, Turkey e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_11

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Abbreviations EO Essential oil IDF International Diabetes Federation MBC Minimal bactericidal concentration MIC Minimum inhibitory concentration TQ Thymoquinone WHO World Health Organization

1  Introduction Medical plants and herbs have been traditionally used in the course of history for their rich content of nutrients and health-promoting activities. About 65–80% of the world population are using medical plants and herbs due to their medicinal effects (Aydin 2015). It was reported that the consumption of dietary supplements is increasing day by day due to their health-promoting activities and it has a multi-­ billion dollar industry (Seeram et al. 2006; Weiss et al. 2006; Aydin 2015). Nigella sativa L. (family Ranunculaceae) is an annual flowering plant (Table 11.1). Botanical classification of Nigella sativa L. (Kooti et al. 2016) Table 11.1. Nigella sativa seeds were found in jars at the Late Bronze Age shipwreck (1300  BC) at Uli Burun, Turkey (Ellis 2007). It is cultivated in Southern Europe, Pakistan, Iran, Northern India, Middle Eastern Mediterranean region, Turkey, Saudi Arabia, and Syria. The flowering and fruiting times of the N. sativa are usually from January to April (Kooti et al. 2016). N. sativa seeds phytochemical profile is defining its nutritional and therapeutic effects in great significance. Numerous scientific studies claimed that N. sativa seeds are nutrient-rich herbs, and therefore it has been used as traditional medicine. The uses of N. sativa seed showed wide range in the world wide in foods (preservative, spice and flavoring agent), painkiller (headache, and back pain), ailment treatments (diabetes, infection, inflammation, hypertension), and pharmaceuticals. Its black trigonal seeds are the source of the active ingredients of the plant. The qualitative and quantitative composition of seed is scientifically proven and its high-value nutrition constituents are providing it for using in functional, nutraceutical and Table 11.1 Botanical classification of Nigella sativa L. (Kooti et al. 2016)

Kingdom Division Order Family Genus Species

Plantae Magnoliophyt Ranunculales Ranunculaceae Nigella Sativa

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pharmaceutical applications. There are several studies analyzed the chemical compounds of different species of Nigella. The nutritional value of black cumin seeds contains a substantial amount of protein, fiber, lipids, carbohydrates, minerals, and vitamins. The studies showed that geographic distribution, cultivation methods and time of harvest may affect the bioactive constituents in the seeds (Mukhtar et al. 2019). Typical chemical composition of the seeds contains oil, carbohydrates, proteins, vitamins, minerals, saponin, fiber and carotenoids (Table 11.2). Active compounds of N. sativa seed have been reported in the literature. The major components of the N. sativa volatile oil changed based on the geographic distribution, cultivation method, time of harvest, and extraction method. Different studies showed that the major bioactive component of the seed is thymoquinone (TQ). About 30–48% of N. sativa essential oil is TQ, which is believed to responsible for most of the medicinal effects (Akgoren Palabiyik and Aytac 2018; Perveen 2019). Other important active compounds reported were thymohyroquinone (THQ), dithymoquinone (nigellone, TQ2), p-cymene (7–15%), carvacrol (6–12%), 4-­terpineol (2–7%), t-anethol (1–4%), sesquiterpene longifolene (1–8%), α-pinene and thymol (Khan et al. 2003; Mukhtar et al. 2019; Perveen 2019; Srinivasan 2018). On the other hand, some researchers reported that TQ is not the major component in the seeds. Nickavar et al. (2003) reported that the major volatile constituents of seed are trans-anethole (38.3%) and p-cymene (14.8%). Another studies reported that p-cymene is the major component of the volatile oil (Ashraf et al. 2006; Harzallah et al. 2011). The difference between those studies might due to the geographic distribution, cultivation method, time of harvest, extraction method and genotype (Ashraf et  al. 2005; Gharby et  al. 2015; Hosseini et  al. 2018a, 2018b; Mukhtar et al. 2019). N. sativa seeds contain fatty acids that are rich in unsaturated fatty acids [eicosadienoic acid (3%), dihomolinoleic acid (10%), oleic acid (20%) and linoleic acid (50–60%)] and saturated fatty acids [palmitic and stearic acids (30% or less)] (Nickavar et al. 2003; Gholamnezhad et al. 2016; El-Hack et al. 2018; Srinivasan 2018). The seed has high-quality nutritional value as it is also rich in essential proteins. The seed contains 8 out of 9 essential amino acids (lysine, leucine, isoleucine, valine, phenylalanine, methionine, threonine, and tryptophan) and some Table 11.2  Typical chemical composition of N. sativa seed (Ramadan 2015; Mukhtar et al. 2019)

Constituent Fixed oil Volatile oil Protein Carbohydrate Alkaloids Saponins Minerals Vitamins

% w/w 35.6–41.6 0.50–1.60 20.8–31.2 24.9–40.0 0.010 0.013 3.7–7.0 1.0–4.0

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non-essential amino acids (arginine, glycine, alanine, cystine, glutamic acid, aspartic acid, proline, serine, and tyrosine) (Al-Jassir 1992; Ramadan 2015; El-Naggar et al. 2017; Mukhtar et al. 2019). N. sativa also contains good amounts of minerals (calcium, phosphorus, magnesium, potassium, sodium, zinc, copper, iron, manganese and selenium) and vitamins (tocopherol, thiamin, phridoxin, niacin, folic acid and riboflavin) (Takruri and Dameh 1998; Mukhtar et al. 2019). Other components of seed include phytosterols (α-sitosterol, and stigmasterol), saponin (α-hederin), crude fiber, flavonoids, two varieties of alkaloids including isoquinoline alkaloids (nigellicimine and nigellicimine-N-oxide) and pyrazole alkaloids (nigellidine and nigellicine) (Al-Jassir 1992; Nickavar et al. 2003; Mehta et al. 2009). Due to the rich phytochemistry of the seed, it has been commonly used in nutritional and pharmaceutical purposes and it has been employed for thousands of years as a spice and food preservative in the food industries. For centuries there has been a great interest in N. sativa seeds especially due to its nutrient-rich chemical composition and pharmacological/biological properties. In this chapter, it was aimed to present and discuss the health-promoting effect of N. sativa seeds based on its valuable nutrient constituents.

2  Health-Promoting Activities of Nigella sativa Seeds N. sativa has been used as a spice or flavoring agent due to its distinctive aroma in different food preparations such as bread, pickles, and salads. It is also been used widely in traditional medicine and pharmacology for its preventive effect on different diseases. Therefore, it has attracted the attention of several researchers. The literature presents many in vivo and in vitro studies about N. sativa and its health-promoting activities (Fig. 11.1).

2.1  Antioxidant Activity of Nigella sativa Seeds Lipid and protein oxidation plays a major role in the quality of meat products as they may have an effect on flavor and discoloration, loss of essential fatty acids, leading to changes in organoleptic attributes, reduced shelf life, and reduced nutrition value of muscle foods (Pranav 2018). In addition, both in food and medicine industry, the use of synthetic antioxidants has several toxicities and side effects (Mukhtar et al. 2019). Different plant sources have attracted attention due to their natural antioxidant activity. In the literature, there are some in vivo and in vitro studies that indicated the antioxidant activity of N. sativa seeds. N. sativa seeds’ bioactive compounds such as TQ, carvacrol, t-anethole, 4-­termpineol, tannins, flavonoids and alkaloids provide the antioxidant activity of the seed (Kooti et  al. 2016). TQ and nigellone reported as the major antioxidant constituents in N. sativa seeds (El-Dakhakhny et al. 2002; Ramadan 2015; Kooti

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Antidiabetic

Anticancer

Antimicrobial

Antiinflammatory

Antioxidant

Anti-hypertensive

Infertility

Health promoting activity Neuroprotective

Antihyperlipidemic

Gastroprotective

Cardioprotective

Fig. 11.1  Summary of the health-promoting activities of Nigella sativa seeds

et al. 2016). In another study, Mukhtar et al. (2019) reported that the antioxidant effect of N. sativa seeds and seed oils linked to tocols (tocopherols and tocotrienols) and phenolics. The effect of N. sativa seed against the negative impact of hyperhomocysteinemia (HHcy) was studied using male weaning Albino Wistar rats (normal and methionine induced HHcy rats). The rats were induced with HHcy, and fed with black cumin oil (100 μL/kg) and TQ (100 mg/kg) for 1 week. It was discovered that there was an increase in their lipid peroxidation, cholesterol levels, plasma triglycerides and the activities of glutathione peroxidase and superoxide dismutase. It was concluded that the level of the serum antioxidant status of rats was increased (El-Saleh et al. 2004). The toxic oxygen metabolites are removed by the antioxidant compounds. On the other hand, excessive amounts of oxygen free radical production may cause ischemia and reperfusion (I/R) during an oxidant injury. Hind limb ischemia was induced to rats and analyzed with the administration of N. sativa seed aqueous extracts to see the extract’s effect on I/R injury. It was observed that N. sativa extract exhibited a protective effect on I/R injury-induced oxidative stress in rat muscle (Hosseinzadeh et al. 2007). In a recent study, free radical scavenging activity (RSA) of N. sativa aqueous extract was studied on cryopreserved buffalo spermatozoa. The extract had antioxidant activity on cryopreserved buffalo sperm as it was also improved the quality of the sperm (Awan et al. 2018). According to the literature, the antioxidant activity of the seed may be related to its tocols and phenolics content.

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2.2  A  nti-inflammatory and Analgesic Effects of Nigella sativa Seeds Inflammation is defending the host from harmful stimuli physiologically, however the impairment of the inflammatory mechanism may lead permanent damage to host tissues and cause the disease progression (Calder et al. 2009). Inflammation leads to several medical diseases such as cystic fibrosis, osteoarthritis, asthma, rheumatoid arthritis, allergies, and cancer. Medical drugs have several adverse side effects including bone marrow depression, gastric ulcer, water and salt retention (Yimer et  al. 2019). Several researchers analyzed the anti-inflammatory effect of N. sativa and reported that due to its bioactive components the seed may be safer with fewer side effects as a natural source. Also, N. sativa was able to inhibit cellular nitric oxide production that initiates toxic oxidative reactions and enzymes called cyclooxygenase (COX) and lipoxygenase (LO) which cause inflammation through prostaglandins and leukotrienes catalyzation (Swamy et al. 2019). Al-Ghamdi (2001) studied the anti-inflammatory effect of black cumin water extract. To produce inflammation in rats, they induced paw edema using Carrageenan (0.05 mL of 1% carrageenan sodium salt). It was reported that N. sativa retained anti-inflammatory effects in carrageenan-induced paw edema. In two different studies, N. sativa phenolics and essential oil were administered to acetic acid-induced mice and carrageenan-induced paw edema, and croton oil-induced ear edema in rats (Ghannadi et al. 2005; Hajhashemi et al. 2004). The analgesic activities of phenolics and essential oil were not able to reverse naloxone treatment. The pain relief effect of N. sativa seed on nociceptive response was studied with mice and compared with a chemical drug (diclofenac sodium). It was reported that N. sativa seed had an analgesic effect on nociceptive, however it was less than diclofenac sodium (Bashir and Qureshi 2010). N. sativa seeds and its callus were analyzed due to their anti-inflammatory effects on mix glial cells of the rat. TQ content of the callus was 12 times higher than seed extract and the results showed that both seed and the callus of N. sativa had moderate anti-inflammatory potential as compared with pure TQ (Alemi et al. 2013). Taka et  al. (2015) analyzed the anti-inflammatory effect of TQ in lipopolysaccharide (LPS)-stimulated BV-2 murine microglia cells. TQ was able to reduce the NO− activity with an IC50 value of 5.04 μM (Taka et al. 2015). Researches had revealed the anti-inflammatory actions of N. sativa seeds and its active components, which might be subjected to the development of a new generations of anti-­inflammatory agents.

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2.3  Anti-cancer Activity of Nigella sativa Seeds Cancer is a major health problem, while in 2019 about 1,762,450 new cases and 606,880 cancer-related deaths were reported in the US (Siegel et al. 2019). There is a growing interest on the consumption of herbs, fruits and vegetables and its relation to the management of cancers. N. sativa seed, its essential oil (EO) and some of the active constituents (i.e., TQ and α-hederin) have demonstrated anticarcinogenic, pro-apoptotic, antioxidative, anti-proliferative, cytotoxic and anti-metastatic effects against various types of cancer cells and animal models (Afrin et  al. 2016; Effenberger et  al. 2010; Kabil et  al. 2018; Khalife et  al. 2016; Majdalawieh and Fayyad 2016; Salim 2010; Shahin et al. 2018; Srinivasan 2018). It was reported that consumption of N. sativa with present chemotherapeutic drugs may be effective in controlling tumor initiation, growth, and metastasis. The anti-tumor activity of Nigella sativa was analyzed using YAC-1 tumor cells to modulate the cytotoxic activity of effector cells (NK). It was reported that aqueous extract of N. sativa increased NK cytotoxic activity against YAC-1 tumor cells, and therefore it was able to demonstrate anti-tumor activity (Majdalawieh et  al. 2010). The terpene-terminated 6-alkyl residues of TQ was tested using MCF-7/ Topo breast carcinoma cells to analyze its anti-cancer activities (Effenberger et al. 2010), whereinTQ induced cell death by apoptosis. In another study, the most aggressive breast cancer (triple-negative breast cancer, TNBC) was studied and TQ was used as eukaryotic elongation factor 2 kinase (eEF-2 K) using TNBC cells and mice (Kabil et  al. 2018). TQ repressed the migration/invasion, cell proliferation, and tumor growth of the eEF-2  K in TNBC cells and reduced the growth of MDA-MB-231 tumors and repressed the eEF-2 K expression in an orthotopic tumor model in mice. In addition, it was reported that TQ was able to interrupt the key signals of tumor growing pathways, beside normal tissues was protected from the toxic side effects of chemotherapeutic agents when TQ used as an adjuvant (Schneider-Stock et  al. 2014). Agbaria et  al. (2015) investigated the anticancer activity of controlled thermal processed N. sativa seeds. It was found that the heating of N. sativa seeds between 50–150 °C produces notably higher anticancer activity that is associated with a higher TQ oil content and inhibits the NF-κB signaling pathway in mouse colon carcinoma (MC38) cells. To increase the efficiency and decrease the toxicity of the anticancer drugs (e.g. topotecan, TP), they were used with other chemotherapeutic agents. Khalife et al. (2016) studied the effect of TQ combination with TP to analyze their synergistic effects on cell proliferation using HT-29 cell lines. The combination of TQ with TP was found as therapeutically active in colon cancer, however their synergism is needed to examine further to reach the molecular level. The effect of N. sativa seed on febrile neutropenia (FN) was analyzed clinically in children (80 patients, 2–18  years-old) with brain tumors. It was observed that children treated with N. sativa seeds showed a decrease in the ratio of FN incidence compared to the control group. Therefore, the life quality of those patients may improve with the N. sativa seed (Mousa et al. 2017). Shahin et al. (2018) analyzed

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the effect of N. sativa and TQ on cell proliferation during hepatocarcinogenesis. Both were administered to hepatocellular carcinoma (HCC) rats which were induced with diethylnitrosamine and found that they had a chemopreventive role in DENA-­ induced HCC rats. The current studies about the anticancerogenic activity of N. sativa are mostly completed in vitro and in vivo. N. sativa may be used as therapeutic agents that regulate the production of tumors in various stages and treatment of many types of cancers. However, further studies are needed for its molecular and cellular mechanisms to underlying the anti-cancer effects of N. sativa.

2.4  Antimicrobial Activity of Nigella sativa Seeds Antibiotic-resistant bacteria are causing serious problems across the world. Recently, particular interest in the antimicrobial activities of N. sativa seed and its active components are on a rise. The antimicrobial activity of N. sativa seeds was researched on various enteropathogenic microbes, including Salmonella thypi, Streptococcus pyogene, Pseudomonas aeruginosa, and Klebseilla pneumonia (Abdallah 2017; Bakal et al. 2017), along with the fungal species such as Candida albicans, Aspergillus niger, Fusarium solani, and Scopnlariopsis brevicaulis (Aljabre et al. 2015; Khan et al. 2003), and pathogenic human viruses, HIV, hepatitis C (HPC), influenza virus (H9N2) and Laryngotrachietis virus (Barakat et  al. 2013; Onifade et al. 2013; Umar et al. 2016; Zaher et al. 2008). Also, it was reported that TQ inhibited the growth of gram-negative and gram-positive pathogenic bacteria (Micrococcus luteus, Bacillus cereus, Listeria monocytogene, Salmonella enteric, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Serovar typhimurium, Helicobacter pylori, Enterococcus faecalis, Vibrio lginolyticus, and Vibrio paraheamolyticus) in a biofilm (Bakal et al. 2017; Chaieb et al. 2011). The antimicrobial effect of N. sativa seeds against Salmonella typhi was analyzed to determine the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the seed. MIC and MBC values of N. sativa seeds to inhibit Salmonella typhi were 45 and 47.5%, respectively (Utami et  al. 2016). N. sativa seeds from different countries were analyzed for its antimicrobial activity and it was found that Indian black seeds had the greatest antimicrobial activity compared to other origins (Sawarkar et  al. 2016). The active constituents of N. sativa seed exhibited significant antimicrobial activity against anaerobic human pathogenic bacteria (C. difficile) which causes diarrhea (Randhawa et al. 2016). Rahman et al. (2017) assessed the antimicrobial activity of N. sativa seed water extract against Staphylococcus aureus, Escherichia coli, Salmonella typhi, Bacillus cereus, Pseudomonas aeruginosa, and Bacillus subtilis compared with the traditional antibiotics. The antimicrobial activity of N. sativa was especially competent against S. aureus followed by B. cereus and B. subtilis. Multidrug resistance of gram-­ negative and gram-positive bacteria was studied in the presence of N. sativa capsule

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and oil (Saleh et al. 2018). It was reported that N. sativa may be used as an alternative of decolonizing agent for gram-positive bacteria in the management of some infectious diseases and may recommend as natural preservatives in food industry. To eliminate the resistance of bacteria against antibiotics, the synergistic effect of phage and phenolic compounds extracted from pomegranate peel, grape seeds and black cumin seeds was analyzed and N. sativa seed had the highest positive effect on phage activity and increased the size of phage plaques (Tayyarcan et al. 2019). In a recent study, the antifungal activity of N. sativa seed against Candida albicans was reported, wherein N. sativa had shown antifungal activity at a concentration of 2000  μg/mL with a maximum zone of inhibition of 25  mm (Nagarajan and Geetha 2018). Zaher et al. (2008) reported on the antiviral activity of N. sativa seeds against Infectious Laryngotrachietis Virus (ILTV). In an animal study, the anti-virus effect of N. sativa was studied using turkeys that were induced with the H9N2 avian influenza virus. N. sativa was able to reduce the pathogenic effects of the H9N2 avian influenza virus in turkeys and supported their immune responses (Umar et al. 2016).

2.5  Antidiabetic Activity of Nigella sativa Seeds It was reported by the IDF’s 9th edition (2019) that currently, 351.7 million people (age 20–64 years-old) have diabetes in 2019 worldwide. Unfortunately, this number is expected to rise to 417.3 million by 2030 and to 486.1 million by 2045. In the same report, it was also mentioned that 760 billion USD was spent on the health cost of diabetes in 2019 and this cost is expected to rise 846 billion USD in 2045. Therefore, to hegde, the high costs and easy accessibility of the natural products, the intention to consume natural products in the management of diabetes is an increasing trend and traditional use of phenolics-rich foods is growing within the pre/diabetic people. Due to the bioactive components in N. sativa seeds and its non-side effects, ease of access and being cheap, increased studies on glucose metabolism is getting attention. N. sativa seeds and turmeric (Curcuma longa) were analyzed for their antidiabetic effects. STZ-induced albino rats were administered with treatments for 6  weeks. Both treatments had an effect on blood glucose level, water, and food intaken decrease and followed by an increase in body weight. The antidiabetic effect of turmeric was higher than N. sativa seeds (El-Bahr and Al-Azraqi 2014). Silver nanorods could be synthesized through N. sativa seeds which could be used in different fields (i.e., display technologies, thermoelectric and electronic devices, optoelectronic devices and biomedicine). The production of nanorods is easier, rapid and eco-friendly when it is synthesized from the plant. It was found that the antidiabetic effect of nanorods may be due to the flavonoid content of synthesized nanorods (Kumar et al. 2015).

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Diabetes induced alloxan rats were fed with N. sativa seeds to analyze its toxic effect and the level of minimal dosage for its anti-diabetic effect (Bensiameur-­ Touati et al. 2017). At low doses, N. sativa seed indicated antihyperglycemic effect. In addition, the antidiabetic effect of N. sativa seeds was studied in hyperlipidaemic albino rats. Rats were administered with a high fed diet and/or N. sativa seeds (Aboul-Mahasen and Alshali 2019). It was observed that blood glucose and pancreatic amylase level was decreased significantly in rats fed with N. sativa seeds. The histopathological analysis showed regeneration of the exocrine and endocrine parts of the pancreatic tissues in hyperlipidemic rats that administered with N. sativa seed. Sathishkumar et al. (2017) analyzed the inhibition of α-amylase activity by N. sativa seeds. The seeds were coated with quercetin or purified polyphenols to regulate the release of phenolic content which increases the effectiveness of the inhibition of α-amylase activity. A detailed study was carried out by Hannan et al. (2019) about the anti-diabetic effect of N. sativa seeds. STZ-induced rats were used to assess the effect of N. sativa seeds on gastrointestinal (GI) motility, intestinal disaccharidase activity and inhibition of carbohydrate digestion and absorption in the gut. In the fasted rats, N. sativa seeds improved GI motility, decreased the activity of α-glucosidase enzymes, reduced glucose absorption, and improved insulin secretion. Pelegrin et al. (2019) reported that taking N. sativa seeds (1 g/day) for 4 weeks did not affect blood glucose level, insulin secretion or insulin sensitivity, however intake of supplement lowered the lipid concentration. Similar results were obtained from Hemadri Reddy et al. (2017) wherein the antidiabetic activity of the N. sativa seed was not observed. It was reported that TQ, the active component of N. sativa, demonstrated antidiabetic activity through body weight, serum glucose and insulin level of the animals (Bule et al. 2020; Younus 2018). The aforementioned studies concluded that N. sativa seeds may be used in the management of diabetes for its effect on the different paths of the carbohydrate mechanism. In addition, the attention of N. sativa seed’s antidiabetic activity is increasing and there are still more in vivo studies that are needed to discover N. sativa seed’s antidiabetic activity.

2.6  C  ardioprotective and Antihypertension Effects of Nigella sativa Seeds Cardiovascular diseases (CVD) cause deaths by an increasing number each year worldwide. It is believed that 1.79 million died due to CVD, and this number will rise to 23.6 million by 2030 and 31% of deaths will be caused by CVD-related diseases (Benjamin et al. 2019). Several diseases affect the development of CVD such as diabetes, obesity, high blood pressure, and plasma lipid concentration. In the literature, there are limited epidemiological and clinical studies and several in vitro

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and in vivo studies have indicated that N. sativa seed consumption may decrease the risk of CVD (Shakeri et al. 2018; Tavakoly et al. 2019). Dehkordi and Kamkhah (2008) recruited people with mild hypertension diseases with a randomized, double-blind, placebo-controlled trial to assess the effect of N. sativa seed water extract supplement. After 8 weeks, it was found that the supplement was able to lower blood pressure significantly. The effect of N. sativa seed powder in hypercholesterolemic male white (HC) rabbits was observed for 8 weeks. The total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels decreased and increased high-density lipoprotein cholesterol (HDL-C) levels were observed in HC rabbits that consumed N. sativa seed powder. The inhibition of plaque formation was observed (Al-Naqeep et al. 2011). The diabetic patient’s heart was evaluated after N. sativa seed capsule intake for a year to explore the effects of its supplementation on cardiac function (Kaatabi et al. 2012). It was found that N. sativa supplementation improves the LV systolic function. N. sativa supplementation had a positive effect on the heart health of type 2 diabetic patients. Mohamad et  al. (2015) investigated the combination effect of N. sativa seeds (50 mg/kg daily) and honey among healthy and hypercholesterolemic subjects for 3 months. The mixture of N. sativa seed and honey decreased TG, TC and increased HDL-C level in hypercholesterolemic subjects. For both groups, the body weight was decreased, and blood pressure was lowered in hypercholesterolemic subjects. The study claimed that a mixture of N. sativa seeds with honey may have beneficial effects in lowering the risk factors of CVD. Similarly, in a more recent study, the mixture of N. sativa seeds (50 mg/kg) and honey was taken by 45 male and female (healthy and hypercholesterolemic subjects) for 3 months. Both genders with hypercholesterolemia disease demonstrated that HDL level increased only in male subjects with hypercholesterolemia. Therefore, it was concluded that the treatment of N. sativa seeds and honey mixture among both genders lowered the lipid profile and relatively lowered the cardiovascular disease risk factors (Mohamad et al. 2014). Both researchers showed that the combination of N. sativa seeds and honey has the potential to decreased risk factors of CVD and in the management of CVD-related diseases for healthy or hypercholesterolemic subjects. The effect of N. sativa supplementation was assessed in subjects with type 2 diabetes based on their lipid profile, mean arterial pressure, and heart rate. Subjects were divided into two groups and the first group was taken 2  g/day of N. sativa supplement and the second group took the only placebo for a year. N. sativa group had a significant reduction in TC, LDL TC/HDL, and LDL/HDL ratios, compared with the respective baseline data and the control group. In addition, the systolic blood pressure, diastolic blood pressure, mean arterial pressure, heart rate and body mass index were decreased in the N. sativa group compared to control (Badar et al. 2017). Hypertensive patients were taken Allium sativum (tablet), N. sativa seeds (soft gelatin capsule), and Hibiscus sabdariffa (dried leaves tea of the plant) for 4 weeks to analyze the effect of those plants as an antihypertensive agent. All of them has

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shown antihypertensive and N. sativa seed gelatin capsule significantly decreased serum creatinine level (AL-Jawad and Al-Attar 2018). Susilowati et al. (2019) analyzed the risk factors of atherosclerosis and oxidative stress of N. sativa administration for 3 weeks to STZ-induced diabetic dyslipidemia rats. I N. sativa seed treatment decreased TC, TG, LDL, and increased HDL levels. Similarly, dyslipidemia is a disease that increased the risk of cardiovascular disorders. Elfouly et al. (2019) analyzed the effect of crushed N. sativa seeds daily for 6 weeks. Total cholesterol, LDL-C and triglyceride levels were decreased significantly in patients that took crushed N. sativa seed. In another study with 50 patients who had nonalcoholic fatty liver disease (NAFLD), supplemention with N. sativa seed (2 g/day) or placebo for 12 weeks was studied. NAFLD is also known to its high relation with coronary heart disease risk factors. It was discovered that N. sativa seed supplementation improved insulin resistance and hepatic steatosis in patients with NAFLD (Darand et al. 2019). Mohtashami and Entezari (2016) conducted a clinical study which is a double-­ blind, cross-over, randomized trial on 51 patients who were fed with a bread that contains N. sativa seed for 2 months. The supplementation of N. sativa seeds did not affect the subject’s blood pressure, blood glucose level or anthropometric parameters, however it was reported that fasting blood glucose level was decreased. When the above research is evaluated, the N. sativa role in therapeutic management is crystallized. The clinical studies about the effect of N. sativa seed on CVD were studied for different lengths of time and the results showed that N. sativa seed may decrease the risk of heart diseases. However, to determine the effective dosage and type of N. sativa seed, more studies need to be conducted putting into the center the dosage, length of intake, and type of N. sativa seeds.

2.7  Antihyperlipidemic Activity of Nigella sativa Seeds The prevalence and severity of metabolic syndromes such as cardiovascular diseases and diabetes (diabetic patients, oxidized LDL formation is enhanced) are increasing. Hyperlipidemia causes an abnormal level of high lipid concentration in the blood. In the literature, there are studies about the antihyperlipidemic effect of N. sativa seed which were reviewed below. Dehkordi and Kamkhah (2008) recruited people with mild hypertension diseases with a randomized, double-blind, placebo-controlled trial to assess the effect of N. sativa seeds supplement. After 8  weeks, the supplement intake significantly decreased total cholesterol and low-density lipoproteins (LDL) level compared to the control group. The effect of powdered N. sativa seeds (PBC) on the risk of hypercholesterolemia was analyzed in Sprague Dawley rats and the rats were fed with PBC for 28 days. It was observed that PBC was able to decrease serum cholesterol, triglycerides (TC), and LDL (Sultan et al. 2011). In another study, PBC was administered to adult male alloxan-induced albino rats for 60  days to assess its effect on cholesterol levels

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(Shalaby et al. 2016). Similarly, it was observed that PBC reduced blood serum TC, TG and LDL, and increased HDL. There were no observed toxic effects on liver function. The effect of N. sativa seed powder was assessed at different concentrations (1, 2 and 3 g/day) in type-2 diabetic patients for 3 months. The lowest concentration of N. sativa supplementation increased the HDL level, but did not affect the TC, TG and LDL-C levels. However, with the intake of 2 g/day the level of TC, TG and LDL-C were decreased, and HDL level was increased. The highest dose of N. sativa supplementation did not affect the antihyperlipidemic activity (Kaatabi et al. 2012). Patients with poor glycemic control (HbA1C > 7%) took N. sativa seed powder capsules for 8 weeks wherein the effect of N. sativa seed powder capsules on the reduction of FBG, PPBG, HbA1C was significantly higher than the standard group. Also, N. sativa seed powder group had lower LDL cholesterol compared to the standard group (Najmi et al. 2012). In another study, subjects with a total cholesterol concentration > 200 mg/dl were asked to rake N. sativa capsules containing 500 mg N. sativa crushed seeds four times a day for 2 weeks. It was observed that the concentration of TC, LDL and TG were decreased in the N. sativa group compared to the placebo group and did not affect the HDL level (Sabzghabaee et al. 2012). Al-Naggar et al. (2017) reported that the seed may lower the cholesterol level through the inhibition of the de novo cholesterol synthesis or stimulation of bile acid excretion. In another reviewed paper, Chrysant and Chrysant (2017) reported that the antihyperlipidemic activity of N. sativa seed may be related through the vasodilation through blockade of Ca2+ voltage channels and reduction of oxidative stress. Ibrahim et  al. (2014) investigated the hypolipidemic effects of N. sativa in menopausal women within 2 months of the intervention study. It was reported that while TC, LDL and TG levels were decreased, the HDL level increased in menopausal women that consumed N. sativa supplement. However, 1 month after the study, the discontinuation of the N. sativa caused a change in lipid profiles back to the pretreatment levels. Sedentary overweight females were recruited to analyze the lipid-lowering effects of N. sativa supplementation with moderate physical activity. The study model was conducted as a randomized, double-blind, controlled trial for 8 weeks and each participant attended to the aerobic training program (3 times/week). N. sativa supplementation with moderate physical activity was significantly lowered total cholesterol (TC), triglyceride, LDL, and body mass index also increased HDL (Farzaneh et al. 2014). The clinical efficiency of N. sativa seed and turmeric (alone or a combination) in males with metabolic syndrome was studied (Amin et  al. 2015). The study was conducted as a double-blind-randomized-controlled trial. The combination decreased LDL, body fat %, fasting blood glucose level, cholesterol, TG, LDL, CRP (c-reactive protein) and raised HDL-cholesterol. The intact seeds (5 g) of N. sativa were put into tea bags with 5 g of coarse milled powder of dry leaf of the Melissa officinalis (Hosseini et al. 2018a). Researchers conducted a randomized open-label controlled clinical trial with subjects who were

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non-alcoholic fatty liver disease (NAFLD). The teabags were consumed every day after infusion of them in 250 mL of boiled water for 10 min for 3 months. The serum level of liver enzymes such as serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were reduced compared to the control group and improved the grade of fatty liver in NAFLD. Fakhria et  al. (2019) developed a new drug including TQ loaded to chitosan vesicle for the treatment of liver disorders. It was discovered that orally consumed TQ loaded chitosan lipid vesicle may be beneficial in the management of hyperlipidemia.

2.8  Gastro-protective Effects of Nigella sativa Seeds Imbalanced hydrochloric acid secretion from the stomach and mucosal damage are associated with gastric and duodenal ulcers. Five to ten percent of the world’s population is suffering due to this chronic illness. In addition, the drugs (i.e., antacids, and H2 antihistamines) used in the treatment of gastric ulcers may cause serious side effects such as hypersensitivity, arrhythmia, and impotence. Therefore, the use of the natural product in the management of gastric ulcers is getting attention (Franco et al. 2015). N. sativa seeds and TQ have been used due to its gastroprotective effects and due to its activity on lowering of gastric acidity and increase the content of gastric mucosal production (Khan et al. 2016). Kanter et al. (2006) observed the cytoprotective effects of N. sativa powdered seeds and TQ using rats with ethanol-induced gastric mucosal damage. After an hour of administration of the N. sativa powdered seeds (500 mg/kg) and TQ (10 mg/ kg), it was observed that N. sativa powdered seeds had greater cytoprotective effects than TQ. Also, both treatments significantly decreased the rate of gastric mucosal injury. The anti- Helicobacter pylori effect of N. sativa seeds and clinically used pharmaceutical drugs (amoxicillin, clarithromycin, and omeprazole) were compared in vivo. It was found that treatment with N. sativa (2 g/day) for 14 days was possessed comparable anti- Helicobacter pylori activity compare to drug therapy for 14 days (Salem et al. 2010). In an intervention study, the combination of N. sativa (6 g/day) and honey (12 g/ day) was investigated due to its effect on gastric Helicobacter pylori infection. Subjects with positive Helicobacter pylori infection were asked to take one teaspoon of combination 3 times/day after meals for 2 weeks. It was observed that the symptoms of the infection were reduced significantly and therefore the combination may be used as an anti-dyspeptic agent (Hashem-Dabaghian et al. 2016). Ahmad et al. (2017) analyzed the effect of TQ on stress-induced gastric ulcers. Wistar albino rats were induced with water immersion restraint (WRS) as a stress factor and following that the rats were administered with TQ orally for 7 days. Acid secretion and gastric juice volume were increased, whereas pH was decreased in

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WRS rats. Histological analysis showed that the gastric mucosa was protected, and the antioxidant activity of the enzymes was increased. The effect of N. sativa seed on the liver injuries was studied using Wistar rats that induced with thioacetamide (TA-liver injury). The treatment of N. sativa seed improved the fibrogenesis in the liver due to TA (Salem et al. 2017). Bukar et al. (2017) investigated the gastroprotective effect of N. sativa seed on Wistar rats after an hour of its administration to rats, their stomach was examined for the mucosal damage. It was found that the groups of rats treated with N. sativa seeds had few vascular congestions compared to the saline-treated group. Therefore, N. sativa seed may be able to prevent gastric ulcers in the stomach.

2.9  Neuro-protective Effects of Nigella sativa Seeds Neurodegenerative disorders are a growing concern nowadays and aging is the main risk of the disease. The consumption of N. sativa seed and its active constituents may decrease the rate of neurodegenerative diseases (Khazdair 2015). Biswas and Guha (2007) investigated the effects of N. sativa seeds on pentylenetetrazole-­ induced albino Holtzman rats (seizure model). N. sativa seed treatment was able to increase sleeping time, however diminished motor coordination and locomotor activity. Also, N. sativa seed-treated group extended the seizure latency and inhibited picrotoxin (a GABAA antagonist). In an intervention study, the effect of N. sativa seed on memory, attention, and cognition was observed for 9 weeks in healthy elderly subjects (Bin Sayeed et al. 2013). The volunteers were asked to take N. sativa seed capsule (500 mg/day) and assessed for their neuropsychological state. There was a significant difference in the score of the logical memory test of the N. sativa-treated group due to its therapeutic effect on memory, attention and cognition. Javanbakht et al. (2013) analyzed the protective effect of N. sativa seed against neurodegenerative diseases using the sciatic nerve of rats. The rats were treated with 400 mg/kg body weight of N. sativa daily for 30 days. The damaged nerve cells and degenerating neurons were decreased in the N. sativa-treated group. In addition, it was reported that the morphology of neurons was improved after N. sativa therapy in the sciatic nerve of rats. To determine the effect of N. sativa seeds on the olfactory epithelium (OE), female albino rats were treated with N. sativa (40 mg/kg/day) for 2 months. The study revealed that N. sativa seed treatment improved the structure and thickness of OE (Eltony and Elgayar 2014). Recently in a review, it was reported that N. sativa and TQ showed protective effects against neurodegenerative diseases such as Parkinson, Alzheimer’s, traumatic brain injury, depression, encephalomyelitis, epilepsy, and ischemia (Samarghandian et al. 2018). These mentioned studies suggest that N. sativa seed or TQ has a neuroprotective potential and may be a candidate as a natural supplement against neurodegeneration-related diseases.

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2.9.1  Fertility Effects of Nigella sativa Seeds Infertility is a reproductive health problem and it was reported that around the world there were about 48.5 million infertile couples (Mascarenhas et al. 2012). There are limited studies analyzed the fertility effect of N. sativa seed in the literature. Mohammad et  al. (2009) investigated the effect of N. sativa (300  mg/kg body weight) on the reproductive system and fertility on adult male albino rats for 60 days. It was observed that N. sativa-induced rats had a significant increase in their weight of reproductive organs and fertility compared to the control group because of their sperm motility and testicular ducts were increased. In a clinical study, the effect of N. sativa (2  g/day) on male infertility was conducted on 40 infertile men with low sperm count or quality for a year. Several analyses (testosterone level, semen parameters, serum follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels) were completed before and after 3 months of the study. It was found that sperm count and activity were significantly higher and sperm viability and normal morphology were significantly increased after the 3 months of treatment with N. sativa. In addition, 3 months of treatment with N. sativa made a significant rise in FSH, LH, and testosterone levels (Marbat et al. 2013). STZ-induced diabetic albino rats were treated with N. sativa seed powder (300  mg/kg body weight) to analyze its effect on luteinizing hormone (LH) and testosterone levels. The level of testosterone and LH was higher in N. sativa seed powder treated group compared to the diabetic control and a healthy control group showed higher levels of testosterone and LH than other groups (Haseena et al. 2015). There is limited information about the fertility effect of N. sativa seed. Therefore, further research about clinical and animal studies need to complete through discover of its potential on infertility management.

3  Potential Toxicity of Nigella sativa Seeds Several studies about the toxicological properties of N. sativa seeds were conducted and it was reported that therapeutic doses of the N. sativa seeds have low toxicity and wide safety margin (Dajani et al. 2016). Bensiameur-Touati et al. (2017) analyzed the subacute oral toxicity at 5 different concentrations of aqueous extract of N. sativa on mice for 6 weeks. For higher doses (21 and 60 g/kg aqueous extract of N. sativa), it was observed that on the week third and fifth only 2 and 3 mice death were reported. For other doses, there were not deaths reported. On the other hand, TQ was reported with a lethal dose (LD50) of 2.4 g/kg bw after an oral acute toxicity test on Swiss albino mice (Badary et al. 1998). Late toxicity caused a significant decrease in the virtual organ weight and glutathione distribution of the hepatic, renal and cardiovascular system. However, the subchronic toxicity analysis after the daily administration of 30, 60, and 90 mg/kg TQ treatment did not cause any deaths or symptom toxicity.

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4  Conclusion N. sativa seed is a traditional medicinal plant used for its low cost, ease of access and lower side effect factors compared to chemical drugs. Due to its high nutritional and phytochemical contents such as phenolics, unsaturated fatty acid found in the fixed and essential oil of the N. sativa seed, it is getting popular as a food ingredient and in medicinal purposes worldwide. N. sativa seed’s medicinal and therapeutic effects on human health based on its extraordinary content of the bioactive constituents. The antioxidant, anti-inflammatory, anticancer, antimicrobial, antidiabetic, cardioprotective, antihyperlipidemic, gastroprotective, neuroprotective, and fertility effects of N. sativa seed were highlighted based on animal and clinical studies. However, clinical and toxicological studies about the use of N. sativa seed are limited, therefore further studies are recommended to evaluate its medicinal effects which could be utilized in the medicine and food industry as a safer nutraceutical and food ingredient. This chapter could be helpful for further research priorities of N. sativa seed to be used in the medicine and food industry for an extensive range of health benefits.

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Schneider-Stock, R., Fakhoury, I. H., Zaki, A. M., El-Baba, C. O., & Gali-Muhtasib, H. U. (2014). Thymoquinone: Fifty years of success in the battle against cancer models. Drug Discovery Today, 19(1), 18–30. https://doi.org/10.1016/j.drudis.2013.08.021. Seeram, N. P., Henning, S. M., Niu, Y., Lee, R., Scheuller, H. S., & Heber, D. (2006). Catechin and caffeine content of green tea dietary supplements and correlation with antioxidant capacity. Journal of Agricultural and Food Chemistry, 54(5), 1599–1603. https://doi.org/10.1021/ jf052857r. Shahin, Y. R., Elguindy, N. M., Abdel Bary, A., & Balbaa, M. (2018). The protective mechanism of Nigella sativa against diethylnitrosamine-induced hepatocellular carcinoma through its antioxidant effect and EGFR/ERK1/2 signaling. Environmental Toxicology. https://doi. org/10.1002/tox.22574. Shakeri, F., Khazaei, M., & Boskabady, M. (2018). Cardiovascular effects of Nigella sativa L. and its constituents. Indian Journal of Pharmaceutical Sciences, 80. https://doi.org/10.4172/ pharmaceutical-sciences.1000447. Shalaby, A., Abdel-Sater, K., El-Din, G., Hassan, A., & Noor, A. (2016). Hepatorenal changes by Nigella satıva seeds powder in diabetic rats. Pakistan Journal of Physiology, 12, 3–7. Siegel, R. L., Miller, K. D., & Jemal, A. (2019). Cancer statistics, 2019. CA: a Cancer Journal for Clinicians, 69(1), 7–34. https://doi.org/10.3322/caac.21551. Srinivasan, K. (2018). Cumin (Cuminum cyminum) and black cumin (Nigella sativa) seeds: Traditional uses, chemical constituents, and nutraceutical effects. Food Quality and Safety, 2(1), 1–16. https://doi.org/10.1093/fqsafe/fyx031. Sultan, M., Butt, M., Ahmad, R., & Suleria, H. A. R. (2011). Supplementation of powdered black cumin (Nigella sativa) seeds reduces the risk of hypercholesterolemia. Functional Foods in Health and Disease, 12, 516–524. https://doi.org/10.31989/ffhd.v1i12.109. Susilowati, R., Ainuzzakki, V., Nadif, M. R., & Diana, A. R. (2019). The efficacy of Nigella sativa L. extracts to reduce cardiovascular disease risk in diabetic dyslipidemia. AIP Conference Proceedings, 2120(1), 70020. https://doi.org/10.1063/1.5115737. Swamy, M.  K., Patra, J.  K., & Rudramurthy, G.  R. (2019). Medicinal plants: Chemistry, pharmacology, and therapeutic applications. CRC Press. Taka, E., Mazzio, E.  A., Goodman, C.  B., Redmon, N., Flores-Rozas, H., Reams, R., Darling-­ Reed, S., & Soliman, K. F. A. (2015). Anti-inflammatory effects of thymoquinone in activated BV-2 microglial cells. Journal of Neuroimmunology, 286, 5–12. https://doi.org/10.1016/j. jneuroim.2015.06.011. Takruri, H. R. H., & Dameh, M. A. F. (1998). Study of the nutritional value of black cumin seeds (Nigella sativa L). Journal of the Science of Food and Agriculture, 76(3), 404–410. https://doi. org/10.1002/(SICI)1097-0010(199803)76:33.0.CO;2-L. Tavakoly, R., Arab, A., Vallianou, N., Clark, C. C. T., Hadi, A., Ghaedi, E., & Ghavami, A. (2019). The effect of Nigella sativa L. supplementation on serum C-reactive protein: A systematic review and meta-analysis of randomized controlled trials. Complementary Therapies in Medicine, 45, 149–155. https://doi.org/10.1016/j.ctim.2019.06.008. Tayyarcan, E. K., Acar Soykut, E., Menteş Yılmaz, O., Boyaci, I. H., Khaaladi, M., & Fattouch, S. (2019). Investigation of different interactions between Staphylococcus aureus phages and pomegranate peel, grape seed, and black cumin extracts. Journal of Food Safety, 39(5), e12679. https://doi.org/10.1111/jfs.12679. Umar, S., Munir, M. T., Subhan, S., Azam, T., Nisa, Q., Khan, M. I., Umar, W., Rehman, Z., Saqib, A. S., & Shah, M. A. (2016). Protective and antiviral activities of Nigella sativa against avian influenza (H9N2) in turkeys. Journal of the Saudi Society of Agricultural Sciences. Utami, A., Pratomo, B., & Noorhamdani. (2016). Study of antimicrobial activity of black cumin seeds (Nigella sativa L.) against salmonella typhi in  vitro. Journal of Medical & Surgical Pathology, 01. https://doi.org/10.4172/2472-4971.1000127. Weiss, D.  J., Austria, E.  J., Anderton, C.  R., Hompesch, R., & Jander, A. (2006). Analysis of green tea extract dietary supplements by micellar electrokinetic chromatography. Journal of Chromatography A, 1117(1), 103–108. https://doi.org/10.1016/j.chroma.2006.03.057.

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Yimer, E.  M., Tuem, K.  B., Karim, A., Ur-Rehman, N., & Anwar, F. (2019). Nigella sativa L. (black cumin): A promising natural remedy for wide range of illnesses. Evidence-­ Based Complementary and Alternative Medicine: ECAM, 2019, 1528635. https://doi. org/10.1155/2019/1528635. Younus, H. (2018). Antidiabetic action of thymoquinone BT- molecular and therapeutic actions of thymoquinone: actions of thymoquinone (H.  Younus ed., pp.  7–17). Springer Singapore. https://doi.org/10.1007/978-981-10-8800-1_2. Zaher, K., Ahmed, W., & Zerizer, S. (2008). Observations on the biological effects of black cumin seed (Nigella sativa) and green tea (Camellia sinensis). Global Veterinaria, 2.

Chapter 12

Nigella sativa Seed Extract in Green Synthesis and Nanocomposite Raya Soltane, Dalila Mtat, Amani Chrouda, Noof Alzahrani, Youssef O. Al-Ghamdi, Hussam El-Desouky, and Khaled Elbanna

Abstract  The World Health Organization (WHO) is providing emphasis on the exploration of medicinal plant species for the benefit of human care and systems. Recently, a great deal of attention has given to the seed and oils of black cumin. Nigella sativa Linn., belong to family Ranunculaceae, is grown in many parts of the world. It is one of the most important medicinal plants because it has multipurpose uses and contains phytochemical components such as alkaloids, terpenoids, saponins, flavonoids, sterols, tannins, and derivatives, showing antimicrobial activity. Scientific research has been published on the bioactivity and medicinal properties of Nigella sativa seeds. Seeds also used as chemical remedies and as traditional pharmacological properties such as antidiabetic, antihypertensive, anticancer and anti-­ inflammatory properties. In the recent years, green syntheses methods are much more important than chemical methods because green methods use the reduction technique using plant extracts instead of chemicals. It is more economical and eco-­ R. Soltane (*) Department of Basic Sciences, Adham University College, Umm Al-Qura University, Adham, Saudi Arabia Department of Biology, Faculty of Sciences, Tunis El Manar University, El Manar, Tunis, Tunisia e-mail: [email protected] D. Mtat Laboratory of Asymmetric Organic Synthesis and Homogeneous Catalysis (UR11ES56), Faculty of Sciences of Monastir Avenue of the Environment, Tunisia, Tunisia, Tunisia A. Chrouda Department of Chemistry, College of Al-Zulfi, Majmaah University, Al-Majmaah, Saudi Arabia Laboratory of Interfaces and Advanced Materials, Faculty of Sciences, University of Monastir, Monastir, Tunisia Laboratory of Analytical Sciences UMR CNRS-UCBL-ENS, Villeurbanne Cedex, France e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_12

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friendly than the chemical method. The present chapter discusses the current knowledge about the green syntheses from Nigella sativa seed extracts and their potential biological activities. Keywords  Nigella sativa · Essential oil · Green synthesis · Green chemistry · Nanoparticles

Abbreviations AgNPs BC GO NsEO PCL PVA

Silver nanoparticles Black cumin Graphite oxide Nigella sativa essential Oil Poly(ε-caprolactone) polyvinyl alcohol

N. Alzahrani Department of Basic Sciences, Adham University College, Umm Al-Qura University, Adham, Saudi Arabia e-mail: [email protected] Y. O. Al-Ghamdi Department of Chemistry, College of Al-Zulfi, Majmaah University, Al-Majmaah, Saudi Arabia e-mail: [email protected] H. El-Desouky Chemistry Department, Faculty of Science, Helwan University, Helwan, Egypt Chemistry Department, Jamoum University College, Umm Al-Qura University, Makkah, Saudi Arabia e-mail: [email protected] K. Elbanna Department of Agricultural Microbiology, Faculty of Agriculture, Fayoum University, Fayoum, Egypt Department of Biology, Faculty of Applied Science, Umm Al-Qura University, Makkah, Saudi Arabia e-mail: [email protected]

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1  G  reen Synthesis Method of Silver Nanoparticles from Nigella sativa Leaf Extract The nanotechnology field has been growing rapidly throughout the world. Synthesis and characterization of nanoparticles are the main objectives followed in this science (Ahmed et al. 2016; Ghorbanpour and Fahimirad 2017; Siddiqui et al. 2019). They have many applications in display technologies, electrical fields and also in micro-electrical mechanical systems (MEMS) (Antariksh Saxena et al. 2010). Silver nanoparticles (AgNPs) is one of the most efficient nanoparticles for potential applications in technologies and biomedicine (Kharissova et  al. 2013). Biologically active medicinal plants have been utilized in nanoscience (Shanker et  al. 2017). Plant-mediated nanoparticles illustrate antimicrobial and anticancer activities as compared to chemically grown nanoparticles (Dauthal and Mukhopadhyay 2016). Nanoparticles can be synthesized, including green synthesis, and may be considered for plants and their derivatives (Khatami et al. 2018). In the chemical methods, reducing agents involved in the reduction of metal ions, and stabilizing agents used to prevent undesired agglomeration of the produced nanoparticles carry a risk of toxicity to the environment. On the other hand, the contents of the produced nanoparticles are thought to be toxic in terms of shape, size and surface chemistry. In the green synthesis method wherein nanoparticles with biocompatibility are produced, these agents are naturally present in the employed biological organisms. Recently, green synthesis of AgNPs from Nigella sativa has emerged and gained importance because it is eco-environmental and effective cost, with lesser toxicity. The seed extract of Nigella sativa contains phytochemicals including phenolics, terpenoids, and flavonoids that may act as reducing agents and can convert metal ions to metal nanoparticles. The green synthesis of AgNPs depends on several parameters such as the concentration of substrate, temperature, reaction time and pH. N. sativa AgNPs showed significant cytotoxic activity on hepatic cancer.

1.1  Green Synthesis of AgNPs Nigella sativa seeds were washed with deionized water. Twenty grams of coarsely ground Nigella sativa seeds were taken and boiled in 100 mL of distilled water and filtered. The filtrate was collected and stored at 4 °C. The seed extracts were taken separately and to this 10 mL of 1 mM silver nitrate solution was added with constant stirring and exposed to different conditions like sunlight irradiation, UV irradiation, and microwave irradiation. The color change of the seed extract from yellow to dark brown indicated that the AgNPs were synthesized (Priti Ranjan et al. 2013).

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2  G  reen Synthesis of Platinum Nanoparticles Using Nigella sativa Seed Extract The unique physicochemical properties and biological activities of metallic nanoparticles give them an important area of research compared to their bulk counterparts. The platinum nanoparticle plays a crucial role in the nano-medical field (Ahmad et al. 2017). Literature has several reports illustrating the use of platinum nanoparticles synthesis from Nigella sativa seed for the treatment of cancer (Athinarayanan et al. 2016; Khalil et al., 2014; Siddiqi and Husen, 2016). It has excellent applications as antimicrobial agents with a strong ability to discouraging the growth of harmful bacteria (El-Sherbiny et al. 2016; El Khoury et al. 2015). Green synthesis of nanoparticles from plant extracts is an important branch in biosynthesis reaction. The wealth, complex, and diverse chemical compounds involved in the seeds of Nigella sativa may provide an exceptional opportunity for the development and innovation of new nanoparticles. As the need for creating efficient, non-toxic and environmentally friendly synthesis methods, biosynthesis of nanoparticles has received significant attention (Das and Brar 2013; David et  al. 2014). Recently synthesis of platinum nanoparticles using Nigella sativa seed extract has been reported (Aygun et al. 2019). Platinum nanoparticles (Pt NPs) were prepared using black cumin seeds extract. The crushed seeds were added to 100 mL ethanol. The seed-ethanol mixture was allowed to stand in the microwave for a certain time to obtain the extract. A 15 mL Nigella sativa extract was added to 85 mL of deionized water, and then PtCl4 was transferred to this mixture. The resulting mixture was stirred at 75 °C for 2 days. The conversion of the colorless solution to black color indicates that PtCl4 was reduced to the nanosize and it is converted to the Pt NPs. To remove unreacted residues, ethanol washing was carried out by a centrifuge for at least three times. Green synthesized of Pt NPs using black cumin seed extract was very effective as potential cancer agents in the pharmaceutical industry.

3  G  reen Synthesis of Reduced Graphene Oxide Using Nigella sativa Seed Extract Graphene has been considered as a superior monolayer of carbon atoms arranged in a two-dimensional honeycomb structure. It has been investigated extensively due to its unique properties in terms of physical, chemical and mechanical properties (Aleiner and Efetov 2006; Jannik et al. 2007; Novoselov et al. 2005; Schedin et al. 2007; Zhang et al. 2005) and is widely used in applications including biotechnology and biomedicine. Many methods of reduction of graphene oxide are considered as harmful processes from an ecological and environmental point of view due to the usage and formation of expensive, toxic reagents. In recent years, scientists have extensively

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worked to develop green and easy reduction methods for expanding the production of graphene derivatives. Reduction techniques via green chemistry have attracted great interest as promising routes to produce reduced graphene oxide (RGO). An efficient green, and novel approach for the preparation of graphene sheets decorated with PdNPs via a one-pot reduction method using Nigella sativa seed extract as a green reduction agent. The reduction of metal ions and stabilization of nanoparticles is related to chemical compositions and the quantity of active component groups present in the extract of Nigella sativa. The graphene oxide (GO) was suspended into distilled water and mixed by an ultrasonic probe. GO suspension was poured into the extract solution and refluxed at 100 °C. The blackish powder was rinsed with distilled water to remove residual contaminants. The RGO was dried and kept in a vacuum oven. Prepared GO was suspended in distilled water. The suspension was placed in a flask and a proper amount of PdCl2 was introduced into the suspension. The flask was refluxed at 100 °C and the aqueous solution of black cumin seed extract was added to the suspension and mixed under the same reflux conditions. The yielded black powder of PdNPs/RGO was filtered and rinsed with deionized water, alcohol, and acetone to remove organic residues (Zengin Kurt et al. 2019). This nanocomposite is considered to be a selective catalyst for hydrogenation of nitroarenes in organic synthesis.

4  G  reen Synthesis of Gold Nanoparticles Using Nigella sativa Essential Oil (NsEO-AuNPs) Gold nanoparticles (AuNPs) have attracted attention owing to range applications in anticancer drug delivery (Brown et  al. 2010), catalysis and medical (Muthu and Wilson 2010), as well as antibacterial activity (Sathishkumar et al. 2009). There is a need to develop non-toxic and eco-friendly procedures for the synthesis of AuNPs. Plants such as coriander leaf, aloe vera, Cinnamomum camphora, lemongrass have been reported as potent biological materials for the synthesis of AuNPs (Badri Narayanan and Sakthivel 2008). Green synthesis of AuNPs have attracted considerable attention and emerged to be a thrust area of research in the field of nanotechnology owing to their various applications. AuNPs present intense optical properties (Geddes et al. 2003), biostability, catalytic activity, anti-HIV activity, anti-angiogenesis (Yamaguchi et  al. 2001; Mukherjee et  al. 2005), and anti-arthritic activities (Kalimuthu et  al. 2010). Currently, N. sativa-mediated biological synthesis of nanoparticles is gaining importance. Several reports have emerged in recent years on nanoparticle synthesis mainly focused on its biomedical applications such as the treatment of infectious diseases and cancer. Green and rapid synthesis of stable gold nanoparticles (NsEO-­ AuNPs) using N. sativa oil was reported (Manju et al. 2016). Two mL of N. sativa essential oil was added to 30 mL of 1 mM aqueous 99 × 10−4 M HAuCl4 solution at 100 °C with stirring and boiled for 1 min. Rapid reduction of

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Au3+ ions to Au0 occurred and the color of the solution becomes pink. The experiment was repeated with different volumes of diluted oil to get purple and violet-­ colored solutions. The synthesized NsEO-AuNPs showed anticancer activity against human lung cancer.

5  G  reen Synthesis of Zinc Oxide Nanoparticles (ZnONPs) Using Nigella sativa Zinc oxide nanoparticles (ZnONPs), as one of the most important metal oxide nanoparticles, are popularly employed in various fields. Among metal oxides, ZnONPs have many applications in cosmetics, paints, and plastic (Velusamy et al. 2015), with antibacterial capacities (El-gammal 2010). ZnONPs have also been used in heavy metal removal from water (Jain et al. 2013), and dental applications (Jayaseelan et al. 2012). Using plant extract to synthesize metal oxide is of significant advantage due to the production of functional molecules that reduce metal ions (Rai and Ingle 2012). ZnO nanoparticles from plants have been synthesized using green chemistry approaches by several workers (Al-Shabib et  al. 2016). Recently, zinc oxide nanoparticles have been green synthesized using: seed extracts of Nigella sativa extracts as a reducing and stabilizing agent (Alaghemand et al. 2018). The synthesis was carried out in a domestic microwave. A 0.05 M aqueous solution of zinc nitrate in 100 mL distilled water was prepared in which 10 mL Nigella sativa seed extract was added to obtain a mixture solution, then put into a domestic microwave oven. Microwave irradiation proceeded at 100% power for 20 min. After microwave processing, the solution was cooled and the resulted precipitate was separated by centrifugation, then washed with deionized water and absolute ethanol, and dried at 80  °C.  Finally, the product was calcined at 800  °C (Al-Shabib et al. 2016).

6  Poly (ε-caprolactone) Based Nanoparticles Loaded with Nigella sativa Essential Oil Nigella sativa seed essential oil is used in the treatment of different diseases such as rheumatism, bronchitis, and asthma (Ahmad et  al. 2013). Nanotechnology can improve drug skin penetration without facing skin (Tomoda et  al. 2012). It was found that poly(ε-caprolactone) polymer is a biocompatible, and safe polymer for in vivo applications (Woodruff and Hutmacher 2010). Poly(ε-caprolactone) based nanoparticles loaded with Nigella sativa seeds essential oil (NSSEO) and indomethacin is used to enhance analgesic and anti-inflammatory effects of indomethacin. Thymoquinone is the most abundant component of Nigella sativa which is mostly

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responsibile of therapeutic properties (Ravindran et al. 2010). Nigella sativa seeds oil encapsulation with indomethacin within the polymeric nanoparticles would enhance the anti-inflammatory activity of indomethacin and decrease indomethacin side effects.

6.1  Nanoparticles Preparation The encapsulation of indomethacin and Nigella sativa seeds oil was performed in two separate phases (Fessi et al. 1989). To prepare the organic phase, 200 mg PCL under the mild heat and magnetic stirring were dissolved in 25 mL acetone then 40 mg indomethacin and 300 mg NSSEO were added to the solution. Five mg PVA was dissolved in 50 mL water with the aid of mild heat and mixed under magnetic agitation. The organic phase containing NSSEO, indomethacin, PCL, and acetone was added dropwise to the aqueous phase. Acetone evaporation was carried out afterward by Rotavapor. On the other hand, indomethacin and NSSEO were encapsulated within poly(ε-caprolactone) polymer by nanoprecipitation method (Badri et al. 2018).

7  S  ynthesis of Nanocomposite-MnO2/BC from Nigella sativa Seed A novel Nigella sativa seed nanocomposite, MnO2/BC, was synthesized by the simple one-step method and utilized for water purification through adsorption and photocatalytic degradation and adsorptive removal of dye from water. MnO2 was incorporated in the carbon framework of black cumin seed powder for the formation of antibacterial MnO2/BC (Nanda et al., 2016). The composite material was developed by mixing globally cultivated black cumin seed powder, having a cellulosic surface with a large number of functional groups and excellent anti-microbial properties (Siddiqui and Chaudhry 2018).

7.1  Preparation of MnO2/BC For the synthesis of black cumin based MnO2 composite, 1.0 g of seed powder was taken in 100 mL of 0.1 M MnCl2 solution to which 100 mL of 0.1 M KMnO4 solution was added. The contents were stirred on a magnetic stirrer at 60 °C, and then cooled to room temperature. The resultant MnO2 precipitate formed in the solution got incorporated into the black cumin seed particle framework, thus the formation of the MnO2/BC composite occurred. The prepared solid compound was separated

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by filter paper then washed severally with deionized water. The pure compound was drained in a hot air oven at 45  °C (Chen et  al., 2013). The synthesis MnO2/BC showed excellent antibiotic activity, therefore, it can be a low-cost potential material for the complete clean-up of water pollutants.

8  Synthesis of Biodiesel from Nigella sativa Seed Oil Biodiesel has gained considerable attention as an alternative fuel due to environmental problems and the depletion of fossil fuels (Tariq et al. 2012). Biodiesel has different physical and chemical properties compared to diesel fuel. It is now produced by transesterification of edible oils such as canola, palm, safflower, soybean, and sunflower in the presence of various catalysts. However, the production of biodiesel using Nigella sativa seed oil as a new feedstock can be interesting. Based on the seed oil properties and the estimated cetane number, N. sativa seed oil can be developed as a biodiesel feedstock. Conversion of more than 93% has been obtained for the transesterification of the oil with methanol upon varying several reaction parameters, including temperature, molar ratio, amount of catalyst and reaction time.

8.1  Biodiesel Synthesis from Nigella sativa Seed Oil For the synthesis of fatty acid methyl ester from N. sativa seed oil with methanol was carried out in a round-bottomed flask fitted with a reflux condenser. After completion of the reaction, the alcohol was distilled, then the residue was centrifuged to complete phase separation. Three phases were formed, with the upper layer containing biodiesel, the middle layer glycerol, and the lower layer catalyst. After separation, the ionic liquid was washed with n-hexane and dried under vacuum at 70 °C. According to the physicochemical characterization and the estimated cetane numbers, the biodiesel produced from N. sativa seed oil can be developed as a potential energy plant (Aghabarari et al. 2014).

9  C  orrosion Inhibition of Iron by a Green Formulation Derived from Nigella sativa Now eco-friendly compounds, especially derived from plant extracts, draw the attention as effective inhibitors of corrosion protection of metallic substrates due to their biodegradability, non-toxic nature, and economical aspects. A formulation based on Nigella sativa oil was investigated for bronze and iron corrosion in a marine environment (Chellouli et al. 2014). A positive effect has reported of extracts

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of Nigella sativa, chamomile (Chamaemelum mixtum), halfabar (Cymbopogon proximus) and kidney bean (Phaseolus vulgaris) on the corrosion inhibition of steel in H2SO4 solution (Abdel-Gaber et al. 2006). The properties of Nigella sativa formulation as iron corrosion inhibitor were tested in an acidic solution simulating acid rain of an industrial atmosphere in an urban area. Films of this formulation on iron-based substrates, with chemical composition and structure similar to historic artifacts, were produced by immersion then submitted to corrosion. Nigella sativa formulation could be applied as a non-toxic and environmental friendly corrosion inhibitor for iron in an acidic medium for the conservation of metallic artifacts in museums and outdoor low carbon steel artifacts (Chellouli et al. 2016).

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Chapter 13

Food Applications of Nigella sativa Seeds Omar Bashir, Nusrat Jan, Gousia Gani, H. R. Naik, Syed Zameer Hussain, Monika Reshi, and Tawheed Amin

Abstract  Black cumin (Nigella sativa L., Family: Ranunculaceae) is an annual herb with a wide range of clinical applications apart from its commercial significance as a spice plant. Black cumin has been considered as one of the most treasured nutrient-rich herbs in history around the world. Its seed is a rich source of nutrients and bioactive components that confer its nutritional uses as well as medicinal and therapeutic potential. This chapter describes the black cumin seed, its vital chemical, and bioactive components and the various processing operations for its oil extraction. The chapter also emphasizes the therapeutical applications of the black cumin seed, therefore, discussing its pharmacological effects against various types of ailments. The role of black cumin in food processing has also been discussed, highlighting its use as a spice, value-added product, antimicrobial agent and an effective food preservative. Keywords  Functional foods · Nutraceuticals · Solvent extraction Antioxidant · Oil

1  Introduction Nigella (Nigella sativa) is an annual herbaceous plant belonging to the family Ranunculaceae. Due to the aroma and pungent bitter taste of the black cumin seeds, they find their use in several cuisines of different food cultures since ancient times, both in whole and ground forms. Black cumin is cultivated worldwide primarily in the Middle East, Mediterranean regions, Southern Europe, India, Pakistan, Syria, Saudi Arabia, and Turkey. India is the largest producer of black cumin in the world. In India, black cumin seeds have been used for thousands of years as a traditional ingredient of innumerable dishes and other spice blends. Besides food uses, it finds

O. Bashir · N. Jan · G. Gani · H. R. Naik · S. Z. Hussain · M. Reshi · T. Amin (*) Division of Food Science and Technology, SKUAST-Kashmir, Srinagar, Jammu and Kashmir, India © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_13

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its use as traditional medicine (Ahmad and Beg 2013). Black cumin has been considered as one of the most treasured nutrient-rich herb in history around the world (Ramadan 2007). The high nutritional profile of black cumin is attributed to the presence of a substantial amount of proteins, carbohydrates, fibres, vitamins, and minerals (Gullu and Gulcan 2013). Black cumin seed is a rich source of amino acids, fatty acids and essential oils (Mamun and Absar 2018). Also, phytochemicals present in black cumin mainly include alkaloids, saponins, and sterols. The seed oils contain basic bioactive components like thymoquinone (TQ), dithymoquinone, and thymohydroquinone (Gullu and Gulcan 2013). Amongst different bioactive constituents, TQ exhibited a wide range of therapeutic benefits (Haseena et al. 2015). Processing of black cumin seeds mainly involves various types of oil extraction processes that are applied on commercial scales. The seed oil of black cumin has been used for a variety of medicinal and food purposes. Studies indicate that black cumin seeds have traditionally been used for the treatment of various types of ailments like asthma, bronchitis, rheumatism, and other inflammatory diseases. The seeds possess the therapeutic potential and have a wide spectrum of activities like anti-diuretic, anti-hypertensive, anti-diabetic, anti-cancer, immune-modulatory, anti-microbial, analgesic and anti-inflammatory, gastro-protective, hepatoprotective, and renal protective effects (Goreja 2003).

1.1  Botanical Description Nigella sativa is an indigenous herb that belongs to plant family Ranunculaceae. It is native to Southwest Asia including Iran, Turkey, Syria, India, and Pakistan. The plant grows to a maximum height of about 40–70 cm and possesses finely divided foliage with pale blue and white flowers. The flowers are delicate with 5–10 petals. The fruit is a large and inflated capsule composed of 3–7 united follicles, each containing numerous seeds. The gynoecium is composed of a variable number of multiovule carpels, developing into a follicle after pollination, with single fruits partially connected to form a capsule-like structure. From the fruit capsules, many small caraway-type black seeds are produced (seed length: 2.5 to 3.5 mm and seed width: 1.5 to 2 mm). The seeds are three-sided and black (Gencler-Ozkan 2011) as shown in Fig. 13.1.

1.2  History and Production of Black Cumin The seeds of Nigella sativa commonly known as Black Cumin/Kalonji/Kalajira are an annual herbaceous plant. The species was first named by Swedish botanist Carl Linnaeus in 1753. Black cumin has the richest and most mystical history among all the plants used in medicine. It is reported that black cumin seeds and their oil were used by Hippocrates to strengthen the liver, to solve problems related to the

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Fig. 13.1  Nigella sativa: (a) unripe capsule (b) flower (c) seeds

digestive system, to treat snake and scorpion stings, abscesses, skin rashes, infections in the head, and the common cold. The black cumin seed oil that was discovered in the remnants of Tutankhamun’s tomb is indicative of its use since ancient times. N. sativa oil was used for treatment by Ibni Sina to stimulate the metabolism and to relieve asthenia and lethargy (Tembhurne et al. 2014). Religious statements also highlight the important properties of black cumin. The oil obtained from this plant is known to have been used by Cleopatra, the Queen of Egypt, for health and beauty (Paarakh 2010). Today, the black cumin seed and its oil are assumed to be important food ingredients and an indispensable source in alternative medicine for the treatment and prevention of various diseases (Lord et al. 2014). Black cumin is widely cultivated throughout southern Europe, Syria, Egypt, Saudi Arabia, Iran, Pakistan, India, and Turkey (Mozaffari et al. 2000). Though it is believed to be a native of Egypt, it is mostly produced in India. India is known to be the largest producer of black cumin in the world where it is commercially cultivated in the regions of West Bengal, Punjab, Jharkhand, Himachal Pradesh, Jammu and Kashmir, Bihar and Assam. Small scale cultivation is also taken at Uttar Pradesh, Rajasthan, Madhya Pradesh, and Tamil Nadu states. The other black cumin producing countries are Syria, Sri Lanka, Bangladesh, Nepal, Egypt, Iraq, and Pakistan. Exact information on its area, production, and productivity in India are not available, but it is estimated to be produced in an area of about 9000 ha area, with an annual production of about 7000–8000 tons. The country consumes most of its cultivated crops for various purposes. Its produce commands premium prices in the global market due to its colour, flavour, and pungency.

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2  Chemical Composition of Nigella sativa Seeds The high nutritional profile of black cumin can be attributed to the presence of a substantial amount of proteins, fibres, minerals, and vitamins (Table  13.1). The reports related to the nutritional composition of black cumin revealed 20–85% of protein, 38.2% of fat, 7.94% of fibre, and 31.9% of carbohydrates. Among various amino acids identified in black cumin, glutamate, arginine, aspartate, cysteine, and methionine are present in significant amounts. Black cumin seeds also contain a sufficient amount of minerals like iron, copper, zinc, phosphorus, calcium, thiamin, niacin, selenium, pyridoxine, and folic acid (Gullu and Gulcan 2013). Phytochemical analysis of black cumin displayed the presence of over hundreds of phytoconstituents which mainly include alkaloids, saponins, sterols, and essential oils. N. sativa seeds usually contain 26–34% fixed oil of which the major fatty acids are linoleic acid (64.6%) and palmitic acid (20.4%). The seed oil is comprised of 0.4%–2.5% essential oils (Mamun and Absar 2018) and with basic bioactive components like TQ, dithymoquinone, and thymohydroquinone (Gullu and Gulcan 2013). Amongst different active constituents reported so far, TQ is found as a major component of the essential oils that exhibits a wide range of therapeutic benefits (Haseena et al. 2015). Table 13.1  Chemical composition of Nigella sativa seeds Constituents Fixed oil

Volatile oil

Composition Linoleic acid (Omega-6), oleic acid, Palmitoleic acid linolenic acid (Omega-3), Myristoleic acid, Dihomolionolenic acid, stearic acid, Eicosadienoic acid, Myristic acid, Arachidic acid, Behanic acid, sterols (β-sitosterol, avenasterol, stigmasterol, campesterol and lanosterol), tocopherols (α, β, and γ) Thymoquinone, retinol (vitamin A), carotenoids (β-carotene) Thymoquinone, p-cymene, Carvacol, α-Pinene, β-Pinene, Longifolene, t-Anethole thymol, Thymohydroquinone, Dithymoquinone (nigellone)

Carbohydrate Glucose, Rhamnose, xylose, arabinose Saponins

α-Hederin (melanthin), Hederagenin (melanthigenin)

Alkaloids

Nigelicine, Nigellimine, Nigellidine

Minerals

Iron, copper, zinc, phosphorus, calcium, thiamin, niacin, pyridoxine, and folic acid

Range 22– 38%

0.40– 1.50%

References Sultan et al. (2009)

Al-Saleh et al. (2006); El-Tahir Kamal and Bakeet (2006) 24.9– Ramadan 40% (2007) 0.013% Ansari et al. (1988) 0.01% Atta et al. (1995) 3.7–7% Gullu and Gulcan (2013)

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2.1  Phytochemical Profile Several bioactive compounds from the seed of N. sativa have been reported; among those, the most important bioactive one is TQ. Other phytochemicals include sterols and saponins, phenolic compounds, alkaloids, novel lipid constituents, fatty acids, and volatile oils (Botnick  et  al., 2012). The essential oil composition of seeds (0.4–0.45%) reported in various studies represented about 40 different compounds, amongst the abundant constituents identified is TQ, thymohydroquinone (THQ), dithymoquinone transanethole, p-cymene limonene, carvone, and carvacrol. The seed oil fatty acid composition (32–40%) mainly comprises of linoleic, linolenic, oleic, palmitoleic, palmitic acids together with arachidonic, eicosadienoic, stearic, and myristic acid (Isik et al. 2017). A new dienoate and two known monoesters along with novel lipids have been isolated from the unsaponified extract of the seed, namely methylnonadeca-15, 17-dienoate, pentyl hexadec-12-enoate, and pentyl pentadec-11-enoate (Mehta et al. 2008). Phytosterols provide nutraceutical and medicinal benefits and are thus considered as the important characteristic compounds for assessing the quality of vegetable oils and food labelling (San Mauro-­Martin et al. 2018). The percentage of the total sterol content of black cumin seed oil is found in the range of 18 and 42% of the unsaponified matter. The major sterols in the seed oil identified are sitosterol, campesterol, stigmasterol, and avenasterol (Matthaus and Ozcan 2011). Steroidal glycosides of new and known structures havebeenisolatedfromN.sativaseedswhichinclude3-O-[β-D-­xylopyranosyl-(1 → 2)-α-Lrhamnopyranosyl-(1  →  2)-β-Dglucopyranosyl]-11-­methoxy-­16,23-dihydroxy-28methylolean-12-enoate, stigma-5,22-dien-3-β-D-­glucopyranoside, and 3-O-[β-D-xylopyranosyl-(1  →  3)-α-L-rhamnopyranosyl-(1  →  4)-β-D-glucopyranosyl]-11-methoxy-16-hydroxy-17-acetoxy hederagenin (Mehta et al. 2009). Vitamins like tocopherols in black cumin exhibit strong free radical scavenging potentials thereby terminating lipids peroxidation (Zaunschirm et al. 2018). The total tocopherol contents of black seed oil reported in varying quantities from diverse sources ranged from 9.15 to 27.9 mg/100 g. Among the foremost tocopherols recognized in black cumin seeds, α, β, and γ forms and tocotrienol are well recognized (Matthaus and Ozcan 2011). Moreover, alkaloids of diverse types have been isolated from the seeds of black cumin, which include novel dolabellane-type diterpene alkaloids: nigellamines A1, A2, B1, and B2 and nigellamines A3, A4, A5, and C (Morikawa et al. 2004) possessing lipid metabolizing property, and indazole class of alkaloids: nigellidine, nigellicine and nigellidine-4-Osulfite (Ali et al. 2008).

3  Extraction and Processing of Nigella sativa Seed Oils Essential oils are aromatic oily liquids which are secondary metabolites of plants. These oils originated from aromatic plants that may be found in all plant organs or leaves, fruits, peel, fruit stem or seeds (Bakkali et al. 2008). There is a wide area of use of essential oils in the pharmaceutical industry, the cosmetic industry as well as

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in the chemical industry (Raeissi and Peters 2005). Nigella seed oil is considered as one of the newer sources of edible oils that have a key role in human nutrition and health (Cheikh Rouhou et al. 2006). Nigella seed oil has been reported to have protective and curative actions (Salem and Hossain 2000). The black cumin seed oil contains high levels of bioactive compounds including phytosterols, high contents of total tocochromanols, β- and γ-tocotrienol and γ-tocopherol, and essential fatty acids (Rudzinska et al. 2016). A great deal of attention has been focused on Nigella seed oil, thus its consumption has increased. Various methods have been employed for oil extraction from the native seeds of black cumin (Babar et al. 2018) such as petroleum ether extraction, n-hexane extraction (Mai Lea et al. 2004), ethanol extraction (Hosseinzadeh et al. 2013), hydrodistillation extraction (Benkaci-Ali et  al. 2007), electric fields/microwave extraction (Bakhshabadi et  al. 2017a, b), supercritical carbon dioxide (SCO2) extraction (Machmudah et al. 2005) and cold pressing extraction (Cheikh-Rouhou et al. 2007). Each of these methods has their characteristic advantages but various disadvantages exist, such as the use of toxic and hazardous solvents, low extraction yields, application of heat in some cases which can compromise the integrity of extracts, the use of costly apparatus and time-consuming methodologies (Kiralan et al. 2014; Soto et al. 2007).

3.1  Solvent Extraction The Soxhlet extraction method is the most generally used and can be considered as the optimum process for Nigella seed oil extraction since the method enhances the yield and quality. The Soxhlet extraction offers many advantages, such as it increases the yield and the extracted oil has a higher percentage of unsaturated fatty acid and a higher n-6/n-3 ratio. In solvent extraction, the oil has been usually produced by a hot solvent extraction method at 40–60  °C (D’Antuono et  al. 2002) and even at 70  °C (Ramadan and Morsel 2004), using the Soxhlet extractor. The hot solvent extraction method could affect the oil properties and may induce partial alteration of the majority of minor constituents that have many functional, antioxidative and pro-oxidative effects (Espin et al. 2000; Koski et al. 2002). In a study, Khoddami et  al. (2011) compared the Soxhlet extraction method with Modified Bligh-Dyer method and n-hexane extraction and found that the Soxhlet extraction method is the best method for extracting oil from Nigella seed.

3.2  Dimethyl Ether Extraction Dimethyl ether (DME) has received much attention as an alternative fuel due to several factors: high cetane value, clean-burning, non-toxic, environmentally friendly, efficient combustion, relatively inexpensive, and potentially renewable

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(Arcoumanis et  al. 2008; Semelsberger et  al. 2006). Unlike similar ethers, DME does not form peroxides on standing (Arcoumanis et al. 2008). Furthermore, observation studies based on inhalation indicated that DME is of low toxicity potential. As an extraction solvent, the residual DME present in food products is expected to be significantly below the lowest no-effect limit and in some cases below the detection limit which makes the extracted material potentially safe for consumption (Anadon et al. 2009). In a study by Subratti et al. (2019), using DME, higher yields of oil was obtained as compared to conventional solvents with thymol being present in a considerable amount, along with the typical fatty acids. Both ground and whole seeds were used for the extraction, with the former yielding a higher quantity of oil. Furthermore, the extraction process is simple, cost-effective, and allows for easy recovery of the DME.

3.3  Cold Press Extraction Demand for oil production by cold pressing is increasing as it produces high nutritional value oil free from residues of solvents. Cold pressed oils do not need refining steps; therefore, they may contain high levels of beneficial phytochemicals and natural antioxidants such as tocols and phenolic compounds (Kiralan et al. 2014; Uquiche et al. 2008). There are different ways to obtain high-quality oils by cold press using different pre-treatments as well (Fathi-Achachlouei et  al. 2019). In a recent study, Mazaheri et al. (2019) reported that blending of black cumin seed and sunflower seed before cold pressing resulted in an improvement in the extraction yields, phenolic contents, amount of pigments, and oxidative stability in the resulting oil mixtures.

3.4  Steam Distillation Extraction The steam distillation is a method for essential oil extraction. It is cheap, flexible, and versatile and does not lead to the decomposition of the essential oil and has the potential for commercialization due to its reliability in producing mass oil production. But it is appropriate to improve the traditional distillation method because of the energy-wasting. The extraction using traditional steam distillation method cannot give the highest purity and quality of Nigella sativa essential oil (Milojevic et al. 2008; Amenaghawon et al. 2014). Steam distillation is the primary method in the essential oil industry to extract the essential oil from herbs, spices, medicinal and aromatic plants for the commercial product (Nickavar et al. 2003; Stoyanova et al. 2003; Milojevic et al. 2008; Amenaghawon et al. 2014). Recently, a study was conducted by Zelelew and Gebremariam (2018) who found that the cumin seeds extracted by steam distillation could give the maximum yields of essential oil at the optimum condition of temperature and time. Antimicrobial activity of the seed

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essential oil was also showed that the seed extract was bacteriostatic at lower concentrations and bactericidal at higher concentrations.

3.5  Microwave-assisted Extraction The microwave-assisted extraction (MAE) method has been defined as an effective tool for the extraction of essential oil (Petrakis et al. 2014). The MAE method is mainly based on heat generation inside the plant and the subsequent increase of inner pressure, causing cellular breakdown and essential oil liberation (Khalili et al. 2018). It offers many advantages, including rapid energy transfer, effective heating, short time extraction, high yield extraction, organic solvent-free, high recovery of bioactive compounds and low operating costs (Gavahian et  al. 2015). MAE has already been used for extraction of essential oil from several aromatic plants in pharmaceutical and food processing, with a high potential for future applications (Qu et al. 2013; Petrakis et al. 2014). Abedi et al. (2017) reported that the MAE method was a viable alternative to HD for the essential oil extraction from Nigella sativa seeds due to the excellent extraction efficiency, higher TQ content, and stronger antioxidant activity.

3.6  Supercritical Fluid Extraction (SFE) Supercritical fluid extraction (SFE) is a quick and selective technique for the extraction of essential oils from aromatic plants especially Nigella sativa (Piras et  al. 2013). The extraction of essential oil components using solvent at high pressure, or SCF, has received significant attention especially in food, pharmaceutical, and cosmetic industries, because it presents an alternative to conventional processes such as organic solvent extraction and steam distillation (Doraiswamy et al. 1999; Eikani et al. 1999). SCF allows a continuous modification of solvent power and selectivity by changing the solvent density (Pourmortazavi et al. 2003). In a study, selective extraction of Nigella sativa seeds with SC-CO2 was investigated to determine the variation in oil composition and to achieve an extract with lower free fatty acid content (Türkay et  al. 1996). In Fullana et  al. (2000) study, an empirical kinetic model was developed for the extraction of Nigella sativa seed oil with supercritical carbon dioxide as a solvent to achieve high yield of the extract. Antioxidant activity of Nigella sativa extract was performed by Machmudah et al. (2005). The SC-CO2 extraction of Nigella sativa oil at different experimental conditions of pressure, temperature and time revealed that the yield, antioxidant capacity, and thymoquinone quantity could vary with the changes in the operating conditions which show that they had significantly influenced the extract yield and composition. Furthermore, Solati et al. (2012) reported that high pressure was recognized to be beneficial for an extract with high yield and antioxidant activity while temperature has shown a

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varying effect and was dependent on the balance between the SC-CO2 density and solute volatility.

4  Traditional Therapeutic Uses of Nigella sativa Seeds Black cumin has been traditionally used as a potential therapeutic agent and a remedy for various diseases. The seeds are believed to be effective against various types of disorders like cough, bronchitis, asthma, chronic headache, migraine, dizziness, chest congestion, dysmenorrheal, obesity, diabetes, paralysis, hemiplegia, back pain, infection, inflammation, rheumatism, hypertension, and gastrointestinal problems such as dyspepsia, flatulence, dysentery, and diarrhea. It has also been used as a stimulant, diuretic, emmenagogue, lactagogue, anthelmintic and carminative as well as it is applied to abscesses, nasal ulcers, orchitis, eczema and swollen joints (Tariq 2008). Black cumin seed oil is used as a local anesthetic as well (Sharma et al. 2005).

5  Pharmacological Activities of Nigella sativa Seeds Various studies have reported that black cumin possesses several medicinal properties and pharmacological actions (Ahmad and Beg 2013). The signficant biological potential and curative properties of black cumin seed confer its application globally. Its beneficial effects on health, especially against diseases such as cancer, diabetes, and cardiovascular disease make it a potential remedy for such chronic disorders (Bamosa 2015; Entok et al. 2014). Table 13.2 presents the main health-promoting and pharmacological activities of Nigella sativa seeds.

6  Food Processing Applications of Nigella sativa Seeds An important issue in the food industry is to preserve food from degradation, mainly by oxidation processes or by a microorganism, during processing, storage, and marketing. The food industry uses synthetic additives, which diminish microbial growth and delay the oxidation of oxidizable materials, such as lipids. However, owing to the economic impact of spoiled foods and consumers’ growing concerns over the safety of foods containing synthetic antioxidants, much attention has been paid to natural bioactive compounds (Viuda-Marto et al. 2011). Nigella sativa seeds have been used for years as a spice and food preservative. Black cumin seeds have been added as a spice to a variety of Persian foods such as yogurt, pickles, sauces, and salads (Venkatachallam et al. 2010). The seeds are used extensively as a spice for flavouring purposes, especially bakery products and cheese. Seeds are used in the

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Table 13.2  Pharmacological activities of Nigella sativa seed Activity Antioxidant activity

Responsible compound(s) Thymoquinone, carvacrol, t-anethole, and 4-terpineol, flavonoids, alkaloids, tannins

Anti-diabetic activity

Thymoquinone

Anti-obesity activity

Thymoquinone

Anti-cancerous activity

Thymoquinone, saponins

Anti-­ hyperlipidemic activity

Anti-­ hypertensive activity

Thymoquinone

Thymoquinone

Impact/Influence/Effects Reduces blood LPO, increases plasma total thiol molecules (TTM). Total anti-oxidant capacity increases, serum and tissue malondialdehyde reduces. Activities of CAT, glutathione peroxidase, and SOD decreases. Prevents gluconeogenesis, reduces the expression of gluconeogenic enzymes and production of hepatic glucose, restores the normal insulin concentrations. Decrease in food intake and body weight, improvements in lipid peroxidation and insulin sensitivity. Increases the macrophage cell number and activation results in death of cancerous cells. Upregulates the expression of cleaved caspase-3 and SMAC in SaOS-2 cells. Decrease in total cholesterol, low density lipoprotein cholesterol and triglyceride, increases high density lipoprotein cholesterol. Reduces the level of hydroxy methyl glutaryl (HMG-CoA) reductase enzyme, stimulating paraoxonase enzyme (PON1). Reduction in blood pressure and hypertension due to antioxidant properties, calcium-channel blockage, and diuretic and hypotensive (soothing heartbeat) functions.

References Awadalla (2012)

Nagi and Mansour (2000) Bayrak et al. (2008) Heshmati and Namazi (2015)

Le et al. (2004)

Darakhshan et al. (2015) Ziaei et al. (2012) Boskabady et al. (2010)

Ahmad and Beg (2013)

Keyhanmanesh et al. (2014)

(continued)

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Table 13.2 (continued) Activity Responsible compound(s) Impact/Influence/Effects References Neuro-protective Thymoquinone, flavonoids Decreases the number of dead Burits and Bucar (2000) activity hippocampal neuronal cells and elevated levels of malondialdehyde. Increases the peripheral blood Benhaddou-­ lymphocytes number. Andaloussi et al. (2010) Modulates critical neuronal Sahak et al. signalling paths. (2016) El-Mahmoudy Thymoquinone Decreases the synthesis of Anti-­ et al. (2002) MCP-1, TNF-alpha, inflammatory interleukin (IL)-1beta and activity cox-2. Inhibition of arachidonic acid Mansour and Tornhamre formation by blocking both (2004) cyclooxygenase and lipoxygenase enzymes. Abdallah (2017) Anti-microbial Thymohydroquinone, Inhibits the growth of activity thymol gram-positive and gram-­ negative bacteria and bacterial biofilm formation. Anti-parasitic Thymoquinone Schistosomicidal properties Assi et al. activity against Schistosoma mansoni. (2016) El-Abhar et al. Gastroprotective Thymoquinone Prevents the appearance of (2003) activity diarrhoea, reduces body weight loss and increases glutathione levels. Parandin et al. Male fertility Thymoquinone Increases in testes and (2012) epididymidis weight, sperm count, blood testosterone concentration, LH and fertility index

preparation of a traditional sweet dish and eaten with honey and syrup as well as for sprinkling on bread (Hamrouni-sellami et al. 2008). Seeds are of importance as a carminative; often they are used as a condiment in bread and other dishes (Burits and Bucar 2000; Ramadan 2007). Volatile and fixed Nigella sativa seeds oil is used worldwide for functional foods and nutraceuticals. Black seed oil have antioxidative properties as it is a rich source of antioxidative components such as phenolic compounds and tocols. There is a high correlation between total phenolic compounds (TPC) and oxidative stability, which causes high resistance to auto-oxidation. TPC is the most important factor for evaluating the quality of oil because TPC have been related to the shelf life, sensorial quality and mainly oxidation resistance of the oil (Asdadi et al. 2014). The black seed oil has been used to improve other vegetable oils’ oxidative stability. For example, black seed oil was added to sunflower oil, and the results showed that sunflower oil

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oxidative stability was improved due to the increased content of TQ and tocopherols (Kiralan et  al. 2016). The peroxide value changes in the individual oils and oil blends showed that the addition of black seed oil to sunflower oil resulted in a significant decrease in peroxide value during storage at room temperature. Peroxide value of sunflower oil reached 166.1 (meq O2/kg oil), while black seed oil had a peroxide value of 33.08 (meq O2/kg oil) after 6  months of storage (Kiralan et al. 2016). Black seed oil could be considered for multi-purpose uses in cosmetic, industrial and pharmaceutical products. For one, they can defend against UV rays, so they apply to such products (Cheikh-Rouhou et  al. 2007). Black seed oil initially has relatively high peroxide value; however, it is stable and its peroxide value does not increase very rapidly during heat treatment and storage. This might be related to its remarkably high polyphenolic content. It should be kept in mind that black seed oil should be refined for edible application due to its initial high peroxide value (Cheikh-Rouhou et  al. 2007). Black seed oil constituents have been utilized for nutritional, supplemental, and functional cosmetics (Kiralan et  al. 2014). Nigella sativa seeds are widely used in Indian cuisines, particularly in mildly braised lamb dishes such as korma. The seeds are added to vegetable and dhal (lentil) dishes as well as in chutneys. The seeds are sprinkled onto naan bread before baking. Although it is the oil that most often used medicinally, the seeds are a bit spicy and are often used whole in cooking curries, pastries, and Mediterranean cheeses. Nigella sativa seed has very little aroma but is carminative, meaning they tend to aid digestion and relieve gases in the stomach and intestines. Nigella seeds are used as a spice and condiment worldwide both in whole and ground form and occasionally in Europe as both a pepper substitute and a spice. In the Middle East Nigella is added to bread dough and is an essential constituent of the Middle East choereg rolls. The dried seeds of Nigella are the major commercial product being used in foods, pickles, baked goods, confectionery, pharmaceutical, and perfumery industries. Owing to antimicrobial activity, it is used in various dairy products like cheeses to preserve its quality and make it microbiologically (Abdallah 2017). It is also used as a preservative in fermented vegetable-based products like pickles.

7  Conclusion The complex synergy of over a hundred chemicals contained in black seed work together to enhance and strengthen the body’s immune system in a way no other singular, naturally occurring substance has ever been known to do. The Nigella plant has been regarded as an effective medicinal and edible herb worldwide, displaying the considerable commercial value in the pharmaceutical and food industries. The plant composition and medicinal properties necessitate further research on other useful and unknown features, so it can be used as a plant-derived medicine to treat various diseases.

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Chapter 14

Nutraceutical Importance and Applications of Nigella sativa Seed Flour Jasmeet Kour, Adil Gani, Vishal Sharma, and Sajad Ahmad Sofi

Abstract  The concept of nutraceutical foods has emerged as a result of several research intercessions. Nigella sativa, commonly known as black cumin, a member of the Ranunculaceae family has widespread abundance across the globe especially in Eastern Europe and West Asia. Out of plants of medicinal importance, it has one of the richest histories since it has been used in the form of medicine having herbal origin by several civilizations. The composition of black cumin seed depends on several factors primarily geographic distribution, harvesting time and agronomic patterns adopted as well. The seeds have been reported to exert positive and beneficial effects on lowering serum lipid profile, triglycerides level and enhancing high-­ density lipoprotein levels. The significance of this seed is also attributed to thymoquinone which is present to a level of 25% in the seed oil. Black cumin is comprising mainly of proteins, carbohydrates, oil in addition to crude fibre and minerals. Iron, phosphorus, and calcium have been reported to be at high levels while calcium, magnesium, zinc, copper, and manganese have been reported in lower amounts. Studies have supported the inclusion of black cumin and its bioactive components daily for the overall improvement of health. Nigella sativa seed has been one of the most important antidiabetic plants highly recommended by traditional practitioners. Crude and purified components of Nigella sativa seeds have been known to impart manifold pharmacological effects including antihypertensive, J. Kour (*) Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Longowal, Punjab, India A. Gani Department of Food Science and Technology, University of Kashmir, Srinagar, Jammu and Kashmir, India V. Sharma Department of Industries and Commerce, District Industries Centre, Kathua, Jammu and Kashmir, India S. A. Sofi Division of Food Science and Technology, Sher-e-Kashmir University of Agricultural Science & Technology, Srinagar, Jammu and Kashmir, India © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_14

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hypoglycemic, antifungal, anti-inflammatory, and immune strengthening. Nigella sativa seed components have been also used in the production of functional c­ osmetic and dietary supplements as well. This chapter reports on the nutraceutical uses and applications of Nigella sativa seed flour. Keywords  Acute lymphoblastic leukemia · Blood glucose · HbA1c · HOMA-IR · Nutraceutical importance · Food applications

Abbreviations ALL Acute lymphoblastic leukemia BCS Black cumin seed BWS Buckwheat seed DM Diabetes mellitus DW Dry weight FBG Fasting blood glucose FDA Food and Drug Administration GAE Gallic acid equivalent HbA1c Glycosylated hemoglobin HDL High-density lipoprotein HOMA-IR Homeostatic model assessment LDL Low-density lipoprotein cholesterol MDA Malondialdehyde NS Nigella sativa SOD Superoxide dismutase STZ Streptozotocin TC Total Cholesterol TG Triglyceride TQ Thymoquinone VLDL Very low-density lipoprotein WHO World Health Organization

1  Black Cumin Seeds Out of all medicinal seeds, black cumin (Nigella sativa L.) is considered to be the most significant one. Egypt holds the privilege to produce the best of the black cumin seeds owing to the optimum conditions in oases for watering till the development of the seed pods. In addition, Black cumin seed is mentioned in the Bible as

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well as in the words of the Prophet Mohammed. Nigella sativa is grown widely prominently in the Mediterranean regions, Middle East, Southern Europe, India, Pakistan, Syria, Saudi Arabia, and Turkey (Khare 2004). These seeds are grown on a widespread basis across the globe and have been known to enhance health for countries especially in the Middle East and Southeast Asia (Ahmad et  al. 2013). N. sativa belonging to the Ranunculaceae family has been able to evolve as a miraculous herb blessed with an enriched religious background due to a wide spectrum of pharmacological attributes (Ahmad et  al. 2013). The seeds are used on a global basis due to their safe, non-toxic, easy availability and effectiveness in comparison with the allopathic medicines (Mills and Bone 2000). It is a small shrub having green leaves bearing rosaceous white and purplish flowers (Aljabre et al. 2015) and the fruit is tiny dark black, whereas the seeds are small and black with a scented odour having a pungent feel and a crunchy texture (Mamun and Absar 2018). These are black and white on the outer and inner side, respectively along with small size with dimensions of 2–3.5 mm by 1–2 mm (Mukhtar et al. 2019). Nigella sativa played a significant role in ancient Egypt which described it as a remedy for various problems and 37 diseases (Ghosheh et al. 1998). Nigella sativa is a flowering plant growing up to 20–30 cm in height and the plantation of this plant is mainly done in Asia and the Middle East (Khare 2004). It has linear leaves with fine divisions up to 2–3 cm long bearing pale blue and white-coloured flowers along with 5–10 delicate petals (El-Hack et  al. 2016). This plant belongs to the Ranunculaceae family which encompasses multiple species, among which Nigella sativa is thoroughly investigated due to its therapeutic value (Aggarwal et al. 2008). The active principles reported in these seeds include thymoquinone, nigellimine-N-­ oxide, nigellicine, nigellidine, nigellone, dithymoquinone, thymohydroquinone, thymol, arvacrol, 6-methoxy-coumarin, 7-hydroxy-coumarin, Oxy-coumarin, alpha-hedrin, steryl-glucoside, tannins, and flavonoids (Sudhir et  al. 2016). The seeds have shown many biological properties which mainly included antiparasitic, antidiabetic and anticancer ones (Padhye et al. 2008). Thymoquinone (TQ), thymohydroquinone, dithymoquinone, thymol, and carvacrol are the principal active components which are pharmacologically also active ingredients (Nasir et  al. 2005). Thymoquinone, produced in 1959 exerts several biological effects and accounted for 18.4%–24.0% of volatile oil (Yuncu et al. 2013). Nigella sativa possesses potent antioxidative properties (Salem 2005). It is one of the most nutrient-rich herbs in history across the globe and various scientific studies are validating the traditionally claimed uses of a small seed of this species (Takruri and Dameh 1998; Ramadan 2007). Black cumin seeds have been also known to exhibit anticancerous, anti-diabetic, anti-inflammatory, antimicrobial and hypotensive characteristics (Ali and Blunden 2003). The seeds have been also acknowledged as a drug used traditionally acting as diuretic and antihypertensive (Zaoui et al. 2000). There is an ample amount of work cited in literature which have explored black cumin as a potent appetite stimulant (Gilani et al. 2004), analgesic (Khan et  al. 1999), antidiarrheal (Gilani et  al. 2001), and anthelmintic as well (Chowdhury et al. 1998).

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Nigella sativa seeds are carminative and relieve gases in the stomach and intestines. The biological activity of the seeds has been attributed to the presence of TQ which is the major component of the essential oil. The therapeutic activity of the seeds has been related to the presence of antioxidants primarily TQ, carvacrol, p-cymene, and 4-terpinol. The plant is known to all Arabian and Islamic countries and carries various colloquial names. These have shown its broad-spectrum therapeutic and several biological attributes primarily anti-inflammatory, antibacterial, analgesic, anthelmintic, antidiabetic and immunomodulatory as well (Ahmad et al. 2013).

2  Compositional Analysis of Nigella sativa Seeds and Flour The plant has been promoted by the ideological belief in the herb as a cure or multiple diseases. Consequently, black cumin has been extensively studied, which justifies its broad traditional therapeutic value. It was in the year 1880 when the first chemical analysis on the black seeds was attempted with the first report published depicting these seeds to comprise 37% oil and 4.1% ash (Greenish 1880). The seeds contain various bioactive components, the most important ones are thymoquinones. The seeds also possess numerous pharmacological properties due to the presence of various constituents among them are thymoquinones, thymol, nigellicine, nigellidine, dithymoquinone, and carvacrol (Shakeri et al. 2018). Seeds are nutritionally rich with plenty of lipids, protein, dietary fibre, vitamins B and E, minerals prominently iron (Srinivasan 2018). The seeds as well as the oil have been a great focus of research and thoroughly investigated due to its unique chemical composition and biological activities. The prominent volatile ingredients in black cumin seed are cuminaldehyde, cymene, and terpenoids (Bettaieb et  al. 2011). The unmatchable nutritional composition of black cumin seeds imparts a typical pungent flavor and promotes their utilization in bread, pickles, and coffee (Javed et al. 2012). Various studies regarding the chemical composition and properties of black cumin seeds have shown that the seeds are credited with the development of enriched food products and require clear criteria for standardization. It was first identified and described by Linnaeus in 1753. Babayan et al. (1978) discovered that Nigella sativa seeds are composed of protein, lipids, carbohydrates, crude fiber and ash to a level of 20–27%, 34.5–38.7%, 23.5–33.2%, 8.4% and 4.8%, respectively. Menounos et al. (1986) reported that the seeds containing esters of fatty acids, free sterols, and steryl esters. The active component comprises the volatile oil having an unsaturated ketone, limonene also called carvene, pinene, and p-cymene (Kapoor 1990). Black cumin seed has a wide variety of chemical constituents, including abundant sources of all the essential fatty acids (Ramadan and Morsel 2002). Proximate analysis of black cumin seeds reported these seeds to be a good source of fibre, carbohydrate, protein, and lipids with pH 5.6. Takruri and Dameh (1998) reported it as a great source of lipids, minerals, crude fibre, and pivotal vitamins as well (Ahmad et al. 2013). Babayan et al. (1978) reported the seeds to contain crude

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protein, lipids, and carbohydrates to a level of 20–27%, 34.5–38.7% and 23.5–33.2%, respectively. The seeds are blessed with 30% oil and volatile oil containing TQ and monoterpenes such as p-cymene and α-piene (El-Kadi and Kandil 1987). Thymoquinone is also reported to be present at a concentration of 52.6 mg/100 g, and 20.13 mg/100 g (Tufek et al. 2015). Black cumin is regarded as an unconventional oilseed, contains proteins, carbohydrates, and fixed oil and volatile oils, alkaloids, saponins, crude fiber, as well as minerals, such as calcium, iron, sodium, and potassium (Gali-Muhtasib et al. 2006). Based on the region, the seeds contain volatile and non-volatile oils, protein, carbohydrates, fiber, alkaloids, tannins, saponins and minerals such as iron, calcium, potassium, magnesium, zinc, and copper along with prominent vitamins such as vitamin A and C, B1, niacin, pyridoxine and folic acid (Gullu and Gülcan 2013). Jan et  al. (2019) investigated the changes in compositional analysis of black cumin seed flour due to roasting (Table 14.1). The moisture content of native black cumin seed flour came out to be 7.66% and there was no significant difference between the moisture content of native, pan-roasted and microwave-roasted seed flour. The lipids content of seed flour came out to be high (21.5%). Ash content was also reported to be in fairly high (6.50%) which indicated a significant amount of mineral content in black cumin seed flour. Protein content was also detected at a significant level (17.1%). Mamun and Absar (2018) reported the nutritional composition of black cumin seed flour prepared from milling black cumin seeds of Bangladesh origin. The pH of flour came out to be 5.63. The moisture, ash, total carbohydrate, starch, total protein, and total lipid contents were 5.5%, 4.69%, 29.1%, 2.55%, 18.0%, and 32.7%, respectively. Lipid content was found to be the highest followed by total carbohydrate content and total protein content. The total protein content being 18.0% was found to be lower than Saudi, Turkey, Iranian and Tunisian seed flour. Regarding the mineral analysis, calcium was found in maximum concentration (579.3 mg/100 g). This was followed by potassium (510 mg/100 g). Iron, magnesium, phosphorus, and sodium were found at the level of 41.8  mg/100  g, Table 14.1  Effect of roasting on the composition of black cumin seed flour Parameter Moisture Lipid Protein Ash Tapped density Bulk density Colour a L b Source: Jan et al. (2019)

Native 7.66 21.5 17.1 6.50 0.55 0.47

Pan-roasted 7.50 22.0 16.9 6.00 0.54 0.43

Microwave-roasted 7.16 22.0 17.0 7.50 0.56 0.43

3.156 5.436 36.36

1.726 6.520 39.36

2.546 16.65 53.01

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218.3  mg/100  g, 91.5  mg/100  g, and 100  mg/100  g, respectively. The secondary metabolites such as alkaloids, flavonoids, saponins, and tannins detected in black cumin seed flour were present to a level of 10.1  mg/100  g, 3.78  mg/100  g, 7.58 mg/100 g, and 2.21 mg/100 g, respectively. Suri et al. (2019) studied the effect of soaking and germination on the compositional analysis of black cumin seed flour. The moisture content of native or unsoaked/ ungerminated seed flour came out to be 8.14%. With soaking and germination, there was an increase in the moisture content due to the rupture in the cell wall of seed leading to absorption of moisture which is well in line with the findings of Handa et al. (2017). Ash content was 2.72% which was higher than the one reported for soaked or germinated flour. It was related to the leaching of minerals in the soaked water during the soaking process. Carbohydrate content of native flour was reported as 39.5% but it decreased with the soaking and germination process which was attributed to the breakdown of carbohydrates. Native black cumin seed flour exhibited protein content of 21.6% which increased with soaking and germination due to the increase in the protease activity during germination. A decrease in phytic acid content and an increase in overall acceptability was also observed in black cumin seed flour with soaking/germination. The phenolic content of native seed flour came out to be 154.8 GAE/100 g DW, whereas overall acceptability was reported as 4.2.

3  Applications of Black Cumin Seed Flour N. sativa has great importance mainly in Islamic countries owing to its numerous benefits (Razavi and Hosseinzadeh 2014). Black cumin is one of the well-known spices as well as a flavouring agent in various food products such as in bread, pickles, yogurt, and sauces. Black cumin seed flour is also known as one of the functional ingredients in various food formulations. It has also a special place in the medical world. The potential of black cumin seed flour for the preparation of various foods scope for its utilization as an added ingredient in the formulation of various functional foods was efficiently explored by Jan et al. (2019). Black seeds are also used as a food additive in the form of flavourings in the bread and pickles owing to their low level of toxicity (Al-Ali et al. 2008). Nigella sativa finds its traditional utilization in many forms such as powder and oil (Heshmati and Namazi 2015). Black cumin seed flour has the potential of increasing the nutritional profile of wheat flour by enhancing fibre, lysine, minerals, and antioxidants (Osman et al. 2015). The water holding capacity of black cumin seed flour can lead to deleterious effects on the water absorption, dough mixing, rheological properties, loaf volume, crumb texture and colour of the finished products (Shelton and D’Appolonia 1985). Black cumin seeds and its processed products such as butter and semi-skimmed flour are embedded with high nutrition and market value as well (Egorova et  al. 2018). It is a thoroughly studied prominent medicinal ingredient, yet its utilization in cosmetics is still under consideration (Awad et al. 2013). This plant is no less than

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a miracle due to its rich history and religious beliefs (Ahmad and Beg 2013). Black cumin usually used as a spice is the most widely used type in agriculture and trade (Yakup 2007). The seeds have long been used in folk medicine in the Middle and the Far East as a traditional medicine for a wide range of illnesses.

4  Applications of Black Cumin Seeds and Seed Flour World Health Organization (WHO) recommends black cumin seed as an herbal remedy (Hussain and Hussain 2016). Several efforts have been made to investigate the utilization of BCS (black cumin seeds) in the form of a natural feed additive to poultry diets for better performance. Hermes et  al. (2009) reported no adverse effects of black cumin seed flour (up to 30  g/kg) on the performance of poultry wherein black cumin seed flour could be utilized to overcome the deleterious effects of hot climatic conditions. Zeweil et al. (2008) reported an increase in plasma total protein, albumin, and globulin with the addition of Nigella sativa to rabbit diets. A major protection mechanism on the food additive induced cardiac disorder was reported by the consumption of black cumin seed flour (4% w/w) and/or an aqueous solution of bees’ honey (2.5 g/kg/day) in rats (El-Kholy et al. 2007). N. sativa (800 mg/kg) incorporation along with exercise (on treadmill, 2 h/day) showed a synergistic effect as N. sativa exercise-induced cardiac hypertrophy led to a reduction in heart rate and well-­ matched electrical activity of the heart to its mass (Al-Asoom et al. 2014). The oral administration of N. sativa (800 mg/kg) increased contractile and vascular functions and reduced oxidative stress in the cardiac tissue. Dogar et al. (2009) confirmed the safe utilization of N. sativa seed flour when children suffering from acute lymphoblastic leukemia (ALL) were fed with N. sativa powder (40 mg/kg) in two doses for a period of 3 months in combination with conventional therapy. It was observed that the side effects produced were lower in comparison with the ones produced by L-asparaginase and medications in the form of daunorubicin, vincristine, and prednisolone. Black cumin seed flour could be an excellent substitute for L-asparaginase, thus proved out to be beneficial as an anti-­ cancerous agent. Al-Naqeep et al. (2011) studied the antiatherogenic effects of black cumin seed flour (1000 mg/kg) and oil (500 mg/kg) on atherosclerosis in rabbits affected by hypercholesterolemia induced by diet. There was a significant reduction in fat deposition in the arterial wall, total cholesterol as well as low-density lipoprotein whereas an increase in high-density lipoprotein was observed. Plague formation reduced considerably as well. Another study confirmed the anticholesterolemic effect of black cumin seed flour was done by Fatima et al. (2007). N. sativa seeds proved to be immensely helpful in curbing common causes of hypothyroidism i.e. Hashimoto’s thyroiditis. Capsules filled with 1.0 g of N. sativa powder were administered to patients two times a day for 8  weeks. There was a noticeable improvement in thyroid functions as well as in body weight, body mass

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index in comparison to the control group. The serum concentration of thyroid-­ stimulating hormone and anti-thyroid peroxidase also decreased. Hence, black cumin flour helped in improving serum lipid profile which made it a significant herbal treatment apart from medication in curing Hashimoto’s thyroiditis related metabolic abnormalities (Farhangi et al. 2016). El Kadi et al. (1990) reported the increase in the ratio of T-lymphocytes helper cells to T-suppressor cells by 72% along with an increase in the number and functions of T-killer cells when humans were fed with whole seed flour at a dosage of 1 g twice a day for 4 weeks. Akhtar and Riffat (1991) analysed the anticestodals action of N. sativa seed flour (40  mg/kg body weight) on children infected with cestodes and reported that black cumin seed flour at a dosage of 40 mg/kg led to a reduction in the number of fecal eggs of parasites such as Ascaris lumbricoides and Taenia saginata in infected people. In another study, black cumin seed flour at a concentration of 60–100  μg/mL led to the significant killing of adult male and female worms (Mohamed et al. 2005). Islam et al. (2016) analysed the effects of supplementing different levels of buckwheat seed (BWS) with black cumin seed (BCS) on the performance, serum lipid profile and intestinal bacterial flora (Escherichia coli, Salmonella, and Lactobacillus sp.) in broiler chicks. Cobb-500 broiler chicks were divided into 4 groups designated as T1 (control); T2 (10% BWS + 1.5% BCS), T3 (20% BWS + 2.5% BCS) and T4 (30% BWS  +  3.5% BCS). There was a significant increase in the final body weight gain of group T2 as compared to the control group. There was no improvement in the growth performance of the chicks. A significant reduction in serum cholesterol and triglyceride concentrations and an increase in HDL-cholesterol concentration was observed. In one of the significant clinical trials, a supplement of N. sativa seed flour (1 g/day) was reported to be beneficial in improving biochemical parameters such as glucose level in blood and lipid profile in menopausal women without affecting their body weights (Ibrahim et al. 2014). Akhtar et al. (2003) studied the effect of N. sativa seed flour on the performance of layers and cholesterol content of egg yolks. Black cumin seed flour was substituted in commercial layer ration at different levels (0.0, 0.5, 1.0 and 1.5%). Twelve experimental units were made out of which 3 units were subjected to each treatment. Two birds were selected randomly and blood samples were collected at periodic intervals of 0, 6 and 12  weeks. With the incorporation of black cumin seed flour, there was an increase in the production of eggs, the mass of eggs, thickness of egg-shell and Haugh unit. There was a reduction in serum triglycerides, low-density lipoprotein, total cholesterol, while an increase in serum high-density lipoprotein cholesterol level was observed. An increase in egg production was related to the inclusion of active ingredients and nutrients present in the black cumin seed flour which enhanced the health and reproductive performance of birds. Similarly increase in the egg mass was also related to the presence of those very nutritional ingredients in the seeds which led to enhanced reproductive performance. The decrease in mortality was also attributed to black cumin seed flour substitution which acted like an antibiotic and a vaccine to boost immunity thereby reducing mortality in poultry birds.

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Guler et al. (2006) incorporated black cumin seeds at 0.5%, 1%, 2% or 3% and avilamycin at 10 mg/kg to the basal diet and feed intake, weight gain on daily basis, feed conversion ratio and carcass characteristics. Daily feed intake remained unchanged after 21 and 42 days. There was a significant difference in daily weight gain between the treatments. The highest weight gain was seen in birds which were fed with a diet comprising 1% black cumin seeds and antibiotic was the highest average daily gain. Feed conversion ratios were improved significantly by supplementation. Similarly, the highest cold carcass, thigh, breast, wing, neck, and liver weights were observed in the 1% black cumin and antibiotic groups. Individuals with Type 2 DM were given N. sativa powder at a dose of 1, 2 and 3 g/day, wherein significant decreases were observed in insulin resistance and β-cell function indicators such as postprandial blood sugar, HbA1c, HOMA-IR, only in individuals who were supplied with 2 g N. sativa powder per day. The most effective dose was determined to be 2  g and this might also confer positive benefits with hypoglycaemic agents (Bamosa et al. 2010). In another study, Type 2 DM patients were given 2 g/day N. sativa powder for 1 year and it was shown that HbA1c values decreased in the intervention group (Bamosa 2015). Ansari et  al. (2010) treated patients suffering from seasonal allergic rhinitis patients with black cumin seeds (250 mg/day) for 2 weeks without producing any side effects. The administration of black cumin seed flour (1 g/day) for two-months led to a reduction in total glycerides (TG), low-density lipoprotein (LDL-C) and total cholesterol (TC) along with an increase in high-density lipoprotein level (Ibrahim et  al. 2014). The intake of black cumin seed flour (500  mg/day) for 6 months concurrent with a statin (10–20 mg/day) on the lipid profiles in patients with stable coronary artery disease decreased the serum levels of LDL, VLDL, TG, and TC which statin alone could not be decreased (Tasawar et al. 2011). The effect of the incorporation of turmeric (Curcuma Longa) and black cumin seed mixture on selected biochemical parameters of streptozotocin (STZ)-induced diabetic rats was evaluated by experimenting on 21 healthy adult albino rats divided into 3 groups. The third group of rats being diabetic was fed with a mixture of turmeric (0.5 g/kg b.wt.) and black cumin seed (1 g/kg b.wt.) orally by once a day for 6 weeks. The blood glucose level, water, and food intake and an increase in body weight were observed in experimental groups as compared to the control one. No effect was observed on proteins. There was a reduction in hypercholesterolemia and hyperlipidemia observed in STZ-diabetic rats administered with black cumin dietary supplementation (El-Bahr et al. 2014). Another breakthrough in exploring the nutraceutical effect of black cumin seed flour was made by Bilal et al. (2008) who reported a remarkable reduction in TC, LDL-C, TG levels and FBG, whereas HDL-C and the insulin levels increased in patients with type 2 diabetes. Desai et al. (2015) explored the potential of black cumin seed flour/powder in inhibiting MDA (malondialdehyde) and SOD (superoxide dismutase). Eighteen rats were divided into 3 groups. The groups signified negative control, diabetic control and rats treated with black cumin powder (300 mg/kg). Rats were also treated with STZ injection (50 mg/body wt.). The inclusion of black cumin seed flour significantly reduced MDA and SOD levels, whereas in the diabetic group, it increased significantly.

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FDA has recommended the utilization of black cumin seeds in the prevention of disease as well as retarding the aging phenomena (Hussain and Hussain 2016). In nutshell, black cumin seeds and their active constituent (TQ) have been proven to deliver beneficial health effects to combat several ailments including inflammatory and auto-immune disorders (Tavakkoli et al. 2017). Consumption of the seed or its supplementation can be taken as a protocol treatment in the eradication of many diseases such as hyperlipidemia and several metabolic disorders (Al-Naggar et al. 2017).

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Bamosa, A. (2015). A review on the hypoglycemic effect of Nigella sativa and Thymoquinone. Saudi Journal of Medicine & Medical Sciences, 3(1), 2–7. Bamosa, A. O., Kaatabi, H., Lebdaa, F. M., Elq, A. M., & Al-Sultanb, A. (2010). Effect of Nigella sativa seeds on the glycemic control of patients with type 2 diabetes mellitus. Indian Journal of Physiology and Pharmacology, 54(4), 344–354. Bettaieb, I., Bourgou, S., Sriti, J., Msaada, K., Limam, F., & Marzouk, B. (2011). Essential oils and fatty acids composition of Tunisian and Indian cumin (Cuminum cyminum L.) seeds: A comparative study. Journal of the Science of Food and Agriculture, 91, 2100–2107. Bilal, A., Masud, T., & Uppal, A. M. (2008). BS5-5 black seed (Nigella sativa) regulates glucose, insulin level and lipid profile in patients with type 2 diabetes. Diabetes Research and Clinical Practice, 79, 19–20. Chowdhury, A. K. A., Islam, A., Rashid, A., & Ferdous, A. (1998). Therapeutic potential of the volatile oil of Nigella sativa seeds in monkey model with experimental shigellosis. Phytotherapy Research, 12, 361–363. Desai, S. D., Saheb, S. H., Das, K. K., & Haseena, S. (2015). Effect of Nigella sativa seed powder on MDA and SOD levels in Sterptozotocine induced diabetic albino rats. Journal of Pharmaceutical Sciences and Research, 7(4), 206–209. Dogar, M. Z. U. H., Adi, H., Akhtar, M. S., & Sheikh, M. A. (2009). Preliminary assessment of efficacy of Nigella sativa seeds in acute lymphoblastic leukemia in local children. Pharmacology, 2, 769–777. Egorova, E.  Y., Reznichenko, I.  Y., & Ermolaeva, E.  O. (2018). Recycling and standardization aspects of Nigella sativa in the food industry. Advances in Engineering Research, 151, 812–819. El Kadi, M., Kandil, O., & Tabuni, A.  M. (1990). Nigella sativa and cell mediated immunity. Archives of AIDS Research, 1, 232–235. El-Bahr, S. M., Nabil, M. T., Korshom, M. A., & EL-Wahab, A. (2014). Influence of combined administration of turmeric and black seed on selected biochemical parameters of diabetic rats. Alexandria Journal of Veterinary Sciences, 41, 9–27. El-Hack, M. E. A., Alagawany, M., Farag, M. R., Tiwari, R., Karthik, K., & Dhama, K. (2016). Nutritional, healthical and therapeutic efficacy of black cumin (Nigella sativa) in animals, poultry and humans. International Journal of Pharmacology, 12(13), 232–248. El-Kadi, A., & Kandil, O. (1987). The black seed (Nigella sativa) and immunity: Its effect on human T cell subset. Federation Proceedings, 46, 1222. El-Kholy, W. M., Hassan, H. A., & Nour, S. E. (2007). The role of black seed and/or bees honey in modulating the heart disorder induced by food additives in male rats. Egyptian Journal of Hospital Medicine, 28, 327–341. Farhangi, M. A., Dehghan, P., Tajmiri, S., & Abbasi, N. M. (2016). The effects of Nigella sativa on thyroid function, serum endothelia growth factor (VEGF)-1, Nesfatin-1 and anthropometric features in patients with Hashimoto’s thyroiditis: A randomized controlled trial. BMC Complementary and Alternative Medicine, 16, 471. Fatima, S., Khan, N., Naz, L., Yasmeen, G., Hajira, B., & Hussain, Z. (2007). Antiatherogenic effect of Nigella sativa L. (Kalonji) seeds in rabbits with experimentally-induced hypercholesterolemia. International Journal of Biotechnology, 437–441. Gali-Muhtasib, H., Roessner, A., & Schneider-Stock, R. (2006). Thymoquinone: A promising anti-­ cancer drug from natural sources. The International Journal of Biochemistry & Cell Biology, 38, 1249–1253. Ghosheh, A. O., Houdi, A. A., & Crooks, A. P. (1998). High performance liquid chromatographic analysis of the pharmacologically active quinines and related compounds in the oil of the black seed (N. sativa L.). Journal of Pharmaceutical and Biomedical Analysis, 19, 757–762. Gilani, A. H., Aziz, N., Khurram, I. M., Chaudhary, K. S., & Iqbal, A. (2001). Bronchodilator, spasmolytic and calcium antagonist activities of Nigella sativa seeds (Kalonji): A traditional herbal product with multiple medicinal uses. Journal of Pakistan Medical Association, 51, 115–120.

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Nasir, Z., Abid, A. R., Hayat, Z., & Shakoor, H. I. (2005). Effect of kalongi (Nigella sativa) seeds on egg production and quality in white Leghorn layers. The Journal of Animal and Plant Sciences, 15, 22–24. Osman, M. A., Alamri, M. S., Mohamed, A. A., Hussain, S., Gassem, M. A., & Abdel Rahman, I.  E. (2015). Black cumin-fortified flat bread: Formulation, processing, and quality. Quality Assurance and Safety of Crops & Foods, 7(2), 233–238. Padhye, S., Banerjee, S., Ahmad, A., Mohammad, R., & Sarkar, F. H. (2008). From here to eternity-­ the secret of pharaohs: Therapeutic potential of black cumin seeds and beyond. Cancer Ther, 6(b), 495–510. Ramadan, M.  F. (2007). Nutritional value, functional properties and nutraceutical applications of black cumin (Nigella sativa L.): An overview. International Journal of Food Science & Technology, 42(10), 1208–1218. Ramadan, M.  F., & Morsel, J. (2002). Characterization of phospholipid composition of black cumin (Nigella sativa L.) seed oil. Food/Nahrung, 46(4), 240–244. Razavi, B. M., & Hosseinzadeh, H. (2014). A review of the effects of Nigella sativa L. and its constituent, thymoquinone, in metabolic syndrome. Journal of Endocrinological Investigation, 37(11), 1031–1040. https://doi.org/10.1007/s40618-014-0150-1. Salem, M. L. (2005). Immunomodulatory and therapeutic properties of the Nigella sativa L. seed. International Immunopharmacology, 5(13-14), 1749–1770. Shakeri, F., Khazei, M., & Boskbady, M. H. (2018). Cardiovascular effects of Nigella sativa L. and its constituents. Indian Journal of Pharmaceutical Sciences, 80(6), 971–983. Shelton, D. R., & D’Appolonia, B. L. (1985). Carbohydrates functionality in the baking process. Cereal Foods World, 30, 437–442. Srinivasan, K. (2018). Cumin (Cuminum cyminum) and black cumin (Nigella sativa) seeds: Traditional uses, chemical constituents, and nutraceutical effects. Food Quality and Safety, 1–16. Sudhir, S. P., Deshmukh, V. O., & Verma, H. N. (2016). Nigella sativa seed, a novel beauty care ingredient: A review. International Journal of Pharmaceutical Sciences and Research, 7(8), 3185–3196. Suri, S., Kumar, V., Tanwar, B., Goyal, A., & Gat, Y. (2019). Impact of Soaking and Germination Time on Nutritional Composition and Antioxidant Activity of Nigella sativa. Current Research in Nutrition and Food Science, 7(1), 142–149. Takruri, H. R. H., & Dameh, M. A. F. (1998). Study of the nutritional value of black cumin seeds (Nigella sativa L). Journal of the Science of Food and Agriculture, 76(3), 404–410. Tasawar, Z., Siraj, Z., Ahmad, N., & Mushtaq, H. (2011). The effects of Nigella sativa (Kalonji) on lipid profile in patients with stable coronary artery disease in Multan, Pakistan. Pakistan Journal of Nutrition, 10, 162–167. Tavakkoli, A., Mahdian, V., Razavi, B. M., & Hosseinzadeh, H. (2017). Review on clinical trials of black seed (Nigella sativa) and its active constituent, thymoquinone. Journal of Pharmacopuncture, 20(3), 179–119. Tufek, N. H., Altunkaynak, M. E., Altunkaynak, B. Z., & Kaplan, S. (2015). Effects of thymoquinone on testicular structure and sperm production in male obese rats. Systems Biology in Reproductive Medicine, 61(4), 194–204. Yakup, K. (2007). Samsun yöresinde ve misir ülkesinde yetiştirilen çörekotu (Nigella sativa L.) Tohumlarinin antioksidan aktivite yönünden incelenmesi. Süleyman demirel Üniversitesi Fen Dergisi, 2(2), 197–203. Yuncu, M., Şahin, M., Bayat, N., & İbrahim, S. (2013). Çörek otu yağının sıçan karaciğer gelişimine etkisi. Gaziantep Medical Journal, 19(3), 180–184. Zaoui, A., Cherrah, Y., Lacaille-Dubois, M. A., Settaf, A., Amarouch, H., & Hassar, M. (2000). Diuretic and hypotensive effects of Nigella sativa in the spontaneously hypertensive rat. Thérapie, 55(3), 379–382. Zeweil, H. S., Ahmed, M. H., El-Adawy, M. M., & Zaki, B. (2008). Evaluation of substituting nigella seed meal as a source of protein for soybean meal in diets of New Zealand land white rabbits. Nutrition and Digestive Physiology, 863–868.

Chapter 15

Nigella sativa Seed Cake: Nutraceutical Significance and Applications in the Food and Cosmetic Industry Jasmeet Kour and Adil Gani

Abstract  Nigella sativa is an annual flowering plant and one of the most significant seeds in the history of medicinal importance. It is popularly used in the bakery industry and medicinal products. It is an annual spicy herb indigenously belonging to Mediterranean regions. The seeds are extracted using a screw press machine to yield oil (32–40%) leaving behind residue meal which is an abundant source of macro as well as micro-nutrients. Macronutrients include protein and fibres, whereas micronutrients include vitamins and minerals. Even after extraction of the oil from the seed, the defatted black cumin seed cake still contains some residual oil rich in bioactive components apart from the main constituents of the meal. The defatted cake is utilized as an animal feed owing to ample protein content and can also act as a high source of phenolic compounds and carbohydrates that could aid in strengthening the immune system. The meal contains most essential amino acids along with crude protein. The production of black cumin defatted seeds is expected to increase shortly due to its extended use as a medical seed. It has been also reported to replace protein in the diets of growing Japanese quail. Despite nutritional as well as economic importance, there is a dearth of literature about the black cumin seed cake. Some of the pivotal works have reported about the rich amino acid profile of the seeds. The meal obtained could be a good alternative for feed for animals. Apart from high crude protein, the defatted cake has been also reported to contain high crude fibres. It has been also reported to be a good replacement for soybean meal. Studies have reported also the utilization of seed cake as a rabbit feed without hampering liver or kidney function. The application of black cumin seeds also finds its prevalence in protective cosmetics owing to its high antioxidative properties.

J. Kour (*) Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Longowal, Sangrur, Punjab, India A. Gani Department of Food Science and Technology, University of Kashmir, Srinagar, India © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_15

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Keywords  Japanese quail · Soybean meal · Rabbit feed · Antioxidants · Nutraceuticals · Cosmetics

Abbreviations BC Black cumin flour BCSM Black cumin seed meal LBW Low birth weight NDPE Net dietary protein energy percent NPU Net protein utilization NSM Nigella sativa seed meal PER Protein efficiency ratio SBM Soybean meal TQ Thymoquinone

1  Black Cumin Seed Cake Nigella sativa, or black cumin seed, is a prominent herb used on the traditional basis in the Middle East and Asia as a medicine to combat various diseases. India is one of the largest producer and exporter of Nigella sativa in the world. Apart from this, the other black cumin main producing countries are Sri Lanka, Bangladesh, Nepal, Egypt, Iraq, and Pakistan. The prominent states known for its commercial cultivation are Punjab, Himachal Pradesh, Madhya Pradesh, Bihar, Jharkhand, Assam, West Bengal and Andhra Pradesh (Malhotra 2012). It is also popular in Egypt, where it is commonly used in bakery products and folk medicine (Al-Okbi et al. 2015). It is native to the Mediterranean regions but now it is also cultivated in other parts of the world including the Middle East, North Africa and Asia (Durrani et al. 2007). It is also well known as a drug prevalent in the medicinal world of traditional India including Unani and Ayurveda (Mukhtar et al. 2019). Nigella has been known to grow wild in countries such as India, Egypt, and Iran where eight species have been recorded (Mozoffarin 1998). Black cumin or Nigella sativa is a valuable medicinal plant and this flowering plant is also called as fennel flower native to South Asia. It belongs to Ranunculaceae family bearing blue coloured flowers which contain small black caraway type seeds. It acts as a powerful diuretic, diaphoretic and digestive remedy as well (Jakubowska et al. 2015). The seeds are used for the treatment of diseases such as cough, eczema, asthma, ascites, hydrophobia, intrinsic hemorrhage, jaundice, diabetes, dizziness and abdominal disorder (Warrier et al. 2004). The constituent which is found in the highest concentration in seed oil is thymoquinone (TQ) (2-isopropyl-5-methyl-1,4-benzoquinone) which has been shown to have anti-neoplastic activities in different types of cancer (Salomi et  al. 1992).

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Black cumin seed comprises volatile and fixed oils that are embedded with ample amounts of health benefits. Fixed seed oil is associated with unsaturated fatty acids, such as linoleic, oleic acid, along with minor amounts of saturated fatty acids, including arachidonic and eicosanoic acids (Hadad et al. 2012). Nigella sativa has high-quality nutritive values concerning nutrients including protein which evaluated by measuring PER (protein efficiency rate), net dietary protein energy percent (NDPE) and net protein utilization (NPU). Nigella sativa seeds can be extracted to produce oil and the meal obtained is seemed to be an advantage in replacing the Nigella sativa seeds itself to feed the animal (Silvia et al. 2012). Black cumin seed cake is obtained using the screw press machine (32–40%) wherein the residual cake obtained is abundantly rich in protein, fibres, vitamins, and minerals. The black cumin cake remained after extraction of oil still contains residual oil in addition to the main constituents of the meal (Al-Okbi et al. 2015). Few studies have acknowledged that this residue even possesses bioactive compounds that can be used as antioxidants. There is a great potential in the black cumin residue to be utilized as an animal feed as a good source of proteins and carbohydrates (Thilakarathna et al. 2018).

2  Characterization of Nigella sativa Cake Nigella seed cake is a useful by-product of the black cumin seed obtained after cold pressing which can be utilized in many fields (Takruri and Dameh 1998). Its high content of protein and carbohydrate increases the significance of this residue. Further investigation is needed to make use of this underutilized residue (Thilakarathna et al. 2018). It can be used as a relatively good source of energy and protein supplement for the human diet (Akhtar et al. 2003). Nigella sativa meal also contains a high level of crude protein (33–84%), crude fibres (54%) and essential amino acids (El-Nattat and El-Kady 2007; Silvia et al. 2012). Nigella sativa seed cake is rich in protein and carbohydrate, amino acids and mineral which makes it a potential source of nutrients for animal feed in altering the soybean meal (El-Nattat and El-Kady 2007). The utilization of Nigella sativa cake as a replacement for seeds for animal feeds efficiently uplifts the economic efficiency (Silvia et al. 2012). It can be also used as a basic ingredient in the formulation of feed diet for growing lambs without any adverse effects, thus, helping to reduce feed costs and more importantly increase the economic efficiency of local industries (Abdel-Magid et al. 2007). Nigella sativa meal contains protein (43%), oil (1%), and carbohydrates (40%) (Osman et al. 2015), where the same components for black cumin protein isolate were 86.9%, 0.04%, and 7.36%, respectively (Osman and Al-Jasser 2004). Al-Okbi et al. (2015) analyzed the composition and amino acid contents of Nigella sativa cake which contained a high level of protein (27.1%). Residual lipids and carbohydrate contents were 12.6% and 40.5%, respectively. The crude fiber was reported as 8.1%and the ash content was 7.3  mg/100  g. Total phenolic content was 42.6  mg

226 Table 15.1  Amino acid content of Nigella sativa cake

J. Kour and A. Gani Amino acid Valine Methionine Isoleucine Leucine Phenylalanine Histidine Lysine Arginine Threonine Aspartic Serine Glutamic Glycine Tyrosine Proline Alanine

Content (mg/100 g) 434 60 119 721 306 777 475 1308 432 1277 926 9620 893 79 234 2233

Source: Al-Okbi et al. (2015) Table 15.2  Composition of Nigella sativa seeds of Indian and Ethiopian origin

Moisture Protein Lipids Fiber Carbohydrate Ash

Indian 4.56 18.4 15.7 7.69 28.1 5.31

Ethiopian 4.37 19.2 16.1 6.03 23.1 4.98

Source: Thilakarathna et al. (2018)

gallic acid equivalent/g dry ethanol extract. Apart from this, identified phenolic compounds were protocatechuic, chlorogenic, vanillic, sinapic, coumaric and cinnamic acid and chrysin (Table 15.1). Black cumin seed cake of both Indian and Ethiopian origin were analyzed for their compositions and mineral content (Table  15.2). Protein and carbohydrates were present in significant amounts in both types of seed cake, followed by lipids and fibres. There was a considerable amount of lipids retaining in the residue of black cumin. Composition of Nigella seed cake of Indian origin revealed that the moisture content, protein, and total carbohydrate were considerably higher than that of Ethiopian type, while the lipids and fibres contents were higher in the Ethiopian origin. Hence, it was concluded that the defatted meal can be developed further as a value-added product and it can be used as a rich source of proteins and carbohydrates (Thilakarathna et al. 2018).

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3  Applications of Black Cumin Seed Cake El-Ayek et al. (1999) substituted the concentrate feed mixture with Nigella sativa cake in growing lambs and concluded that this meal provides 50% of proteins without producing adverse effects on their performance. It can be used as a relatively good protein supplement in the diets of mammals. Nigella sativa seed cake can be a good replacement for imported soybean meal thereby reducing the cost of rabbits feeding (Zeweil et al. 2008). The dried black cumin seeds are of great commercial significance to be used in foods, pickles, baked goods, and confectionery and the pharmaceutical and perfumery industries as well. Nigella seeds are generally dry-­ roasted or fried before being used for a variety of culinary purposes. Ahmad (2005) reported that supplementation of BCSM at 0.5% was more beneficial and profitable in broiler production than 1.0% level. Ihsan (2003) reported that when the broilers fed diets with BCSM, they yielded more profit in comparison with the rations without supplementation. HRS rats, when treated with Nigella sativa cake and its alcohol extract significantly improved the majority of biochemical parameters except for plasma creatinine and creatinine clearance. Plasma calcium level was improved whereas the ethanol extract didn’t produce any noticeable difference. It was noticed that plasma albumin and total protein of HRS rats treated by Nigella sativa meal and its alcohol extract were normalized where they match those of the control normal rats (Al-Okbi et al. (2015). Tekeli (2014) reported that feeding Japanese quail chickens diets with 4% or 8% NSM resulted in the best LBW in comparison with the control. Jamroz and Kamel (2002) found that black cumin seed stimulated the digestive system, which reflects positively in increased absorption and growth performance. This increases the bile flow rate which consequently increases emulsification simulating the pancreatic lipase activity as theorized by Crossland (1980). Mahmoud and Bendary (2014) carried out a study to evaluate digestibility and growth performance in growing Barki lambs and calves upon substitution of soybean and cotton seed meal by Nigella sativa and sesame seed meal. There were insignificant differences in the average daily gain and final body weight of animals upon feeding the two experimental rations. The high nutritional profile of Nigella sativa seed indicates that various extract and paste of Nigella sativa seed could be a wonderful ingredient in hair, skin and oral care cosmetics. Nigella sativa could be the best candidate for treating various fungal and bacterial infections like dandruff, acne, pimples and other skin conditions (Awad et al. 2013). The utilization of Nigella sativa seed powder and honey mix for acne treatment and clear facial was reproted. The various medicinal properties of black cumin seed make it a useful ingredient to be used in the medicinal and cosmetic world. Owing to its high antioxidative properties, black cumin seeds also finds its prevalence in cosmetics in a safe way.

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References Abdel-Magid, S. S., El-Kady, R. I., Gad, S. M., & Awadalla, I. M. (2007). Using cheep and local non-conventional protein meal (Nigella sativa) as least cost rations formula on performance of crossbreed calves. International Journal of Agriculture and Biology, 9(6), 877–880. Ahmad, S. (2005). Comparative efficiency of garlic, turmeric, and kalongi as a growth promoter in broiler. Faisalabad: Department of Poultry Sciences. University of Agriculture. Akhtar, M. S., Nasir, Z., & Abid, A. R. (2003). Effect of feeding powdered Nigella sativa L. seeds on poultry egg production and their suitability for human consumption. Veterimarski Arch, 73, 181–190. Al-Okbi, S. Y., Mohamed, D. A., Hamed, T. E., El-Sayed, E. M., Mohamed, M. S., & Mabrok, H. B. (2015). Protective role of Nigella sativa seed meal and its alcohol extract in hepatorenal syndrome model in rats. Research Journal of Pharmaceutical, Biological, and Chemical Sciences, 6(6), 1355–1363. Awad, E., Austin, D., & Lyndon, A. R. (2013). Effect of black cumin seed oil (Nigella sativa) and nettle extract (quercetin) on enhancement of immunity in rainbow trout, Oncorhynchus mykiss (Walbaum). Aquaculture, 388–391, 193–197. Crossland, J. (1980). Lewis’s pharmacology (5th ed., pp. 656–657). London: Churchill Livingston. Durrani, F. R., Chand, N., Zaka, K., & Sultan, A. (2007). Effect of different levels of feed added black seed (Nigella sativa L.) on the performance of broiler chicks. Pakistan Journal of Biological Sciences, 10(22), 4164–4167. El-Ayek, M. Y., Gabr, A. A., & Mehrez, Z. (1999). Influence of substituting concentrate feed mixture by Nigella sativa meal on animal performance and carcass traits of growing lambs. Egypt Journal of Nutrition and Feeds, 2, 265–277. El-Nattat, W. S., & El-Kady, R. I. (2007). Effect of different medicinal plant seeds residues on the nutritional and reproductive performance of adult male rabbits. International Journal of Agriculture and Biology, 9, 479–485. Hadad, G. M., Salam, R. A., Soliman, R. M., & Mesbah, M. K. (2012). High-performance liquid chromatography quantification of principal ant, oxidants in black seed (Nigella sativa L.) phytopharmaceuticals. Journal of AOAC International, 95, 1043–1047. Ihsan, K. (2003). Effect of different levels of kalongi (N. sativa) seeds on the performance of broilers. M.  Sc. (Hons.) thesis, Department of Poultry Sciences, University of Agriculture, Faisalabad, Pakistan. Jakubowska, M. K., Kraszkiewicz, A., & Krajewska, M. (2015). Possibilities of using waste after pressing oil from oil seeds for energy purposes. Agricultural Engineering, 20(1), 45–54. Jamroz, D., & Kamel, C. (2002). Plant extracts enhance broiler performance. In non-ruminant nutrition: Antimicrobial agents and plant extracts on immunity, health, and performance. Journal of Animal Science, 80(Suppl. 1), E41. Mahmoud, A. E. M., & Bendary, M. M. (2014). Effect of whole substitution of a protein source by Nigella sativa meal and sesame seed meal in ration on performance of growing lambs and calves. Global Journal of Veterinary Medicine and Research, 13(3), 391–396. Malhotra, S.  K. (2012). Nigella. In Handbook of herbs and spices (pp.  392–416). Oxford: Woodhead Publishing Limited. Mozoffarin, V. (1998). A dictionary of plants names (p. 365). Tehran: Farhang Moastar Publishers. Mukhtar, H., Qureshi, A. S., Anwar, F., Mumtaz, M. W., & Marcu, M. (2019). Nigella sativa L. seed and seed oil: Potential sources of high-value components for development of functional foods and nutraceuticals/pharmaceuticals. Journal of Essential Oil Research, 31, 171–183. https://doi.org/10.1080/10412905.2018.1562388. Osman, M. A., & Al-Jasser, M. S. (2004). Nutritional and functional properties of black cumin (Nigella sativa) seed proteins products. Alexandria Science Exchange Journal, 25, 288–295. Osman, M. A., Alamri, M. S., Mohamed, A. A., Hussain, S., Gassem, M. A., & Abdel Rahman, I.  E. (2015). Black cumin-fortified flat bread: Formulation, processing, and quality. Quality Assurance and Safety of Crops & Foods, 7(2), 233–238.

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Salomi, N.  J., Nair, S.  C., Jayawardhanan, K.  K., Varghese, C.  D., & Panikkar, K.  R. (1992). Antitumour principles from Nigella sativa seeds. Cancer Letters, 31, 41–46. Silvia, D., Masturah, M.  F., Axis, Y.  T., Nadiah, W.  A. W., & Bhat, R. (2012). The effects of different extraction temperatures of the screw press on proximate composition, amino acid contents and mineral contents of Nigella sativa meal. American Journal of Food Technology, 7(4), 180–191. Takruri, H. R. H., & Dameh, M. A. F. (1998). Study of the nutritional value of black cumin seeds (Nigella sativa L.). Journal of the Science of Food and Agriculture, 76(3), 404–410. Tekeli, A. (2014). Nutritional value of black cumin (Nigella sativa) meal as an alternative protein source in poultry nutrition. Journal of Animal Science Advances, 4, 797–806. Thilakarathna, R. C. N., Madhusankha, G. D. M. P., & Navaratne, S. B. (2018). Comparison of physico-chemical properties of Indian and Ethiopian origin Black cumin (Nigella sativa) seed cake. International Journal of Food Science and Nutrition, 3(4), 30–31. Warrier, P. K., Nambiar, V. P. K., & Ramankutty, C. (2004). Indian medicinal plants – A compendium of 500 species (Vol. 4, pp. 139–142). Chennai: Orient Longman Pvt. Ltd. Zeweil, H. S., Ahmed, M. H., El-Adawy, M. M., & Zaki, B. (2008). Evaluation of substituting nigella seed meal as a source of protein for soybean meal in diets of New Zealand white rabbits. Nutrition and Digestive Physiology, 863–868.

Chapter 16

Nigella sativa Seeds in Cosmetic Products: Shedding the Light on the Cosmeceutical Potential of Nigella sativa and its Utilization as a Natural Beauty Care Ingredient Fadia S. Youssef Abstract  Nigella sativa L. (black cumin) is highly popular throughout history by being an everlasting source of chemical constituents to which its massive biological and nutritional importance is attributed. Traditionally, Nigella sativa has been described to be the cure of unlimited number of diseases that have recently turned out to be true. Although careful consideration has been given to the therapeutic importance of Nigella sativa seed, its usage in cosmetic science is still to a great extent neglected. Furthermore, there is a great influx in the utilization of herbal drugs in cosmetics exemplified by herbal soap, shampoo, conditioner, as well as in the face and mouth wash. This undoubtedly is attributed to the sound belief in the safety, efficacy and minimal adverse effects of natural products comparable to synthetic chemical ingredients. Thus, in the foregoing chapter, the diverse cosmeceutical properties, external applications as well as the marketed cosmetic products of Nigella sativa will be highlighted. Additionally, this will be correlated with some of its biological activities as anti-inflammatory, anti-aging, antimicrobial, wound healing, anti-acne and antioxidant activities that result in making Nigella sativa a good candidate for many cosmetic preparations. Keywords  Cosmeceutical potential · Nigella sativa · Ranunculaceae · Topical preparations

F. S. Youssef (*) Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_16

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1  Introduction to Cosmetics and Cosmeceutical Preparations The term cosmetic was originated from the Greek word “kosm tikos” that means possessing the power, organization, and talent in decoration. The utilization of cosmetics is as old as mankind since prehistoric ages; a man hunted the animals to be used as food via attracting them through the usage of different decorating colors. Besides, the ancient man graced his body and colored his skin to protect himself from the attack of his enemies and to disseminate fear in them (Fathima et al. 2011). In the modern era, cosmetic products become one of the most principle requirements of human beings all over the globe. The preparation of cosmetic products has passed through a series of modifications. At first, cosmetics were consumed in the form of crude natural products being in the state of crushed fresh leaves, seeds, and roots in addition to the utilization of various extracts obtained from several parts of plants. Following this era, gradual improvement in the shape of cosmetic formulations takes place to be more convenient and charming with elevated interest in chemically-based ingredients rather than naturally-derived constituents (Sudhir et al. 2016). Basically, the cosmetic product can be defined in accordance to the Safety Regulations concerning the consumer products that were launched in 1989 as any product or substance that could be applied externally on teeth, lips, buccal mucosa, nails, hair, epidermal cells and genital organs that exist externally as well. Additionally, these products are administered either to clean, protect, scent, and to keep these external organs in a healthy situation or to improve body appearance, odor, and perspiration aiming to alleviate and prohibit the disease (Aburjai and Natsheh 2003). Cosmetics can be categorized into two main classes namely rinse off and leave on cosmetics. Rinses off preparations include hand wash, shampoo, soaps, and conditioners. Leave on products comprise hair oil, lotion, cream, gel in addition to lip gel, mascara, nail polish, lipstick, deodorant and body spray. Additionally, mouth wash and toothpaste are also considered as cosmetics (Sudhir et al. 2016). Meanwhile, the term cosmeceuticals was disseminated by Albert M. Kligman in the late 1970s. They were considered as cosmetic preparations comprising biologically active constituents. They possess therapeutic and drug-like properties showing valuable topical potential and protection versus degenerative dermal disorders (Singhal et  al. 2011). Actually, a cosmeceutical acts as a bridge linking between pharmaceuticals (corticosteroids and antibiotics) and pure cosmetics (lipsticks). As cosmetics, application of cosmeceuticals is performed topically, however, they possess therapeutic ingredients that beneficially affect the skin biological function triggering therapeutic benefits, skin healing and disease combating properties via the transportation of vital nutrients that are of crucial importance to keep the skin healthy (Grace 2002). The biological role which the cosmeceuticals perform to the skin relies mainly upon the functional constituents they possess (Singhal et al. 2011). The role of cosmeceuticals go beyond gracing and decorating the skin, they effectively decrease skin wrinkling while improving its tone, texture as well as its

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radiance. They exert their role via activating collagen formation, protecting keratin structure, acting as free radicals scavenger, combating inflammation and microbial infection. On the industrial scale, they are considered the fastest developing division in the manufacture of natural body care products owing to the great desire of population among different cultures to keep a youthful manifestation (Saha 2012).

2  P  opularity of Various Medicinal Plants as Herbal Cosmetics Herbal cosmetics are considered to be safe to use when compared to other synthetic beauty products that trigger allergic reactions in the skin manifested by skin itching. A lot of medicinal plants are popular cosmetics and there is a statement that mentioned that there is a plant for every need in each continent that has proven to be true (Aburjai and Natsheh 2003). Many natural products exemplified by vegetable oil are found to be effective in the treatment of skin dryness via reducing the loss of water from the stratum corneum through the formation of an occlusive film. These include castor oil, sunflower oil, cocoa butter (Dweck 1997), mango (Torres-León et al. 2016) and olive oil. Meanwhile, turmeric revealed high efficacy in the alleviation of eczema that is characterized by itching, redness, and swelling. Pea, artemisia, onion and basil are highly effective in relieving pimples, spots as well as acne that severely affects hair follicles in addition to sweat glands triggering black heads and inflammation (Aburjai and Natsheh 2003). Besides, many herbal products revealed high effeciency in counteracting aging that primarily triggered by exposure to UV radiation (Fisher et al. 2002). Aging is mainly due to the imbalance between the degenerative and regenerative power of the skin resulting in wrinkling and thinning of the epidermal layer with concomitant appearance of furrows and lines, particularly during facial expression. Basically, this occurs due to excessive loss in elastin fibers and collagen with decreased circulatory perfusion. Anti-aging natural products that effectively restore the balance between regenerative/degenerative power of the skin in addition to potentiating the production of elastin and collagen include ginseng (Aburjai and Natsheh 2003). Green tea and grape seed exert a potent antioxidant activity evidenced by their free-radical scavenging potential owing to their richness in phenolic hydroxyl moieties that make them good candidates in the manufacturing of many cosmetics (Pietta 2000). Meanwhile, red clover, fenugreek, chamomile, jojoba and licorice roots are potent anti-inflammatory agents that effectively counteract and alleviate many pathological disorders associated with acne, psoriasis, rash, and eczema. Isoflavones obtained from red clover played a crucial role as a sun-protective agent through potentiation of the immune response to counteract photo-suppression (Widyarini et al. 2001). In addition, cucumber, aloe vera and oats are potent skin protective agents (Aburjai and Natsheh 2003).

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Regarding hair care products, products of natural origin are highly popular in this regard, many of them stimulate hair growth, act as hair colorants in addition to acting as anti-dandruff. Grape seeds, ginkgo biloba, rosemary, henna, aloe, and saga exerted a potent proliferation of the hair follicles owing to the proanthocyanidins content of the former, to aloenin in Aloe species and to borneol, camphor, saponins and tannins in Saga and to rosmarinic acid in rosemary (Grindlay and Reynolds 1986; Kobayashi et  al. 1993; Aruoma et  al. 1996; Ortiz et  al. 1997; Takahashi et al. 1998). Many herbal products act as anti-dandruff resolving several biochemical alterations that occur in the scalp comprising the impairment in the desquamation process and reduction in free lipids content as cholesterol, fatty acids, and ceramides that ultimately leads to exposure to microbial infections due to impairing the epidermal water barrier in sufferers (Harding et  al. 2002). Anti-dandruff agents include sage, rosemary, thyme and garlic. Sage effectively controls dandruff and hair falling however, rosemary acts as a conditioner and a hair tonic meanwhile thyme and garlic promote healthy natural hair (Aburjai and Natsheh 2003).

3  Overview on Nigella sativa (Black Cumin) Seed Herbal products possessing medicinal values are considered as gifts from nature and Nigella sativa (Fig. 16.1) is considered as one of these herbs showing promising therapeutic importance displaying more safety, efficacy, and acceptance among people comparable to synthetic drugs (Datta et al. 2012). Nigella sativa L., (black cumin) belongs to family Ranunculaceae, is considered as an annual herb that is cultivated worldwide comprising the Mediterranean basin and Middle-East as well (Naz 2011) showing a vast array of medicinal values. Traditionally, it had been adopted for the alleviation and prohibition of several ailments, in which Prophet Mohammad had mentioned that the black seed could be used to cure every disease except death (Datta et al. 2012). Besides, Nigella sativa was found in folk medicinal reports to be an effective cure for asthma, back pain, infection, bronchitis, chronic headache, chest congestion, cough, chronic headache, diabetes, dizziness, dysmenorrheal, hypertension,

Fig. 16.1  Leaves (a), seeds (b) and flowers (c) of Nigella sativa

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hemiplegia, obesity, paralysis, migraine, and rheumatism. In addition, it was traditionally popular for the relief of many gastrointestinal disorders like diarrhea, dyspepsia, dysentery and flatulence. It showed a pronounced effect as a carminative, anthelmintic, diuretic, emmenagogue and lactagogue as well. Topically, it was employed traditionally as a local anesthetic for curing abscesses, eczema and nasal ulcers (Nadkarni 1976; Sharma et al. 2005; Tariq 2008; Kazmi et al. 2019). On the pharmacological level, black cumin revealed significant antimicrobial and antiviral potential against a panel of micro-organisms in addition to a promising antioxidant, anti-inflammatory, anti-allergic activity via inhibiting cytokines and eosinophil infiltration owing to the presence of thymoquinone. It also displayed anticancer activity, calcium channel blockage activity manifested by its bronchodilator and spasmolytic activities, cardiovascular protective and anti-hypertensive activity. Besides, it revealed amelioration in some of the metabolic disorders as hyperglycemia, hyperlipidemia and obesity (Kalus et al. 2003; Hosseinzadeh and Parvardeh 2004; Büyüköztürk et al. 2005; Dahri et al. 2005; Isik et al. 2005; Salem 2005; Abdulelah and Zainal-Abidin 2007; Farrag et al. 2007; Zaher et al. 2008). Its immunomodulatory potential can be interpreted in virtue of its neutrophil elastase inhibitory activity that relied on the presence of carvacrol, however its hematological behaviour is due to the maintenance of balance between fibrinolysis/thrombus formation via adjusting the endothelial cells fibrinolytic action. Thymoquinone is also responsible for black cumin gastro-protective effect protecting gastric mucosa versus the consumption of absolute alcohol and promoting healing of ulcers (Gilani et al. 2001; Zaoui et al. 2002; Mansi 2005; Sayed-Ahmed and Nagi 2007; Uz et al. 2008; Yaman and Balikci 2010). Thymoquinone isolated from the black cumin was found to exhibit anti-Parkinson’s disease affecting dopaminergic neurons (El-Dakhakhny et al. 2002). Concerning the phytoconstituents predominating in Nigella sativa, many compounds were found that belong to multiple classes which represented mainly by avenasterol-7-ene (1) and avenasterol-5-ene (2), cycloeucalenol (3), citrostadienol (4), cholesterol (5), campesterol (6), nigellone (7), nigellimine-N-oxide (8), nigellicine (9), nigellimine (10), gramisterol (11), 24-ethyllophenol (12), lophenol (13), taraxerol (14), 24-methylenecycloartanol (15), 3-methyllophenol (16), butyrospermol (17), obtusifoliol (18), stigmastanol (19), β-sitosterol (20), amyrin (21), tirucallol (22), β-cycloartenol (23), hederagenin (24), volatile oil and fixed oil. The fixed oil composed of saturated and unsaturated fatty acids. The former is represented mainly by myristic (25), palmitic (26) and stearic acid (27), meanwhile the latter is represented by eicosadienoic (28), arachidonic (29), linoleic (30), oleic (31) and dihomolinoleic acid (32). Regarding its essential oil, it is composed mainly of thymol (33), dithymoquinone (34), thymoquinine (35), nigellone (7), thymohydroquinone (36), carvacrol (37) in addition to α-pinene (38), p-cymene (39), d-citrlnellol (40) and d-limonene (41). A scheme representing Nigella sativa major constituents was represented in Fig. 16.2.

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Fig. 16.2  Nigella sativa major phytoconstituents

4  C  osmeceutical Potential of Nigella sativa and Its Utilization as a Natural Beauty Care Ingredient Although careful consideration has been given to the therapeutic importance of Nigella sativa seed, its usage in cosmetic science is still to a great extent neglected. Certain studies were carried on Nigella sativa that could greatly explain its potential as a cosmeceutical ingredient. Balm sticks with 10% Nigella sativa oil showed significant anti-inflammatory activity when applied topically employing both carrageenan-­induced granuloma pouch and paw edema models as well. It ­effectively

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prohibits both sub-acute and acute inflammation evidenced by the prohibition of edema formation by 60.64%, lowering of leukocytes count by 43.5% with concomitant decreasing in TNF-α concentration by 50% comparard to the control (Dwita et al. 2019). Additionally, Nigella sativa seed oil was found to an effective cure for atopic dermatitis that is a chronic disease accompanied by pruritus and severe skin lesions. Noteworthy to highlight that steroids could effectively alleviate atopic dermatitis, however they cause severe adverse effects and thus natural products were preferred (Chawla and Chauhan 2016). Thus, cream with Nigella sativa seed oil was applied transdermally to assess its efficacy in alleviating dermatitis induced by 1-chloro-2, 4-dinitrobenzene in rats that is compared to a steroidal cream containing betamethasone valerate. Nigella sativa seed oil cream effectively reduced the thickening of the epidermis and inflammatory cells infiltration as evidenced by histological examination with results comparable to betamethasone valerate cream and thus could be used as a natural substitute for attenuation of atopic dermatitis (Chawla and Chauhan 2016). Nigella sativa volatile oil and indomethacin loaded on poly (ε-caprolactone) nanoparticles were formulated in an effort to elevate the anti-inflammatory and the analgesic potential of indomethacin with a concomitant reduction in its dose and adverse effect as well. The nanoprecipitation procedure was employed in the preparation of the nanoparticles showing effective encapsulation efficiency estimated by 70 and 84% for the indomethacin and the Nigella sativa volatile oil, respectively with 14 and 5.63% as drug loading, respectively. This revealed the successful encapsulation of both indomethacin and the Nigella sativa volatile oil that undoubtedly enhance the anti-inflammatory and the analgesic efficacy of indomethacin (Badri et al. 2018). Besides, the topical application of black seed oil significantly reduced pain associated with knee osteoarthritis in the elderly as determined by a crossover clinical trial carried on 40 elderly patients and compared to the effect exerted by oral administration of acetaminophen. This was assessed via pain determination using the visual scale (VAS) in which the treatment response was observed by a reduction in pain scores over 1.5. Black seed oil exerted better activity in pain reduction relative to acetaminophen (Kooshki et al. 2016). Moreover, a blend of Nigella sativa seed oil, red and black raspberry seed oil in addition to corticosteroid, alcohol, and propellant were formulated as a topical spray that effectively treats inflammatory skin disorders with no need to use undecylenic acid and zinc pyrithione as well (Crutchfield 2008). Nigella sativa seed methanol extract exhibited a potent antimicrobial activity versus Propronibacterium acnes as evidenced by its significant zone of inhibition. Consequently, Nigella sativa seed methanol extract was formulated in the form of topical gel and assessed for its ability in the treatment of Acne vulgaris. The gel formulated exhibited acceptable physical appearance, viscosity, pH and homogeneity as well in addition to being simple and highly effective in the curing of acne. Additionally, Nigella sativa seed was incorporated with clotrimazole to exert a

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t­ opical synergistic antifungal potential combating various human pathogenic fungal infections (Bhalani and Shah 2015). Psoriasis represents one of the highly popular chronic inflammatory skin diseases and it is immune modulated with 1–3% prevalence. Topical application of Nigella sativa seed oil was found to be effective in the treatment of psoriasis-like lesions triggered by imiquimod in male albino rats possessing thin skin that could be mainly attributed to its immunostimulatory, antioxidant and anti-inflammatory activities. Imiquimod application resulted in massive inflammation in the epidermis, hyperplasia with degenerative changes in the shape of normal keratinocytes. These alterations were accompanied by a notable presence of collagenous fibers in the dermal layer and positively stained cells of proliferating cell nuclear antigen in all epidermal layers. Nigella sativa seed oil applied topically significantly prohibited psoriasis-like inflammation triggered by imiquimod as manifested by reduction of all dermal and epidermal alterations triggered by imiquimod that suggests its utilization as adjuvant therapy applied topically for the treatment of psoriasis (Bhalani and Shah 2015). Additionally, black cumin constitutes an ingredient in an herbal preparation which was used traditionally to treat moderate and severe psoriasis (DurrDerma). It consists of a mixture of plant-based extracts represented by black cumin, tea tree oil, olive oil, cocoa butter supplemented by vitamins A and B12. DurrDerma preparation effectively ameliorates psoriasis as evidenced by the reduction of the Psoriasis Area and Severity Index (PASI) score in 10 out of 12 examined cases (83%). Its activity greatly relied upon the synergistic anti-oxidative and anti-inflammatory activity of its components (Michalsen et al. 2016). Topical application of Nigella sativa seed oil showed promising results in treating cyclic mastalgia that commonly occurs in women with no optimal remedy and mostly related to menstruation and attributed to hormonal fluctuation and increased estrogen receptors sensitivity owing to increased saturated fatty acid/essential fatty acids ratio. In a randomized blind clinical trial comprising of active and placebo, Nigella sativa seed oil exerted analgesic activity comparable to diclofenac in relieving cyclic mastalgia with no adverse effect, meanwhile it showed superior activity when compared to placebo. The exact mechanism of action of Nigella sativa in alleviating cyclic mastalgia is not investigated but can be partially interpreted in virtue of its major constituents represented mainly by thymoquinone, unsaturated fatty acid, and carvacrol (Huseini et al. 2016). Besides, black seed oil experienced a potent effect in relieving allergic rhinitis when applied topically. Allergic rhinitis is considered to be a chronic inflammatory disorder manifested at any age due to an atopic reaction caused by allergens inhalation. Most drugs could alleviate only the symptoms of allergic rhinitis and mostly experience adverse effects and withdrawal symptoms. This assessment was done clinically on 68 patients who were divided into three groups according to the severity of the allergic rhinitis namely mild, moderate and severe symptoms. After 6 weeks of treatment 100, 68.7 and 58.3% of the mild, moderate and severe cases are symptoms free, respectively meanwhile 25% in both latter groups were improved. Thus, 92.1% of the active groups showed either improvement or symptoms free in addition to 55.2% tolerability to allergen exposure comparable to the

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control group that displayed 30.1% improvement and 20% tolerability (Mohamed Alsamarai et al. 2014). The topical application of black seed effectively ameliorates hand eczema and enhances the life quality of patients when tested clinically in double-blinded clinical trials showing good results as those displayed by Betamethasone. This was manifested by assessing the Dermatol Life Quality Index (DLQI) and the Hand Eczema Severity Index (HECSI) after topical application of the medications twice daily for 28  days. Both Betamethasone and black seed treated groups revealed a fast improvement in hand eczema showing a notable reduction in DLQI and equally reduce the severity of eczema and enhance the life quality (Yousefi et al. 2013). Vitiligo is a dermal disorder that affects melanocytes’ structure and function causing their disruption that eventually reflected on skin pigmentation. The exact etiology is not fully discovered however neural mechanisms, genetic predisposition and biochemical factors as well could play a role in its etiology. Although many drugs were discovered for its curing, no absolute treatment was achieved. Topical application of Nigella sativa seed oil containing cream to face, hands and genital areas to 37 vitiligo patients for 6 months successfully resulted in a notable repigmentation in the affected areas. This strongly recommends the utilization of Nigella sativa as an adjuvant therapy for vitiligo particularly in the sensitive regions as genital areas (Sarac et al. 2019). Another study conducted on 96 patients with vitiligo to compare the activity of both Nigella sativa oil and fish oil in the alleviation of vitiligo. Nigella sativa displayed higher efficacy in vitiligo treatment manifested by a reduction in the size of patient’s lesions compared to the fish oil. This further consolidates the belief of the utilization of Nigella sativa as a cure for vitiligo that is derived from natural products (Ghorbanibirgani et al. 2014). Additionally, a formulation of microemulsion that consists of black seed honey and ethanol extract of propolis showed a significant burn healing potential being superior to silver sulphadiazine cream in its activity with wound contraction estimated by 99.3 and 89.1%, respectively. The former effectively inhibits Staphylococcus aureus with the mean zone of inhibition equals to 26.1 mm as determined by the well diffusion method (Sulaiman et al. 2014). The topical application of black cumin exhibited radioprotective activity manifested by its successful alleviation of nasal mucositis induced in rats by radiotherapy. It effectively reduces inflammatory cell infiltration, the formation of exudates, vascular dilatation and superficial erosion serving as a promising agent in the treatment of acute nasal mucositis triggered by radioactivity (Çanakci et al. 2018). Topical application of Nigella sativa seed oil could act as a good alternative for the treatment of nasal symptoms triggered by aging in the elderly showing better activity when compared to isotonic sodium chloride solution. This was clearly obvious by the improvement of nasal obstruction, dryness, crusting, burning and itching associated with aging triggered nasal symptoms in the elderly with no carryover effects (Oysu et al. 2014). Major constituents of Nigella sativa are familiar by possessing various biological activities that could be formulated in different topical formulations with cosmeceutical potential. Thymoquinone represents a major constituent in Nigella sativa and

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was found to be an effective topical drug that combats inflammation. However it is greatly affected by heat and light in addition to being lipophilic that hinders its utilization, thus it was formulated in different advanced forms to perfectly increase its activity. It was formulated as an optimized nanocarrier preparation that was further assessed for its anti-inflammatory activity using carrageenan-induced paw in rats. The optimized formulation revealed an outstanding activity at a low dose suggesting its higher absorption potential via the stratum corneum (Alotaibi et  al. 2018). Furthermore, it was prepared in the form of liposomes that were loaded with thymoquinone and incorporated into chitosan gel to be topically applied. This modified formulation was compared to normal chitosan gel formulation in which the former showed superior activity compared to the latter approaching that of indomethacin gel in addition to its agreeable skin penetration and rheological characters (Mostafa et al. 2018). The polyphenol-rich fraction of Nigella sativa seeds was investigated for its anti-­ inflammatory and analgesic potential in vivo employing different models namely carrageenan-induced paw edema, acetic acid-induced writhing, croton oil-induced ear edema and formalin, light tail flick as well. Nigella sativa seeds successfully reduced the number of abdominal constrictions in writhing test induced by acetic acid when administered orally in addition to suppressing the nociceptive response in the early and late stages in formalin test with a dose-dependent pattern. In contrast, Nigella sativa seeds revealed no effective analgesia in the light tail-flick test in the animal model and exerted no anti-inflammatory effect in croton oil-induced ear edema when applied topically. It displayed a dose-dependent inhibition to paw edema when administered intraperitoneally only with no observed activity upon oral administration (Ghannadi et al. 2005). Meanwhile, the polyunsaturated fatty acid-rich fraction of black cumin seeds that are rich in octadecenoic acid and/or octadecadienoic acid was found to be highly effective as an antifungal and antibacterial agent and in ameliorating skin disorders, allergic reactions, hemorrhoids, fissures, pain, inflammation as well as septic wounds (Kandil 2010). In addition, sterol rich fraction of Nigella sativa seeds represented mainly by β-amyrin, campesterol, β-sitosterol and stigmasterol was found to be effective in the treatment and prohibition of vaginal diseases, bacterial and fungal infections, allergic reactions, pain and inflammation as well. It was formulated as vaginal suppositories that displayed high efficacy in the treatment of vaginal moniliasis (Cheikh-Rouhou et al. 2008).

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Alotaibi, F. O., Mustafa, G., & Ahuja, A. (2018). Study of enhanced anti-inflammatory potential of Nigella sativa in topical nanoformulation. International Journal of Pharmacy and Pharmaceutical Sciences, 10, 41–51. Aruoma, O., Spencer, J., Rossi, R., Aeschbach, R., Khan, A., Mahmood, N., Munoz, A., Murcia, A., Butler, J., & Halliwell, B. (1996). An evaluation of the antioxidant and antiviral action of extracts of rosemary and Provencal herbs. Food and Chemical Toxicology, 34, 449–456. Badri, W., El Asbahani, A., Miladi, K., Baraket, A., Agusti, G., Nazari, Q. A., Errachid, A., Fessi, H., & Elaissari, A. (2018). Poly (ε-caprolactone) nanoparticles loaded with indomethacin and Nigella sativa L. essential oil for the topical treatment of inflammation. Journal of Drug Delivery Science and Technology, 46, 234–242. Bhalani, U., & Shah, K. (2015). Preparation and evaluation of topical gel of Nigella sativa (KALONJI). International Journal of Research and Development in Pharmacy & Life Sciences, 4, 1669–1672. Büyüköztürk, S., Gelincik, A., Özşeker, F., Genç, S., Şavran, F. O., Kıran, B., Yıllar, G., Erden, S., Aydın, F., & Çolakoğlu, B. (2005). Nigella sativa (black seed) oil does not affect the T-helper 1 and T-helper 2 type cytokine production from splenic mononuclear cells in allergen sensitized mice. Journal of Ethnopharmacology, 100, 295–298. Çanakci, H., Yilmaz, A. A. S., Canpolat, M. S., Seneldir, H., Kir, G., Eris, A. H., Mayadagli, A., & Oysu, Ç. (2018). Evaluation of the effect of topical application of Nigella sativa on acute radiation-induced nasal mucositis. Journal of Craniofacial Surgery, 29, e279–e282. Chawla, R., & Chauhan, A. (2016). Transdermal delivery of Nigella sativa oil for topical application in 1-chloro-2, 4-dinitrobenzene-induced atopic dermatitis in rats. Journal of PharmaSciTech, 6, 51–59. Cheikh-Rouhou, S., Besbes, S., Blecker, C., Deroanne, C., & Attia, H. (2008). Black cumin (Nigella sativa L.) and Aleppo pine (Pinus halepensis mill.) seed oils: Stability during thermal oxidation at 60 C and 100 C. Microbiology Hygiene, 19, 12–20. Crutchfield, C. (2008). Topical steroid spray with botanic seed oils, Google Patents. Dahri, A. H., Chandio, A. M., Rahoo, A. A., & Memon, R. A. (2005). Effect of Nigella sativa (kalonji) on serum cholesterol of albino rats. Journal of Ayub Medical College Abbottabad, 17, 1–6. Datta, A. K., Saha, A., Bhattacharya, A., Mandal, A., Paul, R., & Sengupta, S. (2012). Black cumin (Nigella sativa L.) – A review. Journal of Plant Development Sciences, 4, 1–43. Dweck, A. (1997). African fragranced plants. Cosmetics and Toiletries, 112, 47–56. Dwita, L. P., Yati, K., & Gantini, S. N. (2019). The anti-inflammatory activity of Nigella sativa balm sticks. Scientia Pharmaceutica, 87(3). https://doi.org/10.3390/scipharm87010003. El-Dakhakhny, M., Mady, N., Lembert, N., & Ammon, H. (2002). The hypoglycemic effect of Nigella sativa oil is mediated by extrapancreatic actions. Planta Medica, 68, 465–466. Farrag, A.-R. H., Mahdy, K. A., Abdel Rahman, G. H., & Osfor, M. M. (2007). Protective effect of Nigella sativa seeds against lead-induced hepatorenal damage in male rats. Pakistan Journal of Biological Sciences, 10, 2809–2816. Fathima, A., Varma, S., Jagannath, P., & Akash, M. (2011). A general review on herbal cosmetics. International Journal of Drug Formulation and Research, 2, 140–165. Fisher, G. J., Kang, S., Varani, J., Bata-Csorgo, Z., Wan, Y., Datta, S., & Voorhees, J. J. (2002). Mechanisms of photoaging and chronological skin aging. Archives of Dermatology, 138, 1462–1470. Ghannadi, A., Hajhashemi, V., & Jafarabadi, H. (2005). An investigation of the analgesic and anti-inflammatory effects of Nigella sativa seed polyphenols. Journal of Medicinal Food, 8, 488–493. Ghorbanibirgani, A., Khalili, A., & Rokhafrooz, D. (2014). Comparing Nigella sativa oil and fish oil in treatment of vitiligo. Iranian Red Crescent Medical Journal, 16. https://doi.org/10.5812/ ircmj.4515.

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Gilani, A., Aziz, N., Khurram, I., Chaudhary, K., & Iqbal, A. (2001). Bronchodilator, spasmolytic and calcium antagonist activities of Nigella sativa seeds (Kalonji): A traditional herbal product with multiple medicinal uses. Journal of Pakistan Medical Association, 51, 115–119. Grace, R. (2002). Cosmeceuticals: Functional food for the skin. Natural Foods Merchandiser, 23, 92–99. Grindlay, D., & Reynolds, T. (1986). The Aloe vera phenomenon: A review of the properties and modern uses of the leaf parenchyma gel. Journal of Ethnopharmacology, 16, 117–151. Harding, C., Moore, A., Rogers, S., Meldrum, H., Scott, A., & McGlone, F. (2002). Dandruff: A condition characterized by decreased levels of intercellular lipids in scalp stratum corneum and impaired barrier function. Archives of Dermatological Research, 294, 221–230. Hosseinzadeh, H., & Parvardeh, S. (2004). Anticonvulsant effects of thymoquinone, the major constituent of Nigella sativa seeds, in mice. Phytomedicine, 11, 56–64. Huseini, H. F., Kianbakht, S., Mirshamsi, M. H., & Zarch, A. B. (2016). Effectiveness of topical Nigella sativa seed oil in the treatment of cyclic mastalgia: A randomized, triple-blind, active, and placebo-controlled clinical trial. Planta Medica, 82, 285–288. Isik, A. F., Kati, I., Bayram, I., & Ozbek, H. (2005). A new agent for treatment of acute respiratory distress syndrome: Thymoquinone. An experimental study in a rat model. European Journal of Cardio-Thoracic Surgery, 28, 301–305. Kalus, U., Pruss, A., Bystron, J., Jurecka, M., Smekalova, A., Lichius, J.  J., & Kiesewetter, H. (2003). Effect of Nigella sativa (black seed) on subjective feeling in patients with allergic diseases. Phytotherapy Research, 17, 1209–1214. Kandil, O. (2010). Polyunsaturated fatty acid fractions of Nigella sativa L. seeds, Google Patents. Kazmi, A., Khan, M.  A., & Ali, H. (2019). Biotechnological approaches for production of bioactive secondary metabolites in Nigella sativa: An up-to-date review. International Journal of Secondary Metabolite, 6, 172–195. Kobayashi, N., Suzuki, R., Koide, C., Suzuki, T., Matsuda, H., & Kubo, M. (1993). Effect of leaves of Ginkgo biloba on hair regrowth in C3H strain mice. Yakugaku zasshi: Journal of the Pharmaceutical Society of Japan, 113, 718–724. Kooshki, A., Forouzan, R., Rakhshani, M. H., & Mohammadi, M. (2016). Effect of topical application of Nigella sativa oil and oral acetaminophen on pain in elderly with knee osteoarthritis: a crossover clinical trial. Electronic Physician, 8, 3193. https://doi.org/10.19082/3193. Mansi, K. M. S. (2005). Effects of oral administration of water extract of Nigella sativa on serum concentrations of insulin and testosterone in alloxan-induced diabetic rats. Pakistan Journal of Biological Sciences, 8, 1152–1156. Michalsen, A., Eddin, O., & Salama, A. (2016). A case series of the effects of a novel composition of a traditional natural preparation for the treatment of psoriasis. Journal of Traditional and Complementary Medicine, 6, 395–398. Mohamed Alsamarai, A., Abdulsatar, M., & Hamed Ahmed Alobaidi, A. (2014). Evaluation of topical black seed oil in the treatment of allergic rhinitis. Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 13, 75–82. Mostafa, M., Alaaeldin, E., Aly, U. F., & Sarhan, H. A. (2018). Optimization and characterization of thymoquinone-loaded liposomes with enhanced topical anti-inflammatory activity. AAPS PharmSciTech, 19, 3490–3500. Nadkarni, K. (1976). Indian Materia Medica. Mumbai: Popular Prakashan Ltd.. Naz, H. (2011). Nigella sativa: The miraculous herb. Pakistan Journal of Biochemistry and Molecular Biology, 44, 44–48. Ortiz, G., Terron, M., & Bellido, J. (1997). Contact allergy to henna. International Archives of Allergy and Immunology, 114, 298–299. Oysu, C., Tosun, A., Yilmaz, H.  B., Sahin-Yilmaz, A., Korkmaz, D., & Karaaslan, A. (2014). Topical Nigella sativa for nasal symptoms in elderly. Auris Nasus Larynx, 41, 269–272. Pietta, P.-G. (2000). Flavonoids as antioxidants. Journal of Natural Products, 63, 1035–1042. Saha, R. (2012). Cosmeceuticals and herbal drugs: Practical uses. International Journal of Pharmaceutical Sciences and Research, 3, 59–65.

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Salem, M. L. (2005). Immunomodulatory and therapeutic properties of the Nigella sativa L. seed. International Immunopharmacology, 5, 1749–1770. Sarac, G., Kapicioglu, Y., Sener, S., Mantar, I., Yologlu, S., Dundar, C., Turkoglu, M., & Pekmezci, E. (2019). Effectiveness of topical Nigella sativa for vitiligo treatment. Dermatologic Therapy, e12949. https://doi.org/10.1111/dth.12949. Sayed-Ahmed, M.  M., & Nagi, M.  N. (2007). Thymoquinone supplementation prevents the development of gentamicin-induced acute renal toxicity in rats. Clinical and Experimental Pharmacology and Physiology, 34, 399–405. Sharma, Y., Bashir, S., Irshad, M., Gupta, S. D., & Dogra, T. (2005). Effects of acute dimethoate administration on antioxidant status of liver and brain of experimental rats. Toxicology, 206, 49–57. Singhal, M., Khanna, S., & Nasa, A. (2011). Cosmeceuticals for the skin: An overview. Asian Journal of Pharmaceutical Clinical Research, 4, 1–6. Sudhir, S., Deshmukh, V., & Verma, H. (2016). Nigella sativa seed, a novel beauty care ingredient: A review. International Journal of Pharmaceutical Sciences and Research, 7, 3185–3196. Sulaiman, F., Khiljee, S., Rehman, N. U., Ishaque, S., Masood, M. I., & Anjum, S. M. M. (2014). Formulation of microemulsion containing Nigella sativa honey and propolis and evaluation of its burn healing potential. International Journal of Pharmaceutical Sciences Review and Research, 27, 1–9. Takahashi, T., Kamiya, T., & Yokoo, Y. (1998). Proanthocyanidins from grape seeds promote proliferation of mouse hair follicle cells in vitro and convert hair cycle in vivo. Acta Dermato-­ Venereologica, 78, 428–432. Tariq, M. (2008). Nigella sativa seeds: folklore treatment in modern day medicine. Saudi Journal of Gastroenterology, 14, 105–106. Torres-León, C., Rojas, R., Contreras-Esquivel, J.  C., Serna-Cock, L., Belmares-Cerda, R.  E., & Aguilar, C. N. (2016). Mango seed: Functional and nutritional properties. Trends in Food Science & Technology, 55, 109–117. Uz, E., Bayrak, O., Uz, E., Kaya, A., Bayrak, R., Uz, B., Turgut, F. H., Bavbek, N., Kanbay, M., & Akcay, A. (2008). Nigella sativa oil for prevention of chronic cyclosporine nephrotoxicity: An experimental model. American Journal of Nephrology, 28, 517–522. Widyarini, S., Spinks, N., Husband, A. J., & Reeve, V. E. (2001). Isoflavonoid compounds from red clover (Trifolium pratense) protect from inflammation and immune suppression induced by UV radiation. Photochemistry and Photobiology, 74, 465–470. Yaman, İ., & Balikci, E. (2010). Protective effects of Nigella sativa against gentamicin-induced nephrotoxicity in rats. Experimental and Toxicologic Pathology, 62, 183–190. Yousefi, M., Barikbin, B., Kamalinejad, M., Abolhasani, E., Ebadi, A., Younespour, S., Manouchehrian, M., & Hejazi, S. (2013). Comparison of therapeutic effect of topical Nigella with betamethasone and eucerin in hand eczema. Journal of the European Academy of Dermatology and Venereology, 27, 1498–1504. Zaher, K. S., Ahmed, W., & Zerizer, S. N. (2008). Observations on the biological effects of black cumin seed (Nigella sativa) and green tea (Camellia sinensis). Global Veterinaria, 2, 198–204. Zaoui, A., Cherrah, Y., Mahassini, N., Alaoui, K., Amarouch, H., & Hassar, M. (2002). Acute and chronic toxicity of Nigella sativa fixed oil. Phytomedicine, 9, 69–74.

Chapter 17

Nigella sativa Supplementation in Ruminant Diets: Production, Health, and Environmental Perspectives Yasmina M. Abd El-Hakim, Adham A. Al-Sagheer, Asmaa F. Khafaga, Gaber E. Batiha, Muhammad Arif, and Mohamed E. Abd El-Hack Abstract  Currently, ruminant production faces a vital challenge in the availability of feed resources in the region of production. In most tropics and developing countries, ruminants are limited to grazing on low-quality forages, crop residues, and agro-industrial by-products with very minimal concentrate diets, which negatively affect the animals in achieving their full production potential. Hence, researchers have explored nutritional strategies depending on the natural feed supplements to improve the functions of rumen microflora, enhance fermentation processes and digestion, along with increase nutrients bioavailability and utilization. In particular, plant biologically active compounds are explored owing to the prohibition on the non-therapeutic use of antibiotics as growth promoters together with the critical preference of consumers to high quality and safe animal products. Nigella sativa is

Y. M. Abd El-Hakim Department of Forensic Medicine and Toxicology, Zagazig University, Zagazig, Egypt A. A. Al-Sagheer Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig, Egypt A. F. Khafaga Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, Edfina, Egypt G. E. Batiha National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan Department of Pharmacology and Therapeutics, Faculty of Veterinary Medicine, Damanhour University, Damanhour, AlBeheira, Egypt M. Arif Department of Animal Sciences, College of Agriculture, University of Sargodha, Sargodha, Pakistan M. E. Abd El-Hack (*) Department of Poultry, Faculty of Agriculture, Zagazig University, Zagazig, Egypt e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_17

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among the lesser-known plants that hold promise as a natural additive to improve the production performance of animals. N. sativa is cropped mainly for seed ­production which contains an array of secondary compounds like nigellone, flavonoids, tannins, saponins, resin, and volatile oils that could have a positive impact on animal health. N. sativa meal contains a high protein content of up to 18%. Additionally, the oil of N. sativa has been successfully used in animal feeding. This chapter aims to explore the positive effects of supplementation of ruminant diets with N. sativa on the productivity and health of the animals with a special emphasis on the anti-­methanogenic activity of N. sativa. Keywords  Nigella sativa · Ruminant · Growth · Nutrient digestibility · Methane emissions · Meat quality

1  Introduction and Objectives Nowadays, the consumer is increasingly demanding food safety and quality, especially concerning animal-derived products including red meats and milk because of concerns about animal production systems, like the use of feeds containing synthetic chemicals as growth promoters (Castillo et  al. 2019). So, the trend is the development of production systems that afford consumer safety, sustainability, and respect for animal welfare (Font-i-Furnols and Guerrero 2014). Research in ruminant production is thus dedicating efforts for the development of nutritional interventions, especially for livestock production in difficult environments, to improve the animal product quality (Cherif et al. 2018b). One of the most successful attempts accomplished in the last decade is using natural feed additives like medicinal plants seeds, leaves, roots, extract, oil, or meal. Beneficial effects of medicinal plants in farm animals may arise from the activation of feed intake and the secretion of digestive secretions, immune stimulation, and anti-bacterial effects. Herbs can also contribute to the nutrient requirements of the animals, stimulate the endocrine system and intermediate nutrient metabolism (FrAnKIČ et al. 2009). Also, these plants contain specific phytochemicals that could impact positively on digestion, metabolic utilization, hence further improve the production performance of animals (Makkar et al. 2007). Consequently, these supplements could strongly assist in improving animal productivity and milk production as alternative growth promoters to synthetic chemicals (Campanile et  al. 2008; Valenzuela-Grijalva et al. 2017b; Wang et al. 2009). Nigella sativa, commonly known as black seed, is one of the very important widely grown medicinal plants which used for centuries for the treatment of many complaints and disease (Mariod et al. 2017). Various feeding trials have been previously conducted to establish the functionality of N. sativa supplementation either as

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a meal, seed, or oil in ruminant’s diet on their growth. Initially, N. sativa meal after oil removal can be used as a protein source in animal ration because of its high protein (31–33%) and fat (11–13%) content together with the presence of major minerals such as Ca, P, K, Mg and Na (El-Nattat and El-Kady 2007). But, the oil extraction process could affect the crude protein content. El-Rahman et al. (2011) reported that crude protein in N. sativa meal was 28.8%, but Abdel Magid et al. (2007) reported that N. sativa meal contained 37.4% protein. According to Al-Gaby (1998), the seeds of N. sativa are rich in protein (about 200 g/kg DM), with a glutamic acid dominance (22.4%), aspartic acid (10.05%) and arginine (9.18%). Thus, it could be a very good source of protein. Additionally, Paarakh (2010) claimed that N. sativa seeds contain an array of secondary compounds comprising nigellone, resin, tannins, saponins, flavonoids, and volatile oils that could have a positive impact on animal health. The seed oil of N. sativa contains myristic, palmitic, and stearic as major saturated fatty acids, whereas oleic, linoleic, and linolenic were the main unsaturated fatty acids. The oil contains many bioactive compounds include p-cymene, thymoquinone, α-thujene, longifolene, β-pinene, α-pinene, and carvacrol (Ramadan 2016). Several studies reported the importance of N. sativa as an unconventional source of energy and protein in animal feeding and its capacity to improve the productive performance of different species of ruminants by enhancing the digestibility of nutrients supplied with the diet (Abdel Magid et  al. 2007; Mohamed 2007). For instance, El-Naggar et al. (2019) verified N. sativa oil could be added at 0.2% of the daily feed as a natural growth enhancer of lambs. Also, Cherif et al. (2018a) reported that the addition of 12 g/day N. sativa seeds to the diets, either low or high concentrate, of Barbarine lamb, significantly increased their growth rate with diets. This chapter offers an overview of the effect of dietary N. sativa on ruminant health and production together with the quality of ruminants’ products. Additionally, special emphasis was paid on their impact on ruminal methanogenesis and consequently methane emissions.

2  Effects of Nigella sativa on Growth Performance and Nutrients Digestibility Incorporating natural products in a ruminant diet to improve their growth performance and to reduce cost ration formula in practical feeding is urgently needed. In this regard, many herbs have reported to stimulate rumen micro-flora activity via saving some micro factors to rumen micro-flora including microelements, vitamins, hormones and enzymes which are required to the efficient digestion, absorption and metabolism (Aboul-Fotouh et  al. 2000; Al-Sagheer et  al. 2018) and/or reducing effectively mycotoxins hazards by inhibition fungal growth and aflatoxins production (Allam et al. 1999; Mohamed et al. 2003).

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In this regard, Awadalla (1997) reported that replacement of sunflower meal-­ protein at 0, 50 and 100% by N. sativa meal in the concentrate mixture of growing sheep resulted in no significant differences in all values of nutrients digestibility or the nutritive values in terms of total digestible nutrients (TDN) or digestible crude protein (DCP). Also, Gabr et al. (1998) reported an improvement in nutrients ­digestibility and nutritive values in terms of TDN fed lambs on concentrate feed mixtures in which soybean meal-protein was replaced by N. sativa meal protein at 20% and 40%. Aliam (1999) studied the effect of supplementing four medicinal herbs and plants including garlic, fenugreek, N. sativa, and chamomile to Zaraibi goat ration on their performance. Results showed improvement in digestibility coefficients of dry matter (DM), organic matter (OM), crude protein (CP) together with the nutritive value as TDN and DCP for all additive groups. A ration with N. sativa showed the highest nutritive values, and milk yield is increased in all treatment groups fed by supplementation. Also, El-Ayek et al. (1999) reported that N. sativa meal could participate successfully and economically by about 50% of the protein in formulating concentrate feed mixture in diets of growing lambs without any adverse effects on animal performance. El-Hosseiny et al. (2000) reported that the addition of N. sativa seeds to the diet of suckling and growing lambs significantly increased final body weight (FBW) and daily weight gain (DWG). Also, El-Rahman et al. (2011) indicated that Demeshgi goats fed 20% N. sativa seeds showed significantly high DWG. The high production performance of lamb fed on diet fortified with N. sativa seed could be high linked to its high essential fatty acids content particularly linolenic, oleic, and linoleic acids that are necessary for the body growth (Babayan et al. 1978). Also, the stimulating effect of N. sativa seeds on digestive utilization could be another reason (Ramakrishna Rao et al. 2003). In this regard, the addition of N. sativa seed in feed increased the bile flow rate, which consequently facilitates fat digestion and absorption of fat-soluble vitamins (Crossland and Lewis 1980). In another experiment in growing crossbred (Friesian x local Egyptian bull calves), Abdel Magid et al. (2007) investigated the effect of substituting soybean meal of concentrate feed mixture protein by N. sativa meal at 0, 30 and 60% in the rations. The result showed that the substitution of 30% and 60% soybean meal protein by N. sativa meal showed non-significant improvement of almost nutrients digestibility and nutritive values as TDN.  However, nutritive values as CP were increased with different substitution rations. Also, the substitution of 30% or 60% of soybean meal protein by N. sativa meal significantly improved average daily gain and feed conversion in terms of total dry matter intake (DMI) gain and TDN intake. Notably, relative economic efficiency was higher for calves fed diet with substitution of 30% or 60% of soybean meal protein with N. sativa meal. In a 90 day feeding trial in growing Demeshgi goats, El-Rahman et al. (2011) found that the highest value of DWG was recorded for goats fed ration contained 20% N. sativa meal. Habeeb and El Tarabany (2012) showed that the addition of N. sativa to the diet of Zaraibi kids increased significantly DMI when receiving concentrate mixture and Trifolium alexandrinum hay. The bitter and pungent taste

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of the N. sativa seeds is incurred by their phenolic compounds and essential oils (Aggarwal and Kunnumakkara 2009). The animal could have changed his feeding behavior to dilute this unpleasant taste by consuming more feeds free of these secondary compounds, hence to stimulate the appetite (Gilani et al. 2004). Mahmoud and Bendary (2014) conducted two experiments to investigate the effect of the whole substitution of soybean meal and cottonseed meal by N. sativa meal and sesame seed meal on growth performance, and nutrients digestibility in growing Barki lambs and growing calves ration. The authors evaluated the economic efficiency of such a replacement. Results of chemical composition indicated that N. sativa meal and sesame meal were rich in protein and fat 33.1%, 32.3% and 12.7%, 15.4%, respectively. In both growing Barki lambs and growing calves, N. sativa meal and sesame meal replacement resulted in an increase in DWG and FBW by 125 g and 15 kg, respectively, compared to control group without significant change in digestibility values of DM, OM, CP, ether extract (EE) and nitrogen-­ free extract (NFE). But, crude fiber (CF) recorded lower value of the digestion coefficient, which may be linked to the depressing effect of the higher fat content of N. sativa on rumen flora and consequently fiber digestibility in sheep (Devendra and Lewis 1974). The same tendency was observed by El-Gaafarawy et al. (2003) and El-Rahman et  al. (2011) with sheep fed different levels of N. sativa cake as an unconventional source of protein. In the study of Mahmoud and Bendary (2014) relative economic efficiency was increased by 57% with replacement N. sativa meal and sesame seed meal for cottonseed meal and soybean meal. The effect of the extraction method could modify the effect of N. sativa on nutrient digestibility (Hassan et  al. 2014). N. sativa water extract had no significant effect on in-vitro DM and OM digestion coefficients. But, the addition of alcoholic extract to the diet increased the digestion coefficient. This could be related to two issues. Firstly, low total phenolics content by alcoholic extraction and second, alcohol can dissolve low chain fatty acids which have a great positive effect on increasing ruminant microflora as a source of energy and 60.8% of oil in N. sativa seeds as linoleic acid (Tawffek and Hassan 2014).

3  Effects of Nigella sativa on Milk Production and Composition Kholif and El-Gawad (2001) evaluated the effects of N. sativa seeds dietary supplementation in the diets of lactating goats on milk yield and composition and its effect on Domiati cheese. N. sativa decreased total solids, fat and total nitrogen soluble nitrogen, and pH of the milk. The organoleptic properties of treated cheese were better than the control, except in color, storage period significantly affected organoleptic properties and cheese composition (Fig. 17.1). Nurdin et  al. (2011) evaluated the effect of N. sativa supplementation on milk yield and milk quality in lactating dairy cows suffering subclinical mastitis. The results showed that N. sativa supplementation significantly increased milk yield, milk

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Fig. 17.1  Effect of dietary supplementation of Nigella Sativa on milk yield and composition in ruminants

protein, milk lactose, and significantly decreased mastitis status and did not significant affect milk fat. In a 12  weeks feeding trial in lactating goats, Galbat et  al. (2014) found that rations supplemented with N. sativa, Cuminum cyminum, Trigonella foenum graecum seeds, Carum carvi in equal ratio showed the best improvement of milk production, and milk composition compared with animals fed on the control diet without deleterious effects on the general health of the animals. Also, in a 13 weeks experiment in indigenous lactating cows, supplementation of 80  g/day N. sativa significantly increased milk production (Goswami et al. 2018). The enhancement of milk production and composition with N. sativa dietary supplementation could be highly associated with its modulatory role of rumen ecology by virtue of its antioxidant compound and anti-inflammation potential can increase alveolar cell permeability and can assist in rumen ecological balance. Rumen ecology influences the quality and quantity of dairy cows’ production. For instance, a large microorganism population is responsible to increase volatile fatty acids (VFA) production (acetic acid and propionic acid). Acetic acid is a function of milk fat precursor and propionic acid functions for glucose synthesis. N. sativa helps in establishing better conditions of rumen ecology. As a result, the number of rumen bacteria and total VFA will increase and followed by a reduction of NH3 concentration. A large microorganism population is responsible for increased VFA

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production. When this population increased, it results in the presence of greater production of milk or meat (Kalscheur et al. 2006; Wang et al. 2007).

4  Effects of Nigella sativa on Carcass Traits and Meat Quality Lately, improving the safety, nutritional, and sensory quality of ruminant meat to cope with the rapidly changing requirements of consumers have been the subject of research (Odhaib et al. 2018a). It has been established that nutritional strategy is more effective in enhancing the oxidative stability of meat when compared to exogenous addition of antioxidants because dietary antioxidants are preferentially deposited where they are most needed (Nieto et al. 2012). In addition, dietary intervention remains the most effective strategy to modify the oxidative stability of intact muscle foods, where the use of exogenous antioxidant may be difficult or practically impossible (Adeyemi et al. 2016). Nonetheless, the effects of medicinal plants on livestock product quality are highly variable and inconsistent in the published literature (Nieto et al. 2012; Qwele et al. 2013). Figure 17.2 summarizes the effect of dietary supplementation of N. Sativa on meat quality parameters in ruminants.

Fig. 17.2  Effect of dietary supplementation of Nigella Sativa on meat quality parameters in ruminants

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Dietary supplementation of medicinal plants to livestock has been advocated as an effective strategy for improving production performance (Valenzuela-Grijalva et al. 2017a) of livestock as well as the quality and storage stability of animal products (Karami et al. 2011). The use of medicinal plants is favored due to the h­ azardous effects of synthetic antioxidants on human health (Nieto et al. 2012). In particular, plant phenolics, such as those found in N. sativa, when supplemented in ruminant diets could manipulate rumen biohydrogenation of unsaturated fatty acids, thereby modifying the fatty acid composition of ruminant meat (Yusuf 2014). In Awassi and Karadi lambs, N. sativa supplementation improved killing-out proportions and lean to fat ratio (Hassan 2009; Hassan et al. 2010). This could be linked to the potency of N. sativa to increase the total volatile fatty acid production in the rumen, which caused differences in lipids thickness and its deposition in the animal body (Huck et al. 2000). In a 9-week feeding trial in karadi lambs, Hassan et al. (2011) tested the effect of two levels of N. sativa (0 and 7.5  g/kg DM) supplementation with two levels of rumen non-degradable nitrogen (7 and 10  g/kg DM) on their rations on carcass characteristics. Slaughter body weight, hot and cold carcass weights, and killing out proportion were not significantly affected by both N. sativa supplementation and levels of non-degradable nitrogen. Moreover, no differences in leg cut tissue (lean, subcutaneous fat: intramuscular fat and bone) were recorded. The main wholesale cuts weights were not significantly different among treatments. The differences in fat-tail weights between the N. sativa and non-degradable nitrogen treatments were not statistically significant. Cherif et al. (2018b) reported that N. sativa seeds dietary supplementation significantly increased intramuscular fat in the meat of lamb fed a low-concentrate diet, which is a typical feeding system of the Mediterranean and semi-arid regions. Moreover, the supplementation of N. sativa seeds reduced the lipid oxidation of meat during refrigerated storage, regardless of the feeding system. However, Cherif et al. (2018a) reported that carcass yield was not affected by the addition of N. sativa seeds to high concentrations and low concentrate diets. Odhaib and Sazili (2018) examined the influence of 90 days dietary supplementation of 1% N. sativa seeds on carcass attributes, fatty acid composition, gene expression, lipid oxidation, and physicochemical properties of longissimus dorsi, semitendinosus, and supraspinatus muscles in Dorper lambs. Also, slaughtered and the muscles were subjected to a 7 days postmortem chill storage. The lambs fed N. sativa supplemented diet had greater slaughter and cold carcass weights than the control lambs. Dietary supplements did not affect chill loss, dressing percentage, carcass composition, intramuscular fat, and muscle pH in Dorper lambs. Meat from supplemented lambs had lower cooking and drip losses, shear force, lightness, and lipid oxidation and greater redness compared with the control meat. The impact of dietary supplements on muscle fatty acids varied with muscle type. N. sativa seeds dietary supplementation had no effect on the expression of stearoyl-CoA desaturase and lipoprotein lipase genes in LD and ST muscles in Dorper lambs. N. sativa seeds dietary supplementation diets up-regulated the expression of AMP-activated protein kinase alpha 2 gene in LD and ST muscles and up-regu-

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lated the expression of sterol regulatory element-binding protein 1 in ST muscle in Dorper lambs. Odhaib et  al. (2018a) reported that the supplementation of N. sativa seeds reduced drip loss in different muscles in Dorper lambs. The reduction in drip and cooking losses could be due to the presence of antioxidant compounds in the ­supplements, which reduced the oxidation of myofibrillar proteins during postmortem chill storage. Also, the meat from the N. sativa supplemented lambs had greater redness than the meat from the control lambs. This observation could be due to the antioxidant effect of the phenolics in the supplements, which prevented oxidative deterioration. Additionally, dietary supplementation of N. sativa up-regulated the expression of sterol regulatory element-binding transcription factor 1gene in semitendinosus muscle in Dorper lambs compared with that of the control lambs. This observation could be attributed to the greater concentration of C18:1n-9 and C18:3n-3.

5  Effects of Nigella sativa on Ruminal Fermentation and Methane Production Many herbs and plant extracts have antimicrobial activities against a wide range of bacteria, yeasts, and molds (Castillejos et al. 2006). Recent studies have shown that plant secondary metabolites at lower concentrations could be used to manipulate rumen fermentation favorably. Alterations in rumen fermentation can increase overall animal efficiency by increasing energy digestibility and metabolizability (Olagaray and Bradford 2019). Another important aspect is that high-quality forage is considered as the main livestock diet during the dry season in most of the tropics as well as sub-tropics. Unfortunately, due to the high methanogenic potential (Adjorlolo et  al. 2016), it significantly contributes to the global warming by producing up to 18% of the anthropogenic greenhouse gases, representing 37% of the total anthropogenic methane (CH4) emission (FAO 2006). In addition, unwanted emission of CH4 may account for up to 12% loss of the gross energy of feeds (Eckard et  al. 2010). Interestingly, the inclusion of various medicinal plants in ruminant diets efficiently lowered the CH4 emission (Hariadi and Santoso 2010; Tiemann et  al. 2008). However, a major drawback of implementing diets fortified with some plants aimed at reducing CH4 emissions is the decline in the digestibility of the feeds along with lower productivity of the animals (Beauchemin and McGinn 2006; Broudiscou et al. 2002). The absence of effects on the degradation of substrates in response to certain plant extracts have reduced methane production (Al-Sagheer et  al. 2018; Śliwiński et al. 2002). In this regard, N. sativa seeds are rich of bioactive compounds, such as essential oils (Singh et al. 2005), which are able to impair the biohydrogenation of polyunsaturated fatty acids (PUFA) in the rumen due to the antimicrobial activity of the isoprene moiety of the terpenes (Vasta and Luciano 2011).

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Meziti (2009) reported that the provision of N. sativa seeds in the ruminant diet reduced the allantoin excretion in urine, which could be ascribed to their antimicrobial effect explaining the reduction of microbial N production. Allantoin, is the predominant purine derivative and had been recommended as an indicator of the microbial protein synthesis (Rys et al. 1975). The use of N. sativa meal at 10% and 20% in the feed significantly improved the performance of lambs but increases levels of blood urea nitrogen reflecting an increase in protein degradation in the rumen (Barkah 2017). The high degradation of feed protein in the rumen may result in inefficiency because of the amount of protein degraded before it reaches the intestine to be utilized by the whole body. Cherif et al. (2018a) reported that the addition of N. sativa seeds (12 g/day) to the concentrate of Barbarine lambs significantly increased CP intake and the concentration of ammonia nitrogen (NH3-N), but it decreased protozoa population in the rumen and the concentration of plasma triglycerides. In an in vitro experiment, (Barkah et al. 2019) evaluated the effect of N. sativa meal as a protein source and its combination with the readily available carbohydrates derived from pollard, rice bran, and corn for ruminant diet. The results showed that N. sativa meal has high NH3 concentration in the rumen, especially in the first 4 h and after 6 h of incubation time in rumen fluid in vitro. The combination of N. sativa meal in rations containing corn significantly suppressed NH3 concentration. In addition, N. sativa meal and its combination with corn in the diet produced microbial protein synthesis higher compared with other treatments. The use of N. sativa meal as much as 20% in rations and its combination with the readily available carbohydrates derived from corn can optimize the use of N. sativa meal as a source of feed protein in ruminants by maximizing the utilization of NH3 and VFA to encourage the production of microbial protein synthesis that was important for ruminants. Medjekal et al. (2017) evaluated the anti-methanogenic activity of N. sativa seed powder and their effect on fermentation patterns in an in vitro study using batch cultures of mixed rumen microorganisms. N. sativa seeds do not cause substantial changes in the general profile of fermentation suggesting that this compound does not affect the degradation of the substrate and is not toxic to microorganisms of the rumen to the used dose. But, N. sativa evoked a significant reduction in methane production, 20% less methane than control. The anti-methanogenic activity of N. sativa lipids and sterols could reduce the amount of methane while maintaining significant production of gas. Chowdhury et al. (2018) reported that the in vitro DM and OM degradability of rice straw was high in the presence of ground N. sativa seeds. Odhaib and Sazili (2018) evaluated the effects of different levels of N. sativa seeds (0, 0.5, 1, 1.5 and 2% DM of basal substrate, 60% forage, and 40% concentrate) on the in vitro gas production, rumen fermentation, fatty acids composition and the apparent biohydrogenation of oleic, linoleic and linolenic acids using rumen liquor from Dorper lambs. The volume of gas produced increased as the levels of N. sativa seeds increased up to 1.5% and decreased afterward. Supplementation of N. sativa seeds did not affect in vitro dry matter digestibility, in vitro organic matter

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digestibility, rumen pH, CH4, and NH3-N, total volatile fatty acids (VFA), and the molar proportion of acetate, propionate, and butyrate. In 90 days feeding experiment, El-Naggar et al. (2019) investigated the impact of supplementing two levels of N. sativa oil (0.1% and 0.2%) on rations of growing Ossimi lambs. Results indicated that additions of N. sativa oil didn’t influence feed intake, but it increased average body weight gain and feed efficiency. Nutrients digestibility values and dietary nitrogen utilization were higher with N. sativa oil rations than control. Ruminal fermentation parameters showed comparable pH values among groups. Ruminal NH3-N concentration was lower and TVFA concentration was higher. The study recommended the addition of N. sativa oil at 0.2% of the daily feed as a natural growth enhancer of lambs. The variation of the concentration of NH3-N in the rumen of sheep receiving N. sativa seeds could be the result of the effect of p-cymene which is the main component of oil in N. sativa seeds (Chaves et al. 2008). According to the later authors, p-cymene promotes the microbial deamination activity. In addition, the p-cymene in N. sativa oil has an inhibitory effect on bacterial peptidolysis and deamination processes (Busquet et al. 2006).

6  Effects of Nigella sativa on Ruminant Health and Blood Metabolites Many bacterial diseases affect animals, causing important economic losses in livestock. Subtherapeutic antibiotic use in production animals as antibiotic growth promoters has been implicated as a causativa factor in the development of resistance of bacterial pathogens toward several classes of antimicrobials. Alternatives to antibiotic growth promoters are the use of plant extracts or essential oils as supplements to provide beneficial effects, including direct antibacterial activity and stimulation of the immune system, or enhancement of ruminal digestion. Importantly, the risk of resistance developing to phytochemicals is greatly lower than the risk of resistance against antibiotics (McGaw 2013). In this regard, N. sativa contains many bioactive components with various pharmacological activities that could be highly beneficial to livestock health. For instance, N. sativa seeds meal contains thymoquinone that has antibacterial, diuretic, hypotensive and immunopotentiating activities via increasing the neutrophil percentage and hence increasing the defense mechanism of the body against infection (Kanter et al. 2005). N. sativa contains Zn, Cu, Mn, Mg, Se, vitamin C, vitamin A, vitamin E and folic acid which have a role in enhancing the immune system (Toma et al. 2013). Also, folic acid, Fe and vitamin C content in N. sativa have roles in red blood cell formation, maturation, and hem biosynthesis, absorption, and utilization (Mariod et al. 2017). Animals received N. sativa seeds-containing diets showed a significant reduction of triglycerides level (Abdel-Aal and Attia 1993). Also, Al-Beitawi et  al. (2009)

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reported that feeding crushed and uncrushed N. sativa seed at 3% reduced plasma cholesterol and triglycerides concentrations. The decrease of triglycerides concentration in lambs receiving N. sativa seeds is in line with the findings of El-Saadany et  al. (2008). The reduction in the cholesterol level might be due to the active ­ingredients such as thymoquinone and monounsaturated fatty acids (MUFA) that lower the cholesterol synthesis by hepatocytes and decrease the fractional absorption of cholesterol from the small intestine (Brunton 1996; Tollba 2003). In contrast, Maha et al. (2016) reported that adding of 4.7% of N. sativa oil to the diets of sheep, resulted in a significant elevation in serum total cholesterol, LDL-C, HDL-C, triglycerides concentrations, and the body weights. The rumen atmosphere may destroy the active constituents of N. sativa oil, especially thymoquinone which is considered as an active ingredient responsible for the pharmaceutical interest of the plant. Also, previous study reported that the high ratio of PUFA in the diet is a major lowering plasma cholesterol concentration by dietary means and hence N. sativa seeds containing unsaturated fatty acids may be the cause of reduction of serum cholesterol concentration in monogastric animals (Granner et al. 1988). But in the ruminant animals, although they consume a diet that predominantly contains PUFA as part of plant triglycerols and glycolipids, bacteria in the rumen split off the fatty acids (and sugar) from the glycerol backbone (Hydrolysis process), and the resulting free fatty acids are acted upon by microbial enzymes which convert them ultimately into saturated stearic acid (Biohydrogenation process) (Dawson and Kemp 1970). This suggested that, supplementation of N. sativa oil, to ruminants’ diet, act as additional source of PUFA which later will be converted to saturated fatty acids, passes to the small intestine, and absorbed. Glucose is the most vital carbohydrate in ruminant animal biochemistry. N. sativa seed were known to reduce blood glucose levels in different animal species (Haddad et al. 2003). In this context, Sanad (2000) reported that supplementing concentrate mixture with 50 mg/kg body weight/day N. sativa seeds reduced plasma glucose concentration by 19.8% in buffalo. However, Kaki et  al. (2018) reported that N. sativa seed dietary supplementation (30 g/kg DM) has no effect on the glucose level. El-Ekhnawy et al. (1999) found that N. sativa oilseed led to increasing glucose concentrations in Barki ewes fed the maintenance ration supplemented with 150 and 250 g N. sativa meal. This discrepancy could be related to the concentration effect. Enhancing the immune function of livestock is an effective strategy for combating the repeated global occurrence of criticizing infectious diseases, which cause massive losses to animal husbandry and constitute a health risk to consumers (Gupta et  al. 2016). Growing evidence proposes that diets could modify the immune response in animals (Taranu et  al. 2012). In this regard, the immune-modulatory properties of N. sativa have been reported (Salem 2005). The in vitro immunomodulatory activities of N. sativa extract on the macrophages sheep have been documented (Elmowalid et al. 2013). Also, Odhaib et al. (2018b) reported that dietary supplementation of N. sativa seeds in Dorper lambs for 90 days increased the serum IgA and IgG, but did not affect serum IgM in Dorper lambs. The increase in IgA and IgG could be attributed to the N. sativa antioxidant activity, which can modulate and

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regulate the early activation steps in the immune response like increasing phagocytes capacity against invading pathogens. Moreover, antioxidants protect immune cells and the surrounding tissue, from damage caused by the immune response, which otherwise could harm the animal as much or more than the disease organisms (Burits and Bucar 2000). Kumari and Akbar (2006) tested the clinical efficacy of some herbal preparations (combination of Leptadena reticulata, N. sativa, Foeniculum vulgare, Pueraria tuberosa and Asparagus racemosus) wherein supplementation was effective in curing digestive disorders and early restoration of normal milk production in lactating buffaloes. Korshom et al. (1998) investigated the antitrematodal activity of N. sativa seeds against a ruminant fluke (Paramphistomum) in sheep. EL-Ghousein (2010) reported no difference in RBCs, Hb, and WBCs in ewes fed 10 g/h/d N. sativa seed. The positive hematological impact of medicinal plants is related to their ingredients of folic acid, iron, and vitamin C, which are blood-forming factors that stimulate blood production in the bone marrow (Khattab et al. 2011). Habeeb and El Tarabany (2012) recorded that N. sativa significantly increased RBCs count in Zaraibi kids, while WBCs and PCV were not affected. El-Halim et al. (2014) reported a transient decrease in RBCs count in growing lambs fed N. sativa oil at 2 weeks, but it significantly improved after 6 weeks. Hendawy et al. (2019) reported that dietary supplementation of N. sativa fine powder enhanced red blood cells and white blood cells as well as milk composition of ewes. Zanouny et al. (2013) tested the effect of supplementation with N. sativa seeds (100 and 200 mg/kg bwt/day) on Ossimi male lambs blood biochemistry. N. sativa seeds at both levels significantly increased serum level of total protein, globulin testosterone, and thyroid hormones together with testes parameters, but reduced triglycerides concentrations. The increase in thyroid hormones with N. sativa seeds supplementation may be due to increased metabolism of carbohydrate, fat, and protein, which was reflected in a positive effect on the digestibility coefficient of carbohydrate, fat, and protein. Also, this could be linked to the increase of TDN intake and metabolisable energy as an indicator of energy. There was a positive relationship between energy intake and the concentration of thyroid hormones as reported by Kassab (2007). The increase of total protein and globulin may be due to the increase of the digestibility coefficient of CP expressed as DCP (El-Saadany et al. 2008). The increase in serum globulin concentration may also due to an immune stimulant effect of N. sativa seeds (Mohamed et al. 2003). The decrease of cholesterol concentration as a result of N.a sativa seeds supplementation may be due to the higher content of unsaturated fatty acids in N. sativa seeds (El-Saadany et al. 2008). Testosterone concentrations increased after N. sativa seeds supplementation due to the increase of nutritive values of rations, TDN, ME and DCP, which led to significant improvement in live body weight of lambs and also increasing the growth of different body organs including testes (Zanouny et al. 2013). It may be also, due to the increase in thyroid gland activity. The positive effect of supplementation with N. sativa seeds on reproductive performance may be due to its higher content of fatty acids. N. sativa seeds contained 26.6% oil, in which the major fatty acids in N. sativa seeds are oleic acid and palmitic acid (Nergiz and Ötleş 1993). Linoleic

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and arachidonic acids are essential fatty acids, which considered as a precursor for the biosynthesis of prostaglandin and it increases circulating of gonadotropin ­hormone and stimulates steroid hormones production (testosterone) that is essential for normal reproductive function of male animals (Hanna and Hafez 2018). Kaki et al. (2018) reported that N. sativa seed supplementation (30 g/kg DM) in fattening Sanjabi lambs significantly enhanced antioxidant parameters and lowered alanine aminotransferase and cholesterol concentrations.

7  Conclusion and Health Perspectives Achieving the desired production targets at least cost with little or no hazards on the environment is the hallmark of successful animal production. Dietary supplementation of N. sativa meal, seeds, or oil in ruminant nutrition could have production, environmental, and economic merits. N. sativa showed favorable effects on the growth performance and nutrient digestibility in ruminants. Also, health indicators in ruminant showed a significant improvement with the feeding of diets containing N. sativa. For consumers, it could afford the safety of meat and milk together with the high quality. Environmentally, this plant showed a significant efficiency in reducing methane emissions without cause substantial changes in the general profile of fermentation and does not affect the degradation of the substrate. With the above mentioned characteristics, N. sativa could be advantageously used as feed additives in livestock feeding.

8  Recommendations From the collective studies, one limitation of N. sativa supplementation in the ruminant diet was the rapid degradation in the rumen that could cause inefficiency of protein use. However, the researchers’ efforts overcame this limitation by combination of N. sativa meal with different readily available carbohydrates derived from pollard, rice bran, and corn, because this readily available carbohydrates will be an energy source for rumen microbes which causes the use of ammonia in the rumen by microbes will increase, thereby reducing accumulation ammonia in the rumen. Ammonia will be used by rumen microbes to form microbial protein synthesis. Thus, to achieve the desired effects of using of phytogenic products as dietary supplements, it is not enough to add them to the feed. In the first place, it is a balanced feed ration, animal welfare, and appropriate veterinary prophylaxis that should be taken care of. Also, further studies are warranted to confirm the antimethanogenic activity of N. sativa, establish a dose-response relationship, and examine the stability in a time of the effects, and test if the effects can be reproduced in vivo with animals.

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Chapter 18

Nigella sativa Seeds and Its Derivatives in Poultry Feed Mohamed E. Abd El-Hack, Abdel-Moneim E. Abdel-Moneim, Noura M. Mesalam, Khalid M. Mahrose, Asmaa F. Khafaga, Ayman E. Taha, and Ayman A. Swelum

Abstract  The incidences of antibiotic residue in poultry products and the creation of drug resistance pathogens are prevalent due to the indiscriminate and excessive use of antibiotics in poultry diets. Persistent assessment of antibiotic alternatives has become imperative because of the growing global concerns about food security. Nigella sativa (black cumin) seeds could be considered as a favorable alternative to chemotherapeutic agents in poultry rations. N. sativa is naturally used in human diets and traditional remedies due to the presence of several pharmacologically active substances and a large amount of essential nutrients which gave it its properties as antimicrobial, antioxidant, antinociceptive, antiparasitic, hepatoprotective, analgesic, immunomodulatory, anti-inflammatory and growth-promoting agent. Several reports revealed the growth-enhancing effect of N. sativa under both normal M. E. Abd El-Hack (*) Poultry Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt e-mail: [email protected] A.-M. E. Abdel-Moneim · N. M. Mesalam Biological Application Department, Nuclear Research Center, Atomic Energy Authority, Abu-Zaabal, Egypt K. M. Mahrose Animal and Poultry Production Department, Faculty of Technology and Development, Zagazig University, Zagazig, Egypt A. F. Khafaga Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, Edfina, Egypt A. E. Taha Department of Animal Husbandry and Animal Wealth Development, Faculty of Veterinary Medicine, Alexandria University, Edfina, Egypt A. A. Swelum Department of Theriogenology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_18

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and stress conditions. Alongside improved growth and egg production, feed utilization and feed-to-gain ratio, N. sativa decreased serum and yolk contents of cholesterol which led to improving egg and meat quality. Nutrient utilization, antioxidative status and immune response of birds fed diets with N. sativa have been remarkably increased. Gastrointestinal development and function were also improved while enumeration of pathogenic bacteria was decreased by feeding N. sativa or its derivatives. However, limited studies examined the effect of N. sativa on the chemical composition, antioxidant properties and polyunsaturated fatty acid contents in eggs and meat. The following context presents the effect of N. sativa seeds and its derivatives supplementation in poultry diets as growth promoter alternatives. Keywords  Phytogenic · Phyto-additives · Performance · Egg and meat quality · Antioxidative status · Immune response · Poultry

Abbreviations BWG Body weight gain CP Crude protein CD Crypt depth DFI Daily feed intake DTQ Dithymoquinone DM Dry matter EE Ether extract FA Fatty acids FCR Feed-to-gain ratio HDL-c High-density lipoprotein cholesterol LBW Live body weight LDL-c Low-density lipoprotein cholesterol MDA Malondialdehyde NS N. sativa NSS N. sativa seeds NDV Newcastle disease virus PUFA Polyunsaturated fatty acids ROS Reactive oxygen species TQ Thymoquinone FDA US Food and Drug Administration VLDL-c Very-low-density lipoprotein cholesterol VH Villus height VH: CD Villus height: crypt depth

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1  Introduction In the last two decades, the intensive poultry industry has been suffered from several challenges and stress factors such as environmental changes, overcrowding, nutritional imbalances and contagious infections which down-regulate the immune response of birds. Chemotherapeutic agents (e.g. antibiotics) were used as growth promoters to obviate the harmful effects of these factors. Nevertheless, the redundant use of these agents poses a risk to consumer health. Therefore, it’s necessary to find effective substitutions to antibiotics because of the growing global concerns about meat and egg security. Green feed additives such as probiotic, prebiotic, enzymes and phytogenic additives recently represent the most promising choices (Alagawany et al. 2016, 2017, 2018; Abd El-Hack et al. 2019; Abd El-Moneim and Sabic 2019; Abd El-Moneim et al. 2019; Mahrose et al. 2019). Medicinal herbs, their derivatives and extracts have been used to improve poultry health and growth. Identification of medicinal herbs’ main components and understanding how they work has been of interest recently. Nigella sativa (N. sativa; black seed) is planted in Eastern and South Europe, the Middle East and Asia and considered one of the committed medicinal plants (Khare 2004). N. sativa has been intensively used in many recipes in kitchens of the Middle East and also used as a valuable remedy for several diseases. N. sativa seeds (NSS) were widely used against many health disorders including allergy, bronchial asthma, respiratory distress, lung inflammation, dyslipidemia, obesity, hypertension, immune disorders, dysentery, neurological disorders and gastrointestinal problems (Schleicher and Saleh 2000; Ali and Blunden 2003; Salem 2005; Ahmad et  al. 2013; Abd El-Hack et  al. 2016b; Gökce et  al. 2016). Black cumin seeds and its essential oil have antibacterial properties and can inhibit the growth of a wide range of Gram-negative and Gram-positive bacteria (Saxena and Vyas 1986; Hanafy and Hatem 1991; Kumar and Patra 2017). N. sativa seeds composed of 200–270 g.kg−1 protein, 340–390 g.kg−1 lipids, 240–350 g.kg−1 carbohydrates, 70–80 g.kg−1 crude fiber and 40–50 g.kg−1 ash (Babayan et al. 1978; Bayder 2009). The seeds also contain diversified amount of minerals and vitamins including Zn, Cu, P, Fe and carotene as well as active materials (known as nogelleone) like thymoquinone, thymohdroquinone, thymol, carvacrol, dithymoquinone, α-hedrin, nigellidine and nigellicine-N-oxide (Ahmad et al. 2013; Forouzanfar et al. 2014; Mohammed and Al-Suwaiegh 2016). Selenium, trans-retinol, DL-α-­ tocopherol, and DL-γ-tocopherol are the common antioxidant substances found in N. sativa seeds (Nasir et al. 2005; Al-Saleh et al. 2006). Its oil content of polyunsaturated fatty acids (PUFA) represents double than the monounsaturated fatty acids (MUFA), which helps in the reduction of total cholesterol content. Linoleic acid is the main PUFA (50.3–49.2%), however, the prime MUFA is oleic acid (23.7–25.0%), while palmitic acid (17.2–18.4%) is the major saturated fatty acid (Cheikh-Rouhou et al. 2007; Kumar and Patra 2017). Several studies examined NSS, oils, extracts or its derivatives as feed additives in poultry diets. There is unanimous agreement that feeding NSS (up to 30 g.kg−1) not only has no deleterious effects on poultry performance, but also it may improve productive performance, egg and meat quality,

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b­ iochemical and immunological responses, antioxidative status and mortality rate of birds (Aydin et al. 2008; Khan et al. 2012; Abd El-Hack et al. 2016a, c; Islam et al. 2016; Rahman and Kim 2016; Kumar et al. 2017; Soliman et al. 2017). This chapter reviews the effect of NSS and its derivatives on productive and reproductive performance, egg quality, antioxidative status, bacterial enumeration and immune response of poultry.

2  The Need for Phytogenic Additives in Poultry Feed Poultry is the most reveled human feed nowadays compared to animal meat and considered as the best and cheapest source of quality protein. The overstated exhaustion of poultry meat has promoted producers to keep more poultry breeds (chickens, duck …etc) in well-constructed houses (Ahmed et al. 2018a, b; Farghly et al. 2018; Madhupriya et al. 2018). These days, the poultry feed industry is encountering the defiance of the consciousness among the consumers of poultry meat over the potential antibiotic remains in poultry products and on the danger of bringing about antibiotic impedance in pathogenic microorganisms through antibiotics applied in poultry feeds (Demir et al. 2005; Hajati 2018; Calik et al. 2019). They have guided them across the non-antibiotic feed supplementations, especially phytogenic. Phytogenic feed supplementations or Phyto-additives are considered to be one of the most preferable alternates as growth and immune enhancers (Salaheen et  al. 2015; Hassan et al. 2016; Madhupriya et al. 2018). In spite of the debate on the function of antibiotic use in granting antimicrobial impedance to human pathogens, the European Union released a prohibition on the agreement for antibiotics as growth promoters since 2006 on preventative basics (Castanon 2007; Diaz-Sanchez et al. 2018; Ahmed et al. 2018a, b). In the year 2013, the US Food and Drug Administration (FDA) called for the main industrialists of veterinary important medicines to voluntarily pause distinguishing them as growth promoters in animals and check out the labels such that veterinary oversight is wanted for curative uses (GFI#213; FDA 2013). FDA uninterrupted to support its schedule on enhancing wise use of antimicrobials in animals and issued its definitive judge of Veterinary Feed Directive in 2015, fetching the application of medically substantial antimicrobials in feed under veterinary supervision, so that they are used only when needful to secure the animal health. Later in 2015, the state of California threaded a bill implementing a rigorous prohibition on using medically substantial antimicrobials in animal feeds as growth promotors or disease preventive (Diaz-Sanchez et al. 2018; Ahmed et al. 2018b). Phytogenic feed supplementations could be defined as the complexes or combinations of botanical source integrated into poultry diets to improve productive, reproductive and physiological performance as well as immune status and the quality of meat produced through the enhancement of feed properties, digestibility, nutrient absorption and judgment of pathogens in the poultry gut (Athanasiadou

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et al. 2007; Windisch et al. 2008; Hassan et al. 2016; Gadde et al. 2017; Ahmed et al. 2018a, b; Madhupriya et al. 2018). Phytogenic feed supplementations have been showed to supply antimicrobial, antioxidative, immunogenic and anticoccidial impacts when fed to poultry and rabbits (Hassan et al. 2016; Calik et al. 2019). Phytogenic enhances feed palatability, protects against oxidative damage, improve the gut health through reduced bacterial colony counts, declines fermentation products (including ammonia and biogenic amines), and decreases vigor of the gut-associated lymphatic order (Alagawany et al. 2018; Abd El-Hack et al. 2019; Abd El-Moneim and Sabic 2019; Mahrose et al. 2019).

3  Effects of Nigella sativa and Its Derivatives on 3.1  Productive Performance 3.1.1  Live Body Weight and Body Weight Gain Numerous studies confirmed the growth-promoting effect of N. sativa seeds and reported a significant improvement in live body weight (LBW) with dietary supplementation of N. sativa (Guler et  al. 2006; Al-Beitawi and El-Ghousein 2008; Ashayerizadeh et al. 2009; Erener et al. 2010; Miraghaee et al. 2011; Shewita and Taha 2011; Khan et  al. 2012; Jahan et  al. 2015; Abd El-Hack et  al. 2016a, c; Al-Hothaify and Al-Sanabani 2016; Islam et  al. 2016; Rahman and Kim 2016; Kumar et al. 2017; Soliman et al. 2017; Hassan and Mandour 2018; Arif et al. 2019). Guler et al. (2006) reported that broilers received diets supplemented with 10 g.kg−1 black cumin seeds and the antibiotic recorded the highest LBW compared to the un-supplemented group. Al-Beitawi and El-Ghousein (2008) found that chickens fed 1.5% ground NSS had higher LBW and body weight gain (BWG). Ashayerizadeh et  al. (2009) revealed that broiler chicks fed a diet with 1000  g NSS.ton−1 had a higher LBW when compared with the control and the other treatment groups. Erener et al. (2010) showed that NSS (10 g.kg−1) elevated the LBW at the marketing age compared to the control. Furthermore, Khan et al. (2012) showed that groups fed NSS with levels of 25 and 50 g.kg−1 recorded higher LBW than others fed a diet containing 12.5 g.kg−1 NSS and an antibiotic at 28 and 42 days of age. Linear and quadratic improvements in LBW in quails were achieved with a 30% substitution of soybean meal with NS meal (Abd El-Hack et  al. 2016c). Al-Hothaify and Al-Sanabani (2016) reported that dietary inclusion of 1% NSS recorded the highest LBW, while increasing the inclusion level of NSS did not necessarily cause a higher improvement degree. Contrarily, Soliman et  al. (2017) and Hassan and Mandour (2018) showed that feeding on NSS with levels up to 5.6% and 4.5%, respectively achieved higher LBW compared to the control. Improvement effect of the supplemented NSS in poultry and rabbit diets on BWG has been reported (Osman 2002; El-Bagir et  al. 2006; Guler et  al. 2006;

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Durrani et  al. 2007; Abu-Dieyeh and Abu-Darwish 2008; Al-Beitawi and El-Ghousein 2008; Ashayerizadeh et al. 2009; Erener et al. 2010; Ismail et al. 2010; Khan et al. 2012; Yatoo et al. 2012; Jahan et al. 2015). Osman (2002) demonstrated that broilers fed diets supplemented with NSS oil exhibited higher BWG.  Many studies recommended doses of 10 or 15 g.kg−1 of NSS as a dietary supplement to promote BWG (El-Bagir et  al. 2006; Guler et  al. 2006; Abu-Dieyeh and Abu-­ Darwish 2008; Erener et al. 2010). However, higher concentrations of NSS were included in the broiler diet and achieved greater BWG. El-Bagir et al. (2006) found that the inclusion of NSS in laying hens diet at levels of 10 or 30 g.kg−1 promoted the overall BWG. Durrani et al. (2007) documented that the feeding on NSS at the level of 40 g.kg−1 recorded higher BWG compared to the control. Moreover, in an investigation conducted by Khan et al. (2012), the groups fed NSS at concentrations 25 and 50  g.kg−1 exhibited higher BWG than birds fed diets supplemented with 12.5 g.kg−1 NSS and antibiotic at 28 and 42 days of age. Yatoo et al. (2012) noticed that, compared with the control diet, BWG of all treated-groups was enhanced with the advantage of a 0.5% NSS group. On the other hand, controversial observations related to poultry performance were noticed (Nasir and Grashorn 2010; Al-Mufarrej 2014; Kassu et  al. 2016). However, most of these studies were conducted on the NS meal rather than the NS seeds or oil. Nasir and Grashorn (2010) found no alternations in LBW and BWG by the inclusion of 1% NSS.  The dietary supplementation of NS meal significantly reduced LBW of broiler chicks (El-Sheikh et al. 1998; Akhtar et al. 2003). El-Bagir et  al. (2006) attributed the reduction in BWG due to the high fiber contents of the meal. The growth-promoting effect of NSS may be attributed to one or more of these mechanisms: 1. The presence of pharmacologically active compounds in the oil and the seeds. N. sativa oil and its constituent, thymoquinone (TQ), exhibited hepatoprotective impacts, thus they have been widely used in remedies for gastric mucosal injury and gastrointestinal disorders (Mansour et al. 2002; El-Abhar et al. 2003). 2. High nutritive value of NSS because it’s content of a mixture of essential fatty acids (FA) particularly oleic acids, linolenic, and linoleic as well as fifteen amino acids, eight of them are essential, comprising N. sativa proteins (Takruri and Dameh 1998). 3. Feeding on low doses of NSS increased the serum concentration of thyroxine, thus increasing the metabolic rate (Mandour et al. 1998). 4. Inclusion of NSS in poultry rations increased the flow rate of bile, which resulted in an increase of lipid emulsification that stimulates the activity of pancreatic lipases, which in turn enhances the digestion and absorption of lipids as well as absorption of fat-soluble vitamins (Crossland and Lewis 1980). 5. Better nutrient absorption due to the stimulating impact of NSS on the digestive system that consequently promotes growth performance (Jamroz and Kamel 2002; Kumar et al. 2017).

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6. Antimicrobial effects of NSS active ingredients may be a potential reason for increased performance (El-Kamali et al. 1998; Gilani et al. 2004; Kumar et al. 2017).

3.1.2  Feed Utilization Feed consumption and feed-to-gain ratio are the main assessment factors of poultry growth performances and feed quality. Incorporation of N. sativa seeds in poultry diets could be supportive to improve growth rate and related parameters (Abdel-­ Magid et  al. 2007). However, there was a confliction in the results of researches studied the effect of dietary supplementation of NSS in poultry. Different studies conducted by Denli et al. (2004), Guler et al. (2006), Ashayerizadeh et al. (2009), Khalaji et al. (2011), Miraghaee et al. (2011), Al-Mufarrej (2014), Ghasemi et al. (2014a), Saleh (2014), Al-Hothaify and Al-Sanabani (2016), Kumar et al. (2017), Soliman et al. (2017) and Kadhim et al. (2018) observed insignificant alternations in daily feed intake (DFI) due to dietary supplementation of NS seeds, meal or oil. The increment effect of NSS in DFI was also reported (Osman and El-Barody 1999; Abou-El-Soud 2000; Erener et al. 2010; Ismail 2011; Shewita and Taha 2011; Yatoo et al. 2012). While others found a decrement effect of dietary inclusion of NSS on DFI (Osman 2002; Durrani et al. 2007; Çetin et al. 2008; Abbas and Ahmed 2010; Szczerbinska et al. 2012; Kassu et al. 2016; Rahman and Kim 2016). The prime trend for the NSS effect on DFI has not shown significant changes, for example, Ashayerizadeh et  al. (2009) reported that no statistical alternations for dietary inclusion of 1 g.kg−1 NSS on DFI of broiler chickens were observed. Kadhim et al. (2018) revealed that broilers fed diets with 1% NSS did not show any differences in DFI compared to the untreated birds. Moreover, Ghasemi et al. (2014a), Khalaji et al. (2011) and Al-Hothaify and Al-Sanabani (2016) reported that DFI was not affected in birds fed diets supplemented with 5, 10 and 20 g.kg−1 NSS. Guler et al. (2006) reported that supplementation with NSS at levels 0.5, 1.0, 2.0 and 3.0% of the diets did not significantly affect the DFI of broilers compared against 10 mg. kg−1 avilamycin and control groups during the starter, grower, finisher and overall periods. Soliman et al. (2017) did not find any alternations on DFI after the inclusion of NSS at 1.4, 2.8, 4.2 and 5.6% of broiler diets. However, some reports revealed that DFI of birds fed NSS was increased significantly. Erener et al. (2010) demonstrated that 2 g.kg−1 NSS extracts and 10 g.kg−1 NSS augmented DFI compared to the untreated birds with no differences observed in DFI between NSS and NSS extract. Furthermore, DFI of birds fed 10 g.kg−1 NSS was increased during starter, finisher and overall period compared with the control (Yatoo et al. 2012). Osman and El-Barody (1999) and Shewita and Taha (2011) reported that DFI was elevated in broiler chickens fed a diet supplemented with 2, 4, 6, 8 and 10 g.kg−1 NSS compared with those fed the control diet. The increment in DFI can be attributed to more palatable diets supplemented with NSS, as it has been documented that DFI in birds could be stimulated or depressed by the flavor. As an aromatic plant,

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NSS has the ability to stimulate digestive system activity, enhance appetite of poultry and improve diet palatability which in turn increase the amount of consumed diet (Gilani et  al. 2004). Contrarily, Osman (2002) showed that feeding broiler chicks with a diet supplemented with black seed oil decreased DFI significantly. In agreement, Abbas and Ahmed (2010) reported that diets included with 1 and 2% NSS decreased DFI of broiler chickens significantly. The same levels of dietary NSS reduced DFI of broilers during the finisher or the overall experimental periods (Kassu et al. 2016, Rahman and Kim 2016). Szczerbinska et al. (2012) observed lower DFI in quails consumed diets supplemented with 5.0% NSS as compared with the untreated birds. Also, Durrani et al. (2007) found that birds of experimental groups receiving 20 to 40  g NSS.kg−1 diet recorded a significant reduction in DFI. Çetin et al. (2008) noticed a linear decrement effect on DFI with augmenting values of NSS extract in the rations of partridges. In contrast, Abd El-Hack et al. (2016a) reported that overall DFI of meat-type quails was increased with the supplemental level of NS meal up to 15%, while the addition of 20% NS meal reduced DFI. In another trial, Abd El-Hack et al. (2016c) noticed that Japanese quails fed diets with 40% NS meal showed a reduction in DFI compared with lower levels of NS meal. This confliction in the results may be due to the differences in NS forms added to the diets and the large variations in crude fiber percentages between them. The effects of NSS on the feed-to-gain ratio (FCR) are also conflicting among investigations. Administration of NSS significantly enhanced FCR in several studies (Guler et  al. 2006; Durrani et  al. 2007; Abu-Dieyeh and Abu-Darwish 2008; Ashayerizadeh et al. 2009; Erener et al. 2010; Toghyani et al. 2010; Miraghaee et al. 2011; Yatoo et al. 2012; Ghasemi et al. 2014a; Saleh 2014; Jahan et al. 2015; Kassu et al. 2016; Rahman and Kim 2016; Soliman et al. 2017). Guler et al. (2006) and Erener et al. (2010) reported that FCR was significantly improved in birds fed 10 g NSS.kg−1 diet compared to untreated birds. Abu-Dieyeh and Abu-Darwish (2008) found that broiler chickens received 1.5% of NSS in their diet achieved the lowest FCR than other treatments and control. Al-Hothaify and Al-Sanabani (2016) noticed that dietary inclusion of NSS at 0.5, 1 and 2% of the diet reduced FCR significantly. The same observations were noted by Kassu et  al. (2016) and Rahman and Kim (2016) who reported that dietary levels of NSS (1 and 2%) decreased the overall FCR. Additionally, Soliman et al. (2017) observed a significant reduction in FCR of birds fed diets containing 1.4, 2.8, 4.2 and 5.6% NSS compared to the control birds. The increased feed efficiency and growth performance of poultry fed diets with NSS may be associated with various properties and chemical components of NSS which were abovementioned (Guler et  al. 2006; Abu-Dieyeh and Abu-Darwish 2008; Al-Beitawi and El-Ghousein 2008; Abd El-Hack et al. 2016b, c). In contrast, some studies (Abbas and Ahmed 2010; Nasir and Grashorn 2010; Abd El-Hack et  al. 2016a, c; Hassan and Mandour 2018; Kadhim et  al. 2018) reported that NSS supplementation on poultry diets did not show a significant impact on FCR. Abbas and Ahmed (2010) and Nasir and Grashorn (2010) observed a non-significant effect on FCR of broilers fed diets supplemented with 1% or 2% of NSS. Abd El-Hack et al. (2016a) and Abd El-Hack et al. (2016c) noticed that supplementing NS meal in quail diets with levels up to 40% did not exert reliable

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changes in FCR. The absence of effect of NSS on FCR might be due to the birds fed balanced basal diets and housed in optimal husbandry and management conditions. Lee et al. (2003) reported that healthy and nutritious birds placed in clean, disinfected conditions do not respond well to the treatment with antibiotics. Thus, antimicrobial properties of phytochemicals may have little effects when the diet is well prepared and balanced, and the colonization of pathogenic bacteria is at its minimal level in the intestinal tracts. In brief, birds reared under good management and environmental conditions recorded the maximum feed efficiency and left no or tiny space for the enhancement by NSS inclusion. Some studies reported the negative impacts of NSS on feed efficiency (Al-Beitawi and El-Ghousein 2008; Majeed et al. 2010). Majeed et al. (2010) revealed that overall FCR increased by 3.7% in groups fed diet with 0.25% NSS, while Al-Beitawi and El-Ghousein (2008) reported that broiler feed efficiency was deteriorated by dietary NSS inclusion at high levels. The authors concluded that the relationship between the reduction in feed efficiency and feeding dietary NSS may be dose-dependent. 3.1.3  Nutrient Utilization Improvements in nutrients utilization were reported in response to dietary supplementation of NSS in many reports (Yatoo et  al. 2012; Saleh 2014; Abd El-Hack et al. 2016a; Kumar et al. 2017). Yatoo et al. (2012) observed a significant increase in dry matter (DM) digestibility in diet supplemented with 1% NSS compared with the control. Moreover, Kumar et al. (2017) noticed that nutrient utilization of crude protein (CP) and ether extract (EE) enhanced in broiler chicks fed diets with NSS at levels 0.5% to 2%. Digestibility coefficients of DM, organic matter, EE, CP, and nitrogen-free extract were significantly increased in chicks treated with 10% NS meal when compared with control birds. Contrarily, feeding quails to diet supplemented with 20% Nigella sativa meal significantly reduced digestion of abovementioned nutrients as compared with the other experimental groups (Abd El-Hack et al. 2016a). Saleh (2014) reported that DM and CP digestibility coefficients were significantly increased when broiler chicks were fed diets included 1 mL NSS oil. kg−1 diet compared with the antibiotic and un-supplemented diets, while the digestion of EE was not affected. These benefits of NSS on nutrients digestibility coefficients may be attributed to the increase in activities of digestive enzymes caused by NSS essential oils. Dietary supplementation of NSS may increase the flow of bile (Mahfouz and El-Dakhakhny 1960) and enhance pancreatic lipase activity, which increases the digestion of lipids and absorption of fat-soluble vitamin (Crossland and Lewis 1980). The high nutritive value of NSS may be due to its high content of essential FA mixture, which stimulates the digestive system (Wenk 2003) to cause better performance and nutrients utilization. Furthermore, antifungal and antibacterial properties of NSS (Hanafy and Hatem 1991) resulted in increased availability of nutrients for birds. It has been documented that herbal extracts may stimulate digestive secretions and enzymatic activity as well as increasing digestibility of nutrients probably due to the effect of

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these extracts on the bird’s ecosystem (Bedford 2000), hence practicing beneficial impacts within the digestive tract (Platel and Srinivasan 2004).

3.2  Carcass Traits and Meat Quality Studies revealed that dietary supplementation of NSS enhanced slaughter, thigh and breast weights, abdominal fat and edible internal organs (Guler et  al. 2006; Ashayerizadeh et  al. 2009; Erener et  al. 2010; Saleh 2014; Kumar et  al. 2017). Guler et al. (2006) and Toghyani et al. (2010) observed that administration of 1.0% NSS and antibiotic resulted in marked influences on relative weights of liver, thigh, breast, wings, neck and abdominal fat. Guler et al. (2006), Toghyani et al. (2010) and Erener et al. (2010) revealed that inclusion of NSS in diets enhances the carcass weight, however, non-significant effects on heart weight values were stated. Contrarily, Al-Beitawi et al. (2009) and Al-Beitawi and El-Ghousein (2008) found that feeding on ground or whole NSS at different concentrations did not improve carcass characteristics in broilers. Nasir and Grashorn (2010) and Ismail (2011) attributed the markedly increase in breast percentage to the ability of NSS to influence protein utilization, thereby enhancing breast weight. In the same study, it was reported that dietary supplementation of NSS resulted in enhanced availability of minerals by birds that increases the percentage of ash from meat (Hafez et al. 2009). It was found that feeding NSS and NSS extract did not affect the percentages of dressing, abdominal fat as well as an edible organ (Ghasemi et al. 2014a). Durrani et al. (2007) revealed that the incorporation of NSS to the diets did not significantly affect most of the carcass traits except heart, liver and intestine weights. However, El-Ghammry et al. (2002) revealed diminishing in carcass percentage. Meanwhile, Saleh (2014) noticed that the administration of NSS resulted in a significant increase in breast muscle weight but did not affect liver weight when compared to control and avilamycin-treated birds. The major variations that were noticed in abdominal fat percentage may be attributed to the lipolytic effect of NSS meal, which could be useful for providing physiological activities with energy derived from fat. Abaza et al. (2008) reported that the inclusion of 1 g.kg−1 NSS oil in the chicken rations reduced the abdominal fat percentage. Majeed et al. (2010) revealed that the composition of the diet had no impact on breast, thigh and drumstick final weights. Contrarily, Abbas and Ahmed (2010) noticed that dietary supplementation with 1% NSS significantly reduced dressing percentage when compared with birds fed 2% NSS or un-supplemented diets. Khan et al. (2012) found that dressing percentage did not change on 28 and 42 days of age among NSS feeding and antibiotic groups. Jahan et  al. (2015) did not find significant variations within the average dressing percentage, wing meat, thigh meat, gizzard, heart as well as liver, however, the addition of 1.5% NSS meal to broiler chickens diets resulted in differences in abdominal fat, drumstick meat, breast meat and skin. Ismail (2011) revealed that dietary NSS or NSS extract did not affect the dressing yield, edible internal organs,

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full gut weight, gut length and abdominal fat. Khalaji et al. (2011) reported that NSS supplementation resulted in an increased relative weight of gizzard, but did not affect other carcass traits. The variations in the dressed weights between birds fed NSS and control diets might be due to the discrepancies in feed consumption and the quantity of ingested protein and metabolizable energy which subsequently affect slaughter weight (Ismail 2011). In another study, breast meat color, fat percentage, shear force value and electrical conductivity were not affected by dietary supplementation of NSS (Abbas and Ahmed 2010). The addition of NSS resulted in a significant increase in cooking losses of birds’ meat. Dressing percentage was significantly low in broilers dietary supplemented with 1% whole ground NSS, however, there were no marked influences concerning heart, gizzard, liver and abdominal fat weights by feeding of whole NSS (Abbas and Ahmed 2010). Hafez et  al. (2009) found that giblet and percentage of abdominal fat in broilers were not influenced. Regarding bone percentage, it was higher in the control birds as compared to NSS treated ones. Durrani et al. (2007) reported that the treatment of birds with NSS oil at levels of 0.5 and 1% or NSS at levels of 1 and 2% resulted in greater meat protein and lesser fat content on dry matter basis. In the same study, values tenderness and pH of meat were not influenced. The effect of dietary supplementation of NS on meat quality was summarized in Fig. 18.1.

Fig. 18.1  The effect of dietary supplementation of NS on the meat quality of poultry

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3.3  Chemical Composition and Antioxidant Properties of Meat Chemical composition of meat was improved in birds administrated with NSS diets. Kumar et al. (2017) revealed that DM, EE and CP concentrations in breast and thigh muscle were higher in birds supplemented with 10 g NSS/kg diet than in the control ones. Moreover, broiler chicks fed diets containing 15 g NSS /kg showed high DM and CP percentages in their breast meat while these parameters were not affected in leg meat (Al-Beitawi and El-Ghousein 2008). The latter authors also reported that high levels of NSS (2 and 3%) improved crude fat content in leg and breast meat. The increase in CP and EE of chicken meat could be attributed to the increase in DFI and digestion that elevate the consumed amount of digestible CP and EE (Durrani et  al. 2007; Kumar et  al. 2017). Another explanation to the previous improvement is the elevation in the blood level of thyroid hormones, caused by the treatment with NSS or its essential oils, that involved in energy metabolism and protein synthesis (Hafez et al. 2011). Many scientists have attempted to improve the quality of nutritional aspects of chicken meats by dietary involvement of plant bioactive substances and vegetable oils (Mandal et al. 2014b; Patra 2014). The desirable nutritional characteristics of poultry meat are due to the presence of low lipid content and high levels of PUFA (Mandal et al. 2014a). Fatty acid composition in the muscle, that affects meat quality, depends on the composition of FA and antioxidant substances in diets (Crespo and Esteve-Garcia 2001; Ertas et al. 2005). Limited information is available on the influence of NS seeds or oil on the antioxidant status of birds. Guler et al. (2007) reported that dietary supplementation of NSS at 1, 2 and 3% reduced serum, heart and breast meat concentrations of malondialdehyde (MDA). In the same study, the administration of NSS resulted in a decrease in MDA content of the liver at 5, 10, 20 and 30 g.kg−1. Rahman and Kim (2016) revealed that dietary administration of NSS at doses of 10 and 20 g.kg−1 to broiler chicks diets diminished thigh muscle contents of MDA by 24% and 28% compared to the control as well as enhanced the activity of superoxide dismutase and glutathione peroxidase. Kumar et al. (2017) noticed that administration of NSS attenuated ferric reducing antioxidant power (FRAP) activity in chicken breast meat, thigh and blood. The authors also reported that lipid peroxide values were not affected in thigh and breast meat stored at 4 °C for 1 and 7  days, however, after accounting meat concentration of ether extract (which was higher in NSS-treated birds), peroxide levels in only breast meat tended to reduce in the NSS birds compared to the un-supplemented one. The reduction in lipid peroxidation in tissues of NSS birds was attributed to the scavenger activity of superoxide anion (Tuluce et  al. 2009) or to the presence of antioxidants such as anethole, carvacrol, thymoquinone and 4-terepinol (Guler et al. 2007). Mariod et al. (2009) highlighted that NS meal contains phenolic compounds responsible for its antioxidant activity. These phenolic substances presented in NSS methanol extracts such as p-cumaric acids, hydroxybenzoic and syringic acid possess remarkable antioxidant activities under in vitro trials. The antioxidant properties of NSS were also

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documented by Bassim-Atta and Imaizumi (1998) who stated that ethanol extracts of NSS banned the oxidative damage of triglycerides when added to the corn oil. Fatty acid content in chicken meat is considered as a factor affecting lipid peroxidation and oxidative damage of tissue. However, research conducted to study the impact of NSS on FA profiles in meat is limited. Kumar et al. (2017) mentioned that feeding of NSS enhanced the levels of some unsaturated FA and PUFA that are considered the higher susceptible fractions to be oxidized. Differences in fat deposition in chicken muscles may be due to the differences in lipid oxidation or synthesis rates (Crespo and Esteve-Garcia 2001). Mitigation of FA oxidation in tissue increased concentration of unsaturated FA in meat. The antioxidant activities of NSS may prevent tissue lipids peroxidation, especially PUFA, enhancing their concentrations in meat. Fatty acid profiles could be altered by inclusion of phyto-­ additives with antioxidant activities. For example, total PUFA remarkably elevated in broiler chicks administrated with essential oils of cinnamon (Ciftci et al. 2009).

3.4  Reproductive Performance Studies documented the influence of N. sativa on the reproductive performance of female and male of birds concerning fertility, hatchability, development of the ovarian follicle and semen quality are very limited. In a study of Attia et al. (2008), the authors found that laying Japanese quails fed diets supplemented with NS meal at levels above 33% recorded lower fertility rate and numerically lower hatchability and relative ovary weight percentages. Furthermore, the ovarian follicular development of laying hens was not affected by dietary inclusion of 0.025 and 0.05% NS oil, except weights of the ovary and the largest yellow follicle which were increased. The authors also revealed that oviduct weight and the weights of infundibulum, magnum, isthmus, uterus and vagina proportional to oviduct weight were not significantly changed among the experimental groups. However, Abdulkarim and Al-Sardary (2009) reported that feeding diets containing 0.5% and 1% NS seeds or oil to male broiler breeder birds significantly improved their fertility and hatchability. The same authors noticed enhancement in semen characteristic were enhanced, a decrease in ejaculation time and sperm abnormalities and an increase in ejaculation volume and count, total sperm output, progressive motility, mass motility and viability percentage of sperms. El-Tohamy et al. (2010) revealed that supplementation of NS meal (as a replacement of soybean meal) at a level of 50% in bucks diet reduced the reaction time and sperm motility after 60  min of semen collection. Feeding on NS meal also increased semen volume, total motile sperm, total sperm/ ejaculate, sperm concentration/mL and total function sperm fraction. However, semen pH, latency period, motility grade, motile sperm percentage, abnormal morphology and live sperms were not affected.

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3.5  Egg Production and Quality Many researchers investigated different levels concerning the impact of NSS on laying performance. Egg production and egg weight were significantly elevated by feeding laying hens with 3% NSS (Aydin et al. 2008) and 1.5% NSS (Akhtar et al. 2003). Moreover, Khan et al. (2013) revealed that egg production, egg weight, and egg mass were increased in rural Leghorn chickens received diets with 3, 4 and 5% NSS. The latter authors attributed the enhanced egg production and reproductive performance to the presence of large amounts of macronutrients including crude protein, fat and carbohydrates as well as minerals. However, Hassan and Alaqil (2014) noticed insignificant differences in egg weight, egg mass, egg number and egg production percentage in Hisex laying hens fed diets supplemented with 1, 2 and 4% NSS. Bölükbaşi et al. (2009) observed insignificant changes in egg weight and egg production in LSL laying hens treated with 1, 2 and 3 ml NS oil.kg−1 diet compared with the control birds. In contrast, El-Bagir et al. (2006) observed that feeding on diets supplemented with 1 and 3% NSS diminished egg production without altering egg width and length. The supplied energy from NS oil resulted in an increase in layers weight rather than egg production. The reduction in serum cholesterol might be the reason for the decreased egg production (Akhtar et al. 2003). The diminished levels of serum and yolk cholesterol might be due to the inhibition impact of NSS on HMG-CoA reductase. It was reported that egg production and yolk cholesterol reduced by 20 and 30%, respectively by the administration of synthetic HMG-CoA reductase inhibitor (Elkin et al. 1993). Thus it can be concluded that cholesterol is a prime factor affecting egg production and cholesterol level should be at certain limit for completion of egg formation or production. External and internal egg quality criteria have been affected by dietary inclusion of NS seeds, extract or oil as reported by several investigations. Saleh et al. (2019) revealed that dietary administration of 0.025 and 0.05% NS oil significantly increased yolk width, weight (only in 0.05% NS oil) and colour, eggshell thickness and shell weight (only in 0.05% NS oil), while albumin weight was reduced in 0.05% treated group. Denli et al. (2004) reported that eggshell weight and thickness were increased in layers administrated with 1% NSS extract. Shell thickness and strength were increased by dietary administration of 2 and 3% NSS in laying hens as compared to lower levels (Aydin et al. 2008). Moreover, dietary administration of NSS improved albumin activity of eggs (El-Sheikh et al. 1998; Akhtar et al. 2003), while the yolk index was not altered by the addition of NSS to the diet (El-Sheikh et al. 1998). Bölükbaşi et al. (2009) noticed that dietary inclusion of NS oil had no effect on yolk, albumen and shell percentages, nevertheless, dietary supplementation of 3 mL/kg NS oil diminished the Haugh unit score. The addition of 3% NSS to layer diets reduced yolk cholesterol, triacylglycerols, total lipids and phospholipids by 45, 20, 34 and 11%, respectively. Moreover, Saleh et al. (2019) reported a significant reduction in yolk cholesterol content in birds fed 0.025 and 0.05% NS oil. The reduction in yolk cholesterol content is a greatly eligible effort in order to reduce the amount of cholesterol intake in human food due to its harmful effects on

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Fig. 18.2  The effect of dietary supplementation of NS on poultry egg production and quality

their health. The mode of action by which NSS reduced yolk cholesterol is not entirely known. However, the reduction in yolk cholesterol may be due to the reduced in serum level of cholesterol by the NSS. It is also presumed that NSS may block the de-novo synthesis of cholesterol (El-Bagir et al. 2006). Yolk, albumin and shell relative weights were not affected by supplementation with NS seeds or oil (Bölükbaşi et al. 2009; Khan et al. 2013). The effect of dietary supplementation of NS on egg production and quality was illustrated in Fig. 18.2.

3.6  Blood Biochemistry Many studies were documented concerning the effect of NSS on the blood glucose level. Blood glucose levels were not influenced by NSS in several investigations (Al-Beitawi et  al. 2009; Khalaji et  al. 2011; Ghasemi et  al. 2014b; Kumar et  al. 2017); while El-Kaiaty et al. (2002) observed significant decline in blood glucose levels by 16% in laying hens and Yatoo et al. (2012) who also reported a statistical decrease in serum glucose level by feeding broilers on 1% NSS.  Regarding the impact of NSS on total protein, the concentration of total protein tended to increase in a dose-dependent manner in the NSS-supplemented birds (Kumar et al. 2017). The enhancement in serum level of total protein with NSS administration was observed by Saleh (2014), Yatoo et al. (2012), Khan et al. (2012) and Al-Beitawi and El-Ghousein (2008). Khan et al. (2012) reported higher serum concentration of

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total protein in NSS treated birds than the 1.25% antibiotic-treated or untreated groups. Moreover, Saleh (2014) observed a significant increase in serum values of albumin, total protein and albumin to globulin ratio through feeding 1 mL NSS oil. kg−1 diet compared with avilamycin treated group or non-treated one. Yatoo et al. (2012) showed high levels of serum albumin and total protein with 1% NSS administration as compared with the control. The same findings were noted by Al-Beitawi and El-Ghousein (2008) who showed that the incorporation of 2% NSS in diets of broiler chicks resulted in an increase in total plasma protein level. Hassan et  al. (2007) and Tollba and Hassan (2003) also noticed high serum albumin, total protein and globulin in chickens fed a diet supplemented with high doses of NSS.  The enhancement in serum protein fractions might be attributed to the immuno-­ stimulating action of NSS. Moreover, since the proteins included in NSS diets were more available to chickens, serum protein level was high because serum protein relies on protein availability in the diet (Kumar et al. 2017). In contrast, Shokrollahi and Sharifi (2018) reported that dietary incorporation of 5, 10 or 15 g.kg−1 NSS did not affect serum levels of total protein and albumin. Furthermore, El-Ghammry et  al. (2002) noted that the inclusion of 4  g NSS.kg−1 diet did not affect plasma albumin, globulin and total protein. Hafez et al. (2011) noticed a slightly increase in plasma levels of albumin, globulin and total protein in birds fed NSS diets compared to the control group, but non-significant differences were noted. Hypocholesterolemic and hypolipidemic potential of NSS have been reported in several studies (El-Bagir et al. 2006; Al-Beitawi and El-Ghousein 2008; Bölükbaşi et al. 2009; Hafez et al. 2011; Yatoo et al. 2012; Khan et al. 2013; Shokrollahi and Sharifi 2018; Saleh et al. 2019). Serum cholesterol and triglyceride levels tended to be low in birds fed 10 or 15 g NSS.Kg−1 diet while high-density lipoprotein cholesterol (HDL-c) and very-low-density lipoprotein cholesterol (VLDL-c) were not affected (Shokrollahi and Sharifi 2018). Also, it was reported that cholesterol and LDL-c were linearly reduced with increasing levels (0.5, 1, 1.5 and 2%) of NSS (Kumar et al. 2017). Yatoo et al. (2012) showed that serum concentration of total cholesterol was reduced in birds supplemented with 1% NSS than the control ones. Moreover, serum cholesterol and triglyceride concentrations were reduced in broilers when replacing bacitracin methylene disalicylate by ground NSS, while HDL-c level was elevated (Al-Beitawi et  al. 2009). Al-Beitawi and El-Ghousein (2008) conducted a 49-days trial and noted that using crushed or uncrushed NSS at doses 1.5, 2.0, 2.5 and 3.0% reduced plasma total cholesterol. Hafez et al. (2011) reported that the cholesterol content of plasma was decreased by dietary administration of different levels and forms of NSS. Moreover, in laying hens, Bölükbaşi et al. (2009) showed that feeding on 2 and 3 mL NS oil.kg−1 decreased serum cholesterol level, while triglycerides concentration was not affected. However, Saleh et  al. (2019) reported that triglycerides were reduced in Bovans laying hens received diets supplemented with 0.25 and 0.5  g NS oil.kg−1, while HDL-c was elevated and total cholesterol and LDL-c were not affected. El-Bagir et al. (2006) noted that feeding aged layers on 1 or 3% NSS resulted in a dose-dependent reduction in serum total cholesterol levels. Khan et  al. (2013) revealed that higher doses of NSS (40 and

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50  g.kg−1) in layers diet reduced their serum level of LDL-c, while HDL-c was elevated. In contrast, Khalaji et al. (2011) noted that supplementation of 1% NSS in diets of broiler chickens did not influence the serum cholesterol level. Saleh (2014) assessed the influences of antibiotic (avilamycin) incorporation at level 0.001% against NSS oil at a dose of 1 mL.kg−1 in broiler diets and showed an elevation in plasma level of total cholesterol in NSS group compared to antibiotic and untreated ones. The reduction in serum cholesterol levels in birds fed NSS diets might be attributed to one or more of these reasons: (1) a general decrease in lipid mobilization (Khan et  al. 2013), (2) choleretic activity of NSS constituents that enhance bile production (El-Dakhakhny et al. 2000), (3) the lowering impact of unsaturated FA and thymoquinone on cholesterol biosynthesis by hepatocytes (by reducing the activity of HGM-CoA reductase) or the fractional reabsorption from the small intestine (Brunton 1996; Khan et al. 2013), (4) inhibition impact of β-sitosterol, a sterols found in NSS in a great amount, to cholesterol absorption from the diet (Khan et al. 2012), and (5) sterols also may stimulate the excretion of cholesterol into the intestine to be oxidized by bile acids (Tollba and Hassan 2003). The effect of dietary supplementation of NS on some blood parameters was summarized in Fig. 18.3.

Fig. 18.3  The effect of dietary supplementation of NS on blood parameters of poultry

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3.7  Intestinal Morphology Histomorphology of the small intestine is one of the major indicators for gut health, absorption efficiency and growth performance in broiler chicks. However, there are limited studies examined NSS effects on histomorphometry of intestine. Kumar et al. (2017) found insignificant alternations in crypt depth (CD), villus height (VH), and villus height: crypt depth (VH: CD) ratio in broilers fed diets with 0.5, 1 and 2% NSS. In contrast, Boka et al. (2014) reported an enhancement in intestinal architecture concerning increased jejunal VH and VH: CD ratio and decreased CD of laying hens after dietary inclusion with 2 and 3% NSS. Activation of cellular mitosis and epithelial turnover are directly associated with the increase in VH, and VH: CD ratio (Fan et  al. 1997; Samanya and Yamauchi 2002). Shorter villi and deeper crypts cause poor absorption of nutrients and reduce growth performance (Abd El-Moneim et al. 2019). Dietary supplements that have antibacterial activity can improve intestinal morphology through its ability to diminish the growth of enteric pathogens, reduce its population and decrease toxins production that damage intestinal epithelial cells (Abd El-Moneim and Sabic 2019; Abd El-Moneim et  al. 2019; Abdel-­ Moneim et al. 2019).

3.8  Intestinal Bacterial Enumeration Several plant-origin compounds such as plant extracts, essential oils and whole plant constituents have been considered as antimicrobial agents and gained significant consciousness to be used instead of the known antibacterial feed additives. It has been reported that NSS or their oil exhibited antimicrobial, antiviral, antiparasitic and antibacterial activities against numerous Gram-positive and Gram-negative bacteria (El-Kamali et al. 1998; Durrani et al. 2007; Monika et al. 2013). Active compounds of NSS such as thymoquinone, thymohydroquinone, thymol, carvone and carvene have proved their antifungal and antibacterial activities against a variety of fungi and wide range of infectious bacterial pathogens (Hanafy and Hatem 1991; Harzallah et al. 2012; Ishtiaq et al. 2013; Patra 2014) and their pharmacological influences (Gilani et al. 2004). Moreover, thymoquinone extract of NSS possesses antiparasitic, antiviral, antifungal and antibacterial activities (Abdel Azeiz et al. 2013; Forouzanfar et al. 2014; Ratz-Łyko et al. 2014). Antibacterial potential of NSS essential oils has been also reported (Khan et al. 2012; Boka et al. 2014). The decreasing tendency of bacterial pathogens enumeration in broiler chickens gut administrated with NSS may be attributed to the aforementioned activities of NSS and their constituents. It has been indicated that caecal E. coli and coliform populations were reduced in groups fed NSS at levels 12.5, 25 or 50 g.kg−1 and antibiotic administrated groups, while caecal Lactobacillus counts were not affected (Khan et al. 2012; Kumar et al. 2017). In contrast, lower doses of NSS (10 g/kg) and NSS extract (1 g/kg) did not affect caecal populations of E. coli, coliform and Lactobacillus

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Fig. 18.4  The effect of dietary supplementation of NS on intestinal morphology of poultry

in broiler chickens (Ismail 2011; Khalaji et al. 2011). The effect of dietary supplementation of NS on intestinal morphology was summarized in Fig. 18.4.

3.9  Antioxidative Status Antioxidant activity of NSS and their products are well documented in rats but limited in poultry. The antioxidative characteristics of NS seeds, oil or extract might attributed to (1) its bioactive compounds such as anethole, carvacole, 4-terepinol, thymol, dithymoquinone (DTQ) and TQ (Kruk et al. 2000; Guler et al. 2007), (2) its ability to inhibit generation of eicosanide, and leukotriene B4 thromboxane B2 due to the inhibition of 5-lipooxigenase and cyclooxygenase, respectively (Bassim-Atta and Imaizumi 1998), and (3) its free radicals scavenging activity (Badary et  al. 2003; Ilhan et  al. 2005). It was reported that protective characteristics of NSS against injuries resulted from oxidative stress caused by capturing free radical, reducing production of reactive oxygen species (ROS), including superoxide (O2), hydroxyl (OH) and hydrogen peroxide (H2O2), generated from aerobic respiration, and regulating glutathione activity (Tuluce et al. 2009). Phenolic constituents found in the methanol extracts of NSS such as p-cumaric acids, hydroxybenzoic and syringic acid also showed remarkable antioxidant activities under in-vitro trials (Mariod et  al. 2009). Kruk et  al. (2000) documented that thymol performed as a singlet

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o­ xygen scavenger, while DTQ and TQ revealed superoxide dismutase (SOD)-like activity. In addition, Mansour et al. (2002) noticed that not only TQ but also DTQ acted as superoxide anion quenchers and general free radical scavengers. Furthermore, TQ was found to decrease rats’ serum levels of lactate dehydrogenase and creatine phosphokinase in doxorubicin-induced cardiotoxicity (Nagi and Mansour 2000). Ismail et al. (2010) revealed that not only TQ-rich fraction, but also TQ significantly ameliorated the antioxidant status of plasma through inhibiting hydroxyl radicals formation. Among studies investigated the antioxidant effect of NSS in poultry, Guler et al. (2007) reported that broiler chicks supplemented with 2 and 3% NSS exhibited a lower concentration of MDA in serum, liver, breast muscle and heart muscle compared to those fed 1 and 0.5% NSS and control diets. Sogut et al. (2008) stated that the addition of high NSS levels (3, 5 and 7%) to broiler diets reduced oxidative stress on the liver via decreasing the hepatic lipid peroxidation and increasing antioxidant enzymes activities such as myeloperoxidase, catalase, glutathione-S-­ transferase and adenosine deaminase. In contrast with Guler et al. (2007) findings, Tuluce et al. (2009) noticed that diets incorporated with lower levels of NSS (0.5 and 1%) were able to reduce erythrocyte level of MDA and production of lipid peroxidases and increase glutathione content in treated birds compared to untreated ones. Saleh et al. (2019) revealed that MDA content in the liver of laying hens was numerically reduced when treated with 0.25 and 0.5 g NSS oil.kg−1 compared with the control. In rat and mouse models, the antioxidant mechanisms of NSS oil in protecting mice against carbon tetrachloride (CCl4) (Nagi et al. 1999) and the alkylating agents, including doxorubicin (Al-Shabanah et al. 1998), and cisplatin (Badary et al. 1997), was attributed to presence of TQ.  Moreover, Kanter et  al. (2003) concluded that NSS oil reduced activities of liver enzymes and lipid peroxidation level and contributed to the antioxidant defense system in CCl4-treated rats. Moreover, hepatic antioxidant enzymes (glutathione peroxidase; GPx and SOD) and expression of antioxidant genes (GPx-2, CAT, catalase, and SOD-1) were markedly increased in rats with hypercholesterolemia treated with TQ-rich fraction and TQ (Ismail et al. 2010). Sheikh and Mohamadin (2012) revealed that the levels of MDA in the brain and the activities of enzymatic (glutathione-S-transferase; GST, GPx, CAT, and SOD) and non-enzymatic (vitamin C and GSH) antioxidants were restored close to normal level in rats supplemented with TQ. Besides, the administration of TQ restored the reduction in CAT, GPx, GSH and adenosine triphosphate (ATP) and the increase in blood creatinine, urea-nitrogen, lipid peroxidation and nitric oxide-induced by gentamicin to normal levels. These biochemical data were confirmed by histopathological examination of renal tissues in the same study (Sayed-Ahmed and Nagi 2007). The previous studies concluded that NSS and particularly TQ preserved the activities of the antioxidative enzymes and showed strong protective activity against oxidative stress. The effect of dietary supplementation of NS on the antioxidant status of poultry was illustrated in Fig. 18.5.

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Fig. 18.5  The effect of dietary supplementation of NS on the antioxidant status of poultry

3.10  Immune Response Different studies have investigated the immuno-potentiative activity of NSS. Saleh et al. (2019) reported that antibody titer against Newcastle disease virus (NDV) was significantly increased in Bovans laying hens treated with 250 and 500 g NS oil. ton−1 compared to un-supplemented birds, while antibody titer against avian influenza (H9N1) was not altered. Kumar et al. (2017) clarified that feeding 0.5, 1 and 2% NSS did not affect antibody titer against NDV (vaccinated with live B1 strain and LaSota strain at 5 and 18  days of age) at 28  days of age, but quadratically enhanced the antibody titer at 35 days of age. Moreover, Al-Mufarrej (2014) studied the immune-responsiveness of NSS supplemented broiler chicks and found that inclusion of NSS (1 and 1.4%) stimulated immune response of broilers. The same findings were reported by Ghasemi et al. (2014a). Khan et al. (2012) revealed that incorporation of NSS at levels 4 or 5% into newly evolved crossbred laying hens diets resulted in stimulated immunity against NDV.  Furthermore, the addition of NSS at a dose of 40 g.kg−1 enhanced antibody titers in broiler chicks (vaccinated at 7 and 21, and 11 and 18 days of age) against NDV and infectious bursal disease (IBDV) (Durrani et al. 2007). Al-Beitawi et al. (2009) reported that antibody titer, at 42 days of age, against IBDV and NDV was markedly increased by NSS supplementation when birds inoculated against NDV at 7 and 21 days of age and against IBDV at 14 days of age. The latter authors attributed the enhancement in titers of NDV and IBD to the essential bioactive constituents found in NSS oil like nigellicine, nigellimine, TQ, thymol, and carvacrol. In a study of Shewita and Taha (2011), dietary inclusion of NSS (2–10 g/kg) had no effect on antibody titer against NDV at 14, 24 and 34 days of age, while the weight of immune organs (thymus and bursa)

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was increased markedly by 80–95%. Other studies also reported that the administration of NSS altered the weights of immune organs. Toghyani et al. (2010) noticed that supplementation of NSS at 0.2 and 0.4% in broiler diets enhanced the relative weight of lymphoid organs (thymus and bursa), but not influenced antibody titers against NDV and influenza at 18 and 28 days of age. The same authors also observed that albumin to globulin and heterophil to lymphocyte ratios were not significantly influenced by NSS treatments. Moreover, Hafez et al. (2011) revealed that inclusion of NSS meal or oil in broiler feed markedly improved weights of thymus, spleen and liver. Spleen relative weight was tended to reduce linearly with increasing NSS concentrations, meanwhile bursa index increased linearly (Kumar et  al. 2017). El-Deek et al. (2009) noticed no variations in weights of lymphoid organs, whether in the bursa or spleen, due to dietary supplementation of different levels of NSS.  Yatoo et  al. (2012) also found no significant difference in immunological organ weight. Dietary supplementation of broilers with NSS enhanced the immune response by preventing lipid peroxidation and hepatic damage (Tuluce et al. 2009). Aljabre et al. (2005) revealed that NS volatile oils contain 67 constituents that have many biological activities like antioxidant, antibacterial, and anti-inflammatory that enhanced the immune system (Arslan et al. 2005; Al-Saleh et al. 2006). It was shown that when there were no viral infections, the addition of NSS improves natural killer function along with increasing the ratio of T helper to T suppressor lymphocytes resulting in boosting the cell-mediated immunity (Abdel-Shafi 2013). Another evidence of the ability of NSS to improve the immunity of the bird is its ability to reduce the mortality rate in poultry. Mortality was reduced from 3.5% to 2% by dietary inclusion of 1% powdered NSS in broilers (Nasir et al. 2005) and in layers from 16.67% in the control birds to 4.17% in dietary supplemented one with 1.5% NSS (Akhtar et al. 2003). Furthermore, Hafez et al. (2009) showed that the mortality rate was significantly reduced in NSS fed broilers reared under heat stress conditions compared to control ones. Soltan (1999) revealed that NSS was able to manipulate the microbial population of heat-stressed birds that boost immune response and promote health, thereby reduced mortality rate. In contrast, the results of Ismail (2011) noticed that the NSS administration did not exhibit any statistical impact on the broiler mortality rate. The effect of dietary supplementation of NS on immune status of poultry was illustrated in Fig. 18.6.

4  Conclusions Incorporation of NS seeds, oil or extract in poultry diets as alternative growth promoters revealed numerous advantages due to their antioxidant, antimicrobial and pharmacological properties. Growth performance, laying performance, reproductive performance, nutrient utilization, and egg quality may be improved by the administration of NS products in a dose-dependent manner. Pathogenic bacteria enumeration in chickens’ gut could be decreased by NSS. Hypocholesterolemic and

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Fig. 18.6  The effect of dietary supplementation of NS on the immune status of poultry

hypolipidemic potential of NSS have been well documented in several studies. However, discrepancies among investigations have been reported regarding the enhancement impact of NSS on antibody titer of healthy birds. Several investigations are needed to shed more light on the influence of NSS or their derivatives on poultry reproduction, intestinal architecture and antioxidant properties as well as the FA profile of meat and eggs.

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Salaheen, S., Chowdhury, N., Hanning, I., & Biswas, D. (2015). Organic poultry production with natural feed supplements as antimicrobials. Zoonotic bacterial pathogens and mixed crop-­ livestock farming. Poultry Science, 94, 1398–1410. Saleh, A. A. (2014). Nigella seed oil as alternative to avilamycin antibiotic in broiler chicken diets. South African Journal of Animal Science, 44, 254–261. Saleh, A. A., Kirrella, A. A., Dawood, M. A., & Ebeid, T. A. (2019). Effect of dietary inclusion of cumin seed oil on the performance, egg quality, immune response and ovarian development in laying hens under high ambient temperature. Journal of Animal Physiology and Animal Nutrition, 103, 1810–1817. Salem, M. L. (2005). Immunomodulatory and therapeutic properties of the Nigella sativa L. seed. International Immunopharmacology, 5, 1749–1770. Samanya, M., & Yamauchi, K. E. (2002). Histological alterations of intestinal villi in chickens fed dried Bacillus subtilis var. natto. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 133, 95–104. Saxena, A., & Vyas, K. (1986). Antimicrobial activity of seeds of some ethnomedicinal plants. Journal of Economic and Taxonomic Botany, 8, 291–300. Sayed-Ahmed, M.  M., & Nagi, M.  N. (2007). Thymoquinone supplementation prevents the development of gentamicin-induced acute renal toxicity in rats. Clinical and Experimental Pharmacology and Physiology, 34, 399–405. Schleicher, P., & Saleh, M. (2000). Black cumin: The magical Egyptian herb for allergies, asthma, and immune disorders. Rochester: Inner Traditions/Bear & Co. Shewita, R. S., & Taha, A. E. (2011). Effect of dietary supplementation of different levels of black seed (Nigella Sativa L.) on growth performance, immunological, hematological and carcass parameters of broiler chicks. World Academy of Science, Engineering and Technology, 77, 788–794. Shokrollahi, B., & Sharifi, B. (2018). Effect of Nigella sativa seeds on growth performance, blood parameters, carcass quality and antibody production in Japanese quails. Journal of Livestock Science 9, 56–64. (ISSN online 2277-6214). Sogut, B., Celik, I., & Tuluce, Y. (2008). The effects of diet supplemented with the black cumin (Nigella sativa L.) upon immune potential and antioxidant marker enzymes and lipid peroxidation in broiler chicks. Journal of Animal and Veterinary Advances, 7, 1196–1199. Soliman, E. S., Hamad, R. T., & Ahmed, A. (2017). Prophylactic and immune modulatory influences of Nigella sativa Linn. in broilers exposed to biological challenge. Veterinary World, 10, 1447. Soltan, M. (1999). Effect of diets containing Nigella sativa (black seeds) and or ox bile on growth and reproductive performance of Japanese quail. The Alexandria Journal of Veterinary Sciences, 15, 655–669. Szczerbinska, D., Tarasewicz, Z., Sulik, M., Kopczynska, E., & Pyka, B. (2012). Effect of the diet with common flax (Linum usitatissimum) and black cumin seeds (Nigella sativa) on quail performance and reproduction. Animal Science Papers and Reports, 30, 261–269. Takruri, H.  R., & Dameh, M.  A. (1998). Study of the nutritional value of black cumin seeds (Nigella sativaL). Journal of the Science of Food and Agriculture, 76, 404–410. Toghyani, M., Toghyani, M., Gheisari, A., Ghalamkari, G., & Mohammadrezaei, M. (2010). Growth performance, serum biochemistry and blood hematology of broiler chicks fed different levels of black seed (Nigella sativa) and peppermint (Mentha piperita). Livestock Science, 129, 173–178. Tollba, A., & Hassan, M. (2003). Using some natural additives to improve physiological and productive performance of broiler chicks under high temperature condition. 2. Black cumin (Nigella sativa) or garlic (Allium sativum). Egyptian Poultry Science, 23, 327–340. Tuluce, Y., Ozkol, H., Sogut, B., & Celik, I. (2009). Effects of Nigella sativa L. on lipid peroxidation and reduced glutathione levels in erythrocytes of broiler chickens. Cell Membranes and Free Radical Research, 1, 95–99.

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Chapter 19

Nigella sativa Seeds and Its Derivatives in Fish Feed Mohamed E. Abd El-Hack, Sameh A. Abdelnour, Asmaa F. Khafaga, Ayman E. Taha, and Hany M. R. Abdel-Latif

Abstract  Over the last few decades, the aquaculture sector occupies a privileged place and is considered one of the most rapid sectors in agricultural systems. This increasing in aquaculture production is necessarily needed to increase the production of nutritional feed and intensive aquaculture systems will be more widespread, thus cultured fish will be suffering from different stressors factors as a result of intensification in farm fish. Recently, nutritional scientists used the medicinal plants and their bioactive principles or extracts for many different purposes in aquaculture farms. They exhibited various advantageous properties such as growth-promoting, antimicrobial, immunostimulant, antioxidants, and hepatoprotective activities. Black cumin (Nigella sativa) and its derivatives have some beneficial therapeutic features for farmed fish. Studies concerning the potential effects of N. sativa on fish production and health have been reported. Literature has depicted that growth performance, feed conversion, meat quality can be positively affected by the inclusion of N. sativa or its active principles (i.e., thymoquinone) in fish diet. Additionally, the dietary supplementation of N. sativa or its derivatives in fish diets, not only enhance of the immune responses, improve the biochemical profile in fish blood, protect M. E. Abd El-Hack (*) Poultry Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt e-mail: [email protected] S. A. Abdelnour Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig, Egypt A. F. Khafaga Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, Edfina, Egypt A. E. Taha Department of Animal Husbandry and Animal Wealth Development, Faculty of Veterinary Medicine, Alexandria University, Edfina, Egypt H. M. R. Abdel-Latif Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Alexandria University, Edfina, Behera, Egypt © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_19

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against the invading pathogenic bacteria, but also help to ameliorate the oxidative stress responses of fish towards heavy metal toxicants that may be present in the aquatic environment. This chapter presents a simple synopsis of the consequence and health values of the application of N. sativa and its derivatives, and it investigates the potentials of the production and improvement of fish farms through the application of certain natural therapeutic products. Keywords  Aquaculture · Fish · Nigella sativa · Growth performance · Meat · Immunity · Blood variables · Detoxification

Abbreviations CAT Catalase FCR Feed conversion ratio GPx Glutathione peroxidase Hb Hemoglobin MCH Mean corpuscular hemoglobin MCHC Cell hemoglobin concentration MCV Mean corpuscular volume MPO Myeloperoxidase NSO N. sativa oil NSS Nigella sativa seed PCV Packed cell volume RBCs Red blood cells ROS Reactive oxygen species SGR Specific growth rate SOD Superoxide dismutase USFA Unsaturated fatty acids WG Weight gain

1  Introduction Aquaculture is the most one of the fastest sectors related to the food industry in the world (FAO 2016). Nevertheless, several factors affect the developing of this sector such as intensification, using antibiotics, vaccination, climate change and disease incidences which are considered the important limiting issues in this industry. In the intensively cultured fish, to minimize the invasion of pathogenic microorganisms, the usage of chemicals or antibiotics is extremely desirable, easy and cheaper choice to fish farmers (Bondad-Reantaso et al. 2005). For this issue, it has been recognized that these influences lead not only to develop several antibiotic resistance bacteria

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but also possess their residual effects in the aquatic environment (Jones et al. 2004) and in cultured fish species (Cabello 2006). Recently, great attention has been paid for the use of phytobiotics in aquaculture as an alternative to antibiotic treatments for enhancing productivity, as well as disease control. Nigella sativa (black cumin or black seeds, belongs to Ranunculaceae family), is considered a valuable medicinal herb that is cultivating in most African, and Asian countries. They contain about 36–38% oil, alkaloids, proteins, saponins, carbohydrates and essential oils (0.4–2.5%). The principal active ingredients are thymoquinone, thymohydroquinone, dithymoquinone, thymol and carvacrol (Ali and Blunden 2003). From thousands of years, N. sativa oil (NSO) has been utilized worldwide, as a spice, carminative, feed additive, food preservative, and natural medication for various illnesses in old-style medicine (Merfort et  al. 1997; Nair et al. 2005; Nada et al. 2015). As well, NSO has been illustrated to acquire antimicrobial, antioxidant, anticancer activities and immunostimulatory effects (Salem and Hossain 2000). The possible mechanism(s) of action of Nigella sativa induced protective effects against various disorders are summarized in Fig. 19.1. This chapter highlights the prospective impacts of Nigella sativa and its derivatives (oil, and extracts) on the growth performance, blood hematology and variables, meat quality and immune responses and related gene expression. In addition, the putative protective potency of N. sativa and its derivatives against heavy metal and microbial pathogens in cultured fish species is explored.

Fig. 19.1  The possible mechanisms of action of Nigella sativa against various disorders

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2  Nigella sativa and Bioactive Constituents Many bioactive ingredients have been identified in black seeds. Nickavar et  al. (2003) have demonstrated that black seed is rich in protein (26.7%), lipids (28.5%), carbohydrates (24.9%), fiber (8.4%) and ash (4.8%). They contain various vitamins and minerals like Cu, P, Zn, and Fe. They also contain carotene which is converted by the liver to vitamin A.  The main bioactive ingredients are thymoquinone (30–48%), thymohydroquinone, dithymoquinone, p-cymene (7–15%), carvacrol (6–12%), and thymol (Cheikh-Rouhou et al. 2008). Furthermore, it was documented that black seeds contain two different types of alkaloids; i.e. isoquinoline (nigellicimine and nigellicimine-N-oxide), and pyrazol (nigellidine and nigellicine). They also contain saponin, which is a promising agent with anti-cancer properties (Al-Jassir 1992). Concerning their fatty acid contents, it was reported that black seeds are rich in unsaturated fatty acids (USFA) such as linoleic acid (50–60%), oleic acid (20%), eicodadienoic acid (3%), and dihomolinoleic acid (10%). The saturated fatty acids (palmiti and stearic acid) accounted for about 30% or less (Bourgou et al. 2008). The primary active compounds present in Nigella sativa are presented in Fig. 19.2.

Fig. 19.2  The primary active compounds present in Nigella sativa

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3  B  eneficial Properties of Nigella sativa and Its Derivatives for Fish For a better understanding of the putative impact of N. sativa on the cultured fish physiological and productive functions (growth performance, meat quality, hematological characteristics, and immune response), several studies have been performed. Many investigations showed that the N. sativa seed, extracts, oil or their active compounds are able to enhance the antioxidants as catalase (CAT), and superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities through the inhibition the production of reactive oxygen species (ROS) in the cells. The beneficial effects of Nigella sativa and its derivatives for fish are summarized in Table 19.1 and Fig. 19.3.

3.1  Growth Promoting Activities Numerous investigations have been demonstrated on the growth-promoting activity and immune potentiating characters of N. sativa seeds (John et al. 2007 & Awad et al. 2013). Khatun et al. (2015) demonstrated that feeding Climbing perch (Anabas testudineus) with a diet supplemented different levels of N. sativa oil for 30 days, significantly elevated the weight gain (WG) with the inclusion of 30% dose of N. sativa oil, but feed conversion ratio (FCR) and specific growth rate (SGR) did not change significantly in comparison with the control group. In the rainbow trout diet, Öz et al. (2018) observed a significant enhancement in feed conversion rate, feed intake, protein efficiency and specific growth rates in all groups treated with black cumin oil (0.1–1.3%) in comparison with control. Moreover, Altunoglu et al. (2017) reported that FCR was significantly lowered in the group fed with 0.5 g/kg black cumin extract comparing to the control group. Several exploratory studies recognized that the herbal synergistic action has the potential to enhance the growth and immunity of Nile tilapia, Mugil cephalus, and Japanese flounder (Ji et  al. 2007). Similarly, Abdelwahab and El-Bahr (2012) reported that the combination of N. sativa seeds and Curcuma longa (Turmeric) (0.5% or 1%) in the Asian Seabass diet, help in the improvement of specific growth rate and weight gain compared with the control group. Report from Mohammed and Arias (2016) showed higher average body weight in catfish fed with N. sativa seeds at 5% than that of the control group. N. sativa can beneficially replace protein in the fish diet to provide high-quality protein source with reducing the expensive costs of protein source. Ahmed et al. (2018) evaluated the potential of N. sativa as a protein source in tilapia diets. They documented that N. sativa, when incorporated in tilapia diets (50% or less) as a substitute of soybean meal, did not show undesirable effect on the growth or feed consumption and upgrade the economic efficiency.

Mohammed and Arias (2016) Ahmed et al. (2018)

Öz et al. (2018) Altunoglu et al. (2017) Abdelwahab and El-Bahr (2012)

Author(s) Khatun et al. (2015)

Öz et al. (2017)

Dzpdlat and 2. Meat Duman quality characteristics (2016)

Item 1. Growth promoting activities

Rainbow trout

Barbus grypus

Black cumin oil (11–14 g/kg diet)

N. sativa 50% or less of soybean meal as a substitute Black cumin oil (0.6%)

Tilapia

Catfish

Asian seabass

Rainbow trout

Dose Inclusion of 30% dose of N. sativa oil for 30 days Black cumin oil (0.1–1.3%) Fed with 0.5 g/kg black cumin extract Combination of N. sativa seeds and Curcuma longa (turmeric) (0.5 or 1%) N. sativa seeds at 5%

Fish species Climbing perch (Anabas testudineus) Rainbow trout

Significantly prolong the shelf life with elevated the sensory quality than the control groups They suggested that this oil could be used as a real antimicrobial, antioxidants and enhanced the meat quality of fish. No impact on the sensory parameters The shelf life of rainbow trout was (23) when stored in ice at 2 °C in all treated groups Produced a significant reduction in the total bacteria count (Enterobacteriaceae) in rainbow trout (P  100 > 100 1.8 > 100 > 100 > 100 3.0 7.0

E. coli 62.0 > 100 > 100 41.0 > 100 > 100 > 100 > 100 0.8

Chloramphenicol was used as positive control

growth and aflatoxin production of A. parasiticus and results showed that NSEO had strong activity against A. parasiticus (MIC90: 2.75; MFC: 6.25 mg/mL). This study showed that NSEO can be used as a natural inhibitor in low-level foods to protect food from fungal and toxin contamination. Viuda-Martos et al. (2011) studied the effectiveness of the Egyptian NSEO on three bacterial spices (Listeria, Pseudomonas, and Serratia) and they found it only against L. innocua (inhibition zones of 31 mm). Jrah Harzallah et al. (2011) compared the antibacterial properties of the Tunisian NSEO and its bioactive compounds (thymoquinone). The results showed the NSEO had a stronger antibacterial activity against S. mitis, S. mutans, S. constellatus and G. haemolysans (MIC  =  2.13  mg/mL) than thymoquinone, which mean the different compounds in the essential oil working in a synergistic manner. This emphasizes the importance of using whole oil (or crude extract) of seeds in pharmacological research.

28  Food Applications of Nigella sativa Essential Oil Table 28.4 Antioxidant activities of Nigella sativa essential oil and its main constituents. (Bourgou et al. 2010)

Tested compound Nigella sativa essential oil p-cymene γ-Terpinene Thymoquinone β-Pinene Carvacrol Terpinen-4-ol Longifolene Quercetin

443 Inhibition of DCFH oxidation 1.0 > 200 > 200 1.0 > 200 190.0 > 200 > 200 0.1

Quercetin was used as positive control A = IC50 values (μg/mL); b = IC50 values (μM)

4.2  Antioxidant Activity Lipid oxidation is a crucial factor affecting food quality during food processing, marketing, and storage. In the past years, the antioxidant activity of Nigella sativa seeds and NSEO had been widely investigated (Adamu et  al. 2010). Table  28.4 shows the antioxidant activities of the main constituents in NSEO by DCFH-DA assay and it was confirmed that the in vivo antioxidant activity of the essential oil is mainly due to the action of thymoquinone (Bourgou et al. 2010). It is reported that carvacrol and thymol in NSEO also contribute to antioxidant activity in vitro (Houghton et al. 1995). As reported by Cascella et al. (2018), Nigella sativa could be a useful compound for preventing and treating cerebral ischemia and neurodegenerative diseases, due to it has a significant antioxidant effect and could represent effective neuroprotective activity. In addition, Viuda-Martos et al. (2011) compared antioxidant properties of five essential oil of the species widely cultivated in Egypt. Accordingly, they found that black cumin oil has the strongest antioxidant activity, and its free radical scavenging was 95.89% at 50 mg/mL. Sultan et al. (2009) studied fixed and essential oil from Nigella sativa and they found NSEO (80.2%) showed higher antioxidant capacity than fixed oil (32.3%) by DPPH· radical scavenging test. NSEO inhibited DPPH· radical formation wherein the mean value of IC50 (μM) was found to be 515 (Erkan et  al. 2008). In another experiment using NSEO, the IC50 value of thymoquinone and carvacrol tested by DPPH· assay was found to be 460, 211 and 28.8 mg/mL, respectively (Burits and Bucar 2000).

4.3  Nutritional Value As an edible and medicinal plant, Nigella sativa is generally used in numerous forms, such as spice or food ingredient. In different types of meals, NSEO was applied as a bread or cheese flavoring and spice. According to the report (Ahmad

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Compound Protein Carbohydrates Crude fiber Ash

Quantity (%) 26 25 8.4 4.8

et al. 2013), Nigella sativa seeds contain protein (26%), carbohydrates (25%), crude fiber (8.4%), ash (4.8%) with high amounts of minerals (Cu, P, Zn, and Fe) and carotene (Table 28.5). Tulukcu (2011) indicated that the crude oil extracted from Nigella sativa is considered a functional oil due to its rich content of omega-6 and omega-9 fatty acids including linoleic (54.-70%) and oleic (15–24%), respectively. Meanwhile, very low toxicity of NSO have been observed in rat and mice (Zaoui et al. 2002). Mahmoudvand et al. (2014a) evaluated the cytotoxic effects of various extracts of NSEO and thymoquinone on murine macrophages by MTT assay and they observed a dose-dependent response. In this stage, NSEO showed a less cytotoxic effect on murine macrophages and the value of IC50 was 641.6 μg/mL, while thymoquinone as active constituent showed higher cytotoxic effect (IC50 = 36.3 μg/ mL). In another study, Dollah et  al. (2013) studied the toxicity of Nigella sativa powder on liver function, but they did not see significant changes in liver enzyme blood levels. The same result was reported by Khader et al. (2009), who extracted NSO with no significant side effects on liver or kidney function. Zaoui et al. (2002) also confirmed the low toxicity of NSO by high LD50 values, and suggested a wide margin of safety for therapeutic doses of NSO. From the above results, it can be concluded that Nigella sativa and its oils are comparatively safe as a dietary supplement or a remedy for drugs, but there is still lack of systematic evaluation.

5  Stability of Nigella sativa Oil Oxidation is a big problem of NSO during food processing, marketing and storage since the oil is sensitive to oxidation. Oxidation not only deteriorates the taste and aroma of the oil but also leads to the formation of hazardous compounds. Literature has reported that the progress of lipid oxidation of edible oils is influenced by many factors such as light, heat treatment, oxygen availability and the presence of antioxidants (Grosshagauer et al. 2019). Thus, the mechanism and prevention method of oxidation must be considered. Lipid oxidation of NSO is a complex chemical reaction sequence that is prone to enzymatic and non-enzymatic oxidative degradation. The happening of enzymatic oxidation (hydrolysis) of oil typically requires the presence of water and enzymes (such as lipoxygenases, esterase and cyclooxygenases) in the oil phase, wherein the enzymatic oxidation will lead to the production

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of hydro-peroxides, which may cause cell damage (Ahmed et  al. 2016). Nonenzymatic oxidation can be divided into auto-oxidation and photo-oxidation. According to the Choe and Min’s report (2006), auto-oxidation is mediated by autocatalytic formation and reaction of free radicals (radical forms of acylglycerols) or triplet oxygen (3O2) with oils, whereas the photo-oxidation is mediated by ultraviolet or singlet oxygen (1O2). The automatic oxidation of NSO could be accelerated by free fatty acids, metals such as iron, monoacylglycerols and diacylglycerols, and thermo-oxidic compounds. The study from Mohammed et al. (2017a), pointed out that NSO is susceptible to oxidative degradation and the peroxide value (78.5 mEq O2/kg oil) of NSO showed significant increases with 24 days. A similar finding was reported by Ramadan and Mörsel (2004), who reported that NSO showed increasing in the peroxide value up to 64.5 mEq O2/kg after 21 days. On the other hand, NSO will oxidize and NSEO will volatilize during storage. Fortunately, the challenges of using NSO can be achieved to some extent by suitable encapsulation technology.

6  Encapsulation of Nigella sativa Essential Oil and their Potential Applications in Food NSO is rich in monounsaturated fatty acids (very sensitive to oxidation). A small amount of essential oil (NSEO) in NSO should also be prevented from volatilization. Besides that, there are some problems with low solubility and a weird smell that cannot be fully applied. Luckily, the incorporation of NSO or NSEO into a shell of wall material for protection can significantly improve physical properties, such as dispersibility, solubility, turbidity, viscosity, and thus promote bioactivity, in comparison with the free NSO (Blanco-Padilla et al. 2014). Some encapsulation systems studied about NSO and its main founding are listed in Table 28.6. Nowadays, various technologies for the encapsulation of bioactive compounds have been investigated; however, only a few techniques, namely spray drying and freeze-drying, could be used in the food industries (de Souza Simões et al. 2017), because of its high efficiency, low cost and suitable equipment that is readily available (Dobry et al. 2009). Meanwhile, there are also some other ways to encapsulate NSO at laboratory scale (Fig. 28.4), such as nanoprecipitation (Badri et al. 2018), high-energy methods (Periasamy et al. 2016; Sharif et al. 2017) and layer-by-layer (Konuk Takma and Korel 2019). Spray drying (Fig.  28.5) is the most common and economically practicable method used in the manufacture of powdered form for food applications (Carneiro et al. 2013). In general, there are three main steps in the spray drying process for microencapsulation: preparation of the dispersion, homogenization of the emulsion, and atomization of material into the drying chamber (Gharsallaoui et al. 2007). The

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-

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+

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Charge Neutralization Charge Resaturation Charge Reversal

Fig. 28.4  Layer by layer method. (Miladi et al. 2014)

wall material content, oil concentration, ratio, and spray dried inlet air temperature are the main parameters affecting the final encapsulated powder (Jafari et al. 2008). Mohammed et  al. (2017b) investigated the effect of process conditions on the microencapsulation of spray-dried NSO by the response surface methodology (RSM). The results showed that lipid oxidation of NSO could be significantly reduced by optimizing the microencapsulation conditions. The highest encapsulation efficiency reached 92.7%. Edris et al. (2016) also used the spray drying technique to encapsulate Nigella sativa oleoresin into the powder. The results showed that the encapsulation efficiency of the volatile oil fraction reached 86.2%, which means that it can be used in the manufacture of foods and nutraceuticals. There are some other techniques to encapsulate NSEO.  Sharif et  al. (2017) used different ratios of NSEO with modified starch, under high-speed homogenization, to formulate nanoemulsion. The nanoemulsion exhibited monomodal size distributions with a mean droplet diameter below 200 nm and showed stability against phase separation and coalescence for 4 weeks of storage at room temperature. Badri et al. (2018) used the nanoprecipitation method (Fig. 28.3) to design NSO nanocapsule by nanoprecipitation. The results showed that the size of the prepared nanoparticles did not change significantly within 30  days of the stability study (change interval 250–270 nm). Negut et al. (2018) prepared Fe3O4 nanoparticles containing NSO by co-precipitation method in NH4OH solution. In brief, given excellent physical properties of NSEO after encapsulation, they have the potential to be used in novel food products as a food preservative, and antioxidants in different forms (emulsion, powder, and packaging). Konuk Takma and Korel (2019) prepared the film using antimicrobial chitosan and alginate containing NSO and investigated the effect of NSO packaging film on the quality and shelf-life of chicken breast meats stored at 4 °C for 5 days. The results showed that the samples stored in the active packaging had Lower Total Aerobic Mesophilic (TAMC) and Psychrotrophic Bacteria Counts (PBC) in comparison with the control group. Shaaban et al. (2015) prepared a water-based microemulsion system containing NSEO and its antibacterial activity against six pathogenic bacteria was evaluated. Results showed that NSEO microemulsion was highly effective against

Layer-by-layer (LbL)

Nanoprecipitation

Spray drying

Ultrasonication (Fig. 6) Spray drying

Spray drying

NSEO

NSEO

NSO

NSO

NSO

NSO

High speed homogenization

Encapsulation techniques Emulsification

NSEO

Core material NSEO

Powder

Emulsion

Powder

Nanoparticles

Film

Nano-emulsions

Form Microemulsion

Modified starch and Powder maltodextrin

Maltodextrin DE10 and sodium caseinate

Sodium caseinate and maltodextrin DE10 Whey proteins

Poly (ε-caprolactone)

Alginate and chitosan

Modified starch

Wall materials Tween 20

Main conclusions The EO emulsion was more effective than eugenol emulsion against S. aureus, E. coli, B. cereus, L. monocytogens Emulsions showed shear thinning phenomena and stability towards coalescence during storage (4 weeks at 25 °C) Results indicated that active film containing NSEO has the potential to maintain safety and quality of chicken meat. NSEO were successfully encapsulated that would boost the anti-inflammatory and analgesic effects of indomethacin. The application of microencapsulation had a significant protection effect on the oil oxidation Emulsion droplets keep stable at least 8 days upon storage at 4 °C Encapsulated NSO showed increased stability and resistance content of bioactive compounds, and antioxidant activity, as well as changes in fatty acid composition than un-encapsulated NSO The results of chemical and sensory analysis suggest that NSO microcapsules can be used for producing functional yogurt

Table 28.6  Encapsulation systems of Nigella sativa oil, Nigella sativa essential oil and thymoquinone

(continued)

Abedi et al. (2016)

Anandan et al. (2017) Mohammed et al. (2017a)

Tarhini et al. (2017)

Konuk Takma and Korel (2019) Badri et al. (2018)

Sharif et al. (2017)

References Shaaban et al. (2015)

28  Food Applications of Nigella sativa Essential Oil 447

Thymoquinone

Thymoquinone

Core material Nigella sativa oleoresin

Wall materials Gum Arabic and tween 80

Solvent evaporation PLGA (poly method (dl-lactide-coglycolide) High-pressure Sorbitol, tween 80 homogenization and thimerosal

Encapsulation techniques Spray drying

Table 28.6 (continued)

Nanostructuredlipidcarriers (NLC)

Nanoparticles

Form Powder

Main conclusions The powder, which encapsulates a functional oleoresin, can be used in fortification of different processed foods, dairy or nutraceutical products. PLGA encapsulated TQ nanoparticle with sustained release property has preserved antioxidant as well as anti-microbial activity NLCs could be a promising vehicle for the oral delivery of TQ and improve its gastroprotective properties

Abdelwahab et al. (2013)

Nallamuthu et al. (2013)

References Edris et al. (2016)

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Fig. 28.5  The process diagram of spray drying

S. aureus, B. cereus, and S. typhimurium even at the lowest tested concentration (100  μg/well). Sharif et  al. (2017) indicated encapsulated NSEO showed higher bactericidal activity against 2 Gram-positive pathogenic bacterial strains as compared to pure NSEO. Abedi et al. (2016) found out that NSO microcapsules can be employed to produce functional yogurt because of its high stability, proper chemical and sensory properties. Despite this fact, there are still only a few studies carried out to reveal their potential application as functional food products.

7  Conclusion Interest in new sources of oilseeds has recently grown. This chapter concludes that among the various seed oils, NSEO containing several phytochemicals that have been considered all over the world for having a positive impact on human health. Particularly, the essential oil in Nigella sativa displays considerable commercial value thanks to its strong antimicrobial and antioxidant activities. These outstanding properties of NSEO have the potential to play a remarkable role as a novel source of food preservatives or antioxidants. However, there are still some physical shortcomings like lipid oxidation, low water solubility and volatility. The recent advancement in encapsulation offers the great opportunity to design novel carriers to deliver NSEO and to control its release. Therefore, further research is needed to be conducted to find new applications of Nigella sativa and to transfer NSEO encapsulation from laboratory-scale to industrial scale.

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Chapter 29

Health-Promoting Activities of Nigella sativa Essential Oil Mahmoud Alagawany, Shabaan S. Elnesr, Mayada R. Farag, Mohamed E. Abd El-Hack, Asmaa F. Khafaga, Khan Sharun, Gopi Marappan, and Kuldeep Dhama

Abstract  Nigella sativa is an important herb that has been used by traditional medicine for the treatment of different illnesses. N. sativa oil is a potential source of natural metabolites like phenolic compounds, flavonoids, alkaloids, saponins, and fatty acids that have potential health benefits. Among the bioactive compounds, thymoquinone is the most important and extensively studied component of black cumin. Thymoquinone possesses several properties like anticonvulsant, antidepressant, anxiolytic, antipsychotic, and memory enhancer that can be utilized in the treatment of neurodegenerative diseases. The other active compounds like thymohydroquinone, dithymoquinone, carvacrol, thymol, nigellidine, nigellicine, M. Alagawany (*) · M. E. Abd El-Hack (*) Department of Poultry, Faculty of Agriculture, Zagazig University, Zagazig, Egypt e-mail: [email protected]; [email protected] S. S. Elnesr Department of Poultry Production, Faculty of Agriculture, Fayoum University, Fayoum, Egypt e-mail: [email protected] M. R. Farag Forensic Medicine and Toxicology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt A. F. Khafaga Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, Edfina, Egypt K. Sharun Division of Surgery, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India G. Marappan Division of Avian Physiology and Reproduction, ICAR-Central Avian Research Institute, Bareilly, Uttar Pradesh, India K. Dhama Division of Pathology, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_29

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nigellimine-­ N-oxide and alpha-hedrin also possess well established biological activities that can be used for managing different diseases and disorders. Some of the biological activities of N. sativa include antioxidant, antimicrobial, ­anti-­inflammatory, anti-cancer, anti-parasitic, anti-neurodegenerative, antibabesial, diuretic, hepatoprotective, antidiabetic, antihypertensive agents, antidiarrheal, analgesic, and anthelmintic fertility-enhancing activity. Currently, studies are being undertaken to explore the utility of these seeds as an additive to improve the growth performance and immunity in animals and poultry. The present chapter describes the beneficial application and health-promoting activities of Nigella sativa essential oil and its active ingredients when used in animal and human diets. Keywords  Thymoquinone · Antineurodegenerative · Antiparasitic · Animal · Human · Health benefits · Biological activity

Abbreviations BSO Black seed oil CCl4 Carbon tetrachloride DPPH 2,2-diphenyl-1-picrylhydrazyl MDA Malondialdehyde MRSA Methicillin-resistant Staphylococcus aureus PUFA Polyunsaturated fatty acids ROS Reactive oxygen species TQ Thymoquinone

1  Introduction Medicinal plants are considered as a complementary means to control more diseases, especially in developing countries, for the reason that they have pharmacological properties. Indeed, the discovery of the vast majority, traditional drugs is based on the physiological, therapeutic and chemical actions of the bioactive components of many medicinal herbs. The herbal medicine continues to be a primary ideology in many populations today and a very common practice in different parts of the world. N. sativa as one of the most important herbs that possess therapeutic potential towards several medical conditions, and hence, it is of substantial medicinal value. It prevents or treat different diseases and boost the overall health status. Nigella sativa is an annual flowering plant that is grown almost all over the world. N. sativa is known as black seed, black caraway, black cumin, nigella, Roman

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coriander, nutmeg flower, fennel flower, kalonji, Kalo jeera, Hak Jung Chou and Habbat al-barakah. Several articles have mentioned that the N. sativa extracts have antimicrobial (against several germs) and anti-inflammatory properties as well as antioxidant properties due to its antiradical activities (Abdel-Wahhab and Aly 2005). This chapter aims to review the valuable efforts of scientists for a brief period to explore the medicinal activities and therapeutic potential of Nigella sativa essential oil related to more diseases. The information provided in this work will benefit general practitioners, medical students and the community. In addition, the aim of this chapter is to provide comprehensive and recent information about the nutritional and pharmaceutical benefits of Nigella sativa essential oil in feeding humans, animal and poultry as well.

2  Bioactive Phytochemicals in Nigella sativa Essential Oil A wide range of investigations has been conducted concerning the curative properties and biological activities of black cumin oil (Ahmad et  al. 2013). Globally, Nigella sativa oil is used in the treatment of various diseases in some countries. Its favorable effects on health, especially against ailments such as cardiovascular disease, diabetes and cancer have been highlighted (Shafiq et al. 2014; Bamosa 2015). The volatile oil content in Nigella sativa ranges from 0.4% to 2.5% (Sultan et al. 2009). The volatile oil contains active basic constituents such as dithymoquinone, thymohydroquinone and thymoquinone (Güllü and Gülcan 2013). Thymoquinone (TQ) is the most important component of black cumin studied by researchers. The TQ concentration is from 20.1 to 52.6  mg/100  g (Tüfek et  al. 2015). Many constituents were identified in the essential oil, but the major ones were TQ (27.8–57.0%), carvacrol (5.8–12%), p-cymene (7.1–15.5%), limonene (4.3%), 4-terpineol (2.0–7.0%), t-anethole (0.25–4.0%), longifoline (1–8%), oleic acid (23.4%), palmitic acid (12.5%) and linoleic acid (55.6%) (Burits and Bucar 2000; Nickavar et al. 2003). Also, El Tahir et al. (1993) reported that the volatile oil contained 18.4–24.0% TQ, and 46% monoterpenes (e.g. α-pinene and p-cymene). In addition to other compounds such as campesterol, (+)-citronellol, β-sitosterol, citronellyl acetate, stigmasterol, and α-spinasterol, and fatty acids (oleic, myristic, palmitic, stearic and palmitoleic acids) (Rastogi and Mehrotra 1993). Randhawa and Al-Ghamdi (2002) stated that the active compounds in N. sativa oil include thymohydroquinone, TQ, dithymoquinone, carvacrol, thymol, nigellidine, nigellicine, nigellimine-N-oxide, and alpha-hedrin. Wajs et  al. (2008) illustrated that the major components of the N. sativa essential oil were monoterpenes (87.7%) and their oxygenated derivatives (9.9%), while sesquiterpenes (0.7%) and their oxygenated derivatives (0.3%) constituted only a small fraction. Cheikh-­ Rouhou et al. (2007) revealed that N. sativa oil is a potential source of natural phenolic compounds. Farag et al. (2014) indicated that N. sativa oil contains more than 52 metabolites including 10 flavonoids, 6 phenolics, 10 alkaloids, 8 saponins, and 18 fatty acids that might be utilized for their health benefits.

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3  B  iological and Pharmaceutical Activities of N. sativa Essential Oil Most of the aromatic plants exhibit various pharmaceutical activities and since N. sativa is an aromatic plant, the different parts of black cumin are being used as a health-promoting agent in traditional medicine. They are used as a diuretic, antihypertensive agents (Zaoui et al. 2000), antidiarrheal (Gilani et al. 2001), analgesic (Khan et al. 1999), anthelmintic (Chowdhury et al. 1998) and antibacterial agents (El-Kamali et al. 1998), stimulate the appetite and digestion (Gilani et al. 2004). Many pharmacological properties have been traditionally attributed to N. sativa oil. It presents a potent and therapeutically interesting activity on the respiratory, endocrine, immune and cardiovascular systems (Gilani et al. 2004). N. sativa oil is believed to have antioxidant, antidiabetic, antihyperlipidemic, antihypertensive, anticancer, antimicrobial, antiviral, antitumor, antibacterial, analgesic, anticonvulsant, antihyperlipidemic and anti-inflammatory effects (Shafiq et al. 2014). Due to these effects, N. sativa oil has been used in the treatment of many diseases such as diarrhea, asthma, dyspepsia, dysentery, icterus, fever, hemorrhoids apoplexy, and digestive, liver, kidney cardiovascular, and respiratory diseases (Forouzanfar et al. 2014). Most of these activities have been attributed to the major component of the essential oil of the seeds (Ali and Blunden 2003). Pharmacologically active components of the oil are dithymoquinone, TQ, thymol and thymohydroquinone (Ghosheh et al. 1999). Thymohydroquinone and TQ are active components of the oil that have several pharmacological activities such as antioxidant, antihypertensive and anti-inflammatory effects (Khan et  al. 1999). Varol (2008) recommended the application of topical N. sativa oil to increase speed wound healing. Thymoquinone is testified in rats to prevent oxidative injury in different in vivo and in vitro studies (Daba and Abdel-Rahman 1998; Mansour et al. 2002). It has been suggested to act as an antioxidant and was described to avoid membrane lipid peroxidation in the tissues (Mansour et  al. 2002). These effects seem to be correlated with inhibition of eicosanoid generation (namely thromboxane B2 and leucotrienes B4) through inhibiting cyclooxygenase and 5-­lipooxygenase (Hosseinzadeh et al. 2007a).

4  Antioxidant Effects The animal’s body contains defense mechanisms against the free radicals found in most cells. Free radicals in different types of interacting elements cause the interaction of unsaturated lipids, proteins and amino acids. The balance between antioxidants and free radicals can be recovered from an external supply of antioxidants (Divya 2015; Khafaga and Bayad 2016a, b). High concentration of phytochemicals in N. sativa oil could be responsible for their preventing impacts in various degenerative diseases. Thus, the antioxidant properties of N. sativa oil have a full range of

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perspective applications in human health care. N. sativa oil and its main active constituent (TQ) and other components (carvacrol, 4-terpineol, carvone and anethole) have high antioxidant activity and marked reactive oxygen species (ROS) scavenging potency (Kruk et al. 2000; Badary et al. 2000). N. sativa oil demonstrated stronger radical scavenging action against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical compared with the synthetic antioxidants (Hassanien et al. 2015). Thymoquinone can decrease ROS creation indirectly and prevent lipid peroxidation (Al-Majed et al. 2006). Thymoquinone possesses strong antioxidant properties, wherein oral administration of TQ protected numerous organs against oxidative damage induced by a ariety of free radical generating agents (Baynes 1991; Houghton et al. 1995). Ghadlinge et al. (2014) stated that the histological and biochemical results showed that N. sativa oil has the potential to safeguard the liver tissue against oxidative damage and could be used as a defensive drug against substances induced liver damage. N. sativa oil and its fractions (glycolipids, phospholipids and neutral lipids) presented potent in vitro radical scavenging activity, which is correlated well with their content of polyunsaturated fatty acids (PUFA), phospholipids, and unsaponifiable (Ramadan et al. 2003). Thymoquinone protects liver tissues from the harmful effects of toxic metals and attenuates lipid peroxidation in the liver after exposure to chemicals like carbon tetrachloride (Kapoor 2009). Furthermore, it has been mentioned that N. sativa oil promotes the antioxidant enzymes (like GSH-PX and SOD) activity, and it decreases the lipid peroxidation of the biological membranes thanks to their antioxidant properties and elimination of ROS (Butt and Sultan 2010). The treatment with N. sativa oil decreased lipid peroxidation and oxidative stress, and it stimulates endogenous antioxidant enzymes during the hyperoxia process in the lungs (Tayman et al. 2013). The treatment with N. sativa oil reduced tissue malondialdehyde (MDA) and protein carbonyl levels, but it prevented the inhibition of glutathione peroxidase (GSH-Px), Superoxide dismutase (SOD), and catalase (CAT) enzymes (Kanter et al. 2008). Also, TQ prompted protection of isolated hepatocytes against tert-butyl hydroperoxide induced toxicity evidenced by reducing leakage of alanine aminotransferase and alkaline phosphatase (Daba and Abdel-Rahman 1998). N. sativa oil protected against hepatotoxicity coinciding with improvement in serum lipid profile (Nagi et al. 1999; El-Dakhakhny et al. 2000a), decreasing the elevated lipid peroxide and liver enzyme levels, and boosting the reduced antioxidant enzyme levels (Kanter et al. 2003). The treatment with N. sativa oil prevented carbon tetrachloride (CCl4)-induced liver fibrosis in rabbits with the improvement of the antioxidant status (Turkdogan et al. 2001). Stimulation of polymorphonuclear leukocytes with TQ showed protective action against superoxide anion radical either generated biochemically, photochemically or derived from calcium ionophore, signifying to its potent superoxide radical scavenger (Nagi and Mansour 2000). Constituents of N. sativa oil such as TQ, dithymoquinone, and thymol have a role in neutralizing ROS (Kruk et al. 2000). According to Ghedira (2006), TQ inhibits non-enzymatic lipid lipoperoxidation in liposomes. Also, the carvacrol, 4-terpineol and t-anethole have the significant scavenging

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Genetic factors

Inhibition: Activation:

Oligomer s

Clearance IDE, NEP

AP P

βSecretase

C99

Gsk3β

Aβ42

Plaques

α- Secretase

Synaptic loss Non- Amyloidogenic path

Hyperphosphorylation of tau Tangles Metabolic dysfunction

Evaluation of intracellular level of Ca+2

Mitochondrial dysfunction

Membrane damage

Free radicals

Neuronal death

Fig. 29.1  The antioxidant mechanism of Nigella sativa (thymoquinone); thymoquinone protected against lipid peroxidation, DNA damage, protein destruction, and enhanced apoptotic cell death

influence on free radicals. Salem (2005) showed that black cumin oil augments the glutathione and the antioxidant defense system in the renal cortex. Moreover, the protective effect of N. sativa oil against the hepatonephrotoxicity and oxidative changes (augmentation of DNA damage and peroxidation lipid, and reduction of the rate of SOD) induced during aflatoxicosis, can be attributed to its free radical scavenging effect (Abdel-Wahhab and Aly 2005). Thymoquinone may act as an antioxidant agent and prevents membrane lipid peroxidation in tissues (Mansour et al. 2002). The action mechanism is still largely unknown, but it seems these effects may be correlated to inhibition of eicosanoid generation, namely leucotrienes B4 and thromboxane B2, and membrane lipid peroxidation (Hosseinzadeh et al. 2007a). The antioxidant mechanism of TQ is summarized in Fig. 29.1.

5  Anti-cancerous Effects N. sativa oil has been reported to possess potent anticancer properties and cancer-­ ameliorating effects. Both the oil and its active ingredients expressed anti-tumor properties toward different cancers (Salem 2005). The oil components are claimed to exhibit anticancer activity by preventing genetic changes in normal cells or through the death of cancer cells (Shafiq et  al. 2014). Thymoquinone has

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anticancirogenic, antimutagenic and antioxidant properties. The positive effects of N. sativa oil against cancer types possibly occurs via antioxidant effects, because oxidative stress has an effective role in the formation and development of different cancer types, and TQ increases the activity of the antioxidant enzymes such as superoxide, dismutase, catalase and glutathione peroxidase (Badary et  al. 2000). Thymoquinone has potent anticancer and chemosensitizing properties, which are mediated by its ability to promote apoptosis, arresting the cell cycle and by the generation of ROS. It also enhances the immunity and has fewer side effects compared to the traditional anticancer agent (Mahmoud and Abdelrazek 2019). Another recently identified active compound of N. sativa is alpha-hederin, which is a pentacyclic triterpene saponin that has potential anti-cancer activity. Alpha-hederin has the capacity to induce apoptosis of cancer cell lines in a dose-dependent manner (Adamska et al. 2019). Thymoquinone took from N. sativa extract exhibited significant anticancer properties against lung cancer cells, and prohibited cell proliferation by about 90% (Shafiq et al. 2014). N. sativa extracts represent a promising treatment for breast cancer may due to their effects on inactivating a breast cancer cell line (MCF-7) (Shafiq et al. 2014). N. sativa oil components can decrease DNA damage and inhibit carcinogenesis in colon tissues exposed to toxic agents. TQ prevents the creation of 5-lipoxygenase products, 5-hydroxeicosa-tetraenoic acids, which are essential for colon cancer cells. In addition, TQ may be effective on the cell without the other, because the effect depends on the type of colon cancer cell, whereas it was found that TQ affects the HCT-116 colon cancer cells without affecting the HT-29 colon cancer cells. N. sativa oil exerts inhibitory effects on colon carcinogenesis in rat and anti-­ proliferative effect on the colonic epithelium (Salim and Fukushima 2003). TQ is a potential anticancer agent that can be used for the treatment of genitourinary cancer. TQ produces its anticancer activity by reducing the cell viability, inhibition of renal cancer cells colony formation and induction of apoptosis. The compound also triggers the production of reactive oxygen species and superoxides along with the activation of the apoptotic and autophagic cascade that contributes to its anti-cancer activity (Liou et al. 2019). The mechanism of anticancer action of N. sativa oils may correlate with antioxidant properties of the active ingredients found such as TQ, or due to its cytotoxic and immunosuppressive effects (Islam et al. 2004). N. sativa oil is known to exert insulin-sensitizing actions and lipid-modulating properties (Le et al. 2004), which extend to levels of triglyceride, cholesterol, and prostaglandin in rats (Kocyigit et al. 2009). Furthermore, the same active constituents of N. sativa are known to affect tumor insulin growth factors by the suppression of kinase signaling pathways (Yi et al. 2008). The seed extracts of N. sativa produces anti-hyperglycemic activity in type 2 diabetic model rats by delaying/decreasing carbohydrate digestion, decreasing the glucose absorption from the intestines and by enhancing glucose utilization that is mediated by increased insulin release (Hannan et al. 2019). The anticancer mechanism of TQ is illustrated in Fig. 29.2.

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Induce cancer cell death

Induce apoptosis

Induce autophagy

DNA damage Telomere attrition

Induce apoptosis

ROS

Prevent cellular damage

ROS

Prevent cancer formation

ERK 1/2 MMP 2,9

Suppress EMT process

TWIST1 STAT3

MAPK

PI3K/Akt Inhibit cancer cell migration& invasion

Inhibit carcinogenesis

Fig. 29.2  The anticancer mechanism of Nigella sativa (thymoquinone); thymoquinone caused apoptosis in malignant cells via production of reactive oxygen species (ROS), DNA cleavage, telomeric attrition, autophagy induction, immunomodulation. Also, thymoquinone regulates the epithelial to mesenchymal transition (EMT) with inhibition of cancer metastasis

6  Antimicrobial Activity The alarming increase in bacterial strains resistant to existing antimicrobial agents has demanded alternative strategies. Medicinal plants are now considered as a substitute treatment because of their secure choice in several diseases. Among them, N. sativa is a promising traditional herb having rich medical background. N. sativa essential oil acts as antibacterial agents against a wide spectrum of bacterial strains, wherein N. sativa essential oil encloses quantities of phenolic compounds (p-cymene, carvacrol, and TQ), which might be the reason of the antimicrobial potential of this oil (Hassanien et  al. 2015). Thymoquinone have abroad antimicrobial spectrum including Gram-positive and -negative bacteria, parasites fungi and viruses (Forouzanfar et al. 2014). Hosseinzadeh et al. (2007b) indicated that the essential oil of black cumin is effective against E. coli and S. aureus bacteria. The black cumin essential oil exhibited antifungal activity against Curvularia lunata and Aspergillus species (Agarwal et al. 1979b) as well as against pathogenic yeast Candida albicans (Hanafy and Hatem 1991). The presence of biologically active compounds such as α-phellandrene, α-thujene, limonene, 2(1H)-naphthalenone, TQ, α-pinene, myristicin in N. sativa oil contributed the antimicrobial activity of the oil (Gerige et al. 2009). The antimicrobial and resistance modifying activity of N. sativa essential oil is due to the presence of compounds such as TQ, carvacrol, and p-cymene (Mouwakeh et al. 2019).

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N. sativa oil can be used as a natural antibacterial agent to treat the infection caused by multidrug-resistant bacteria (Saleh et al. 2018). Carvacrol, and p-cymene are the two bioactive compounds that can be used as a potent efflux pump inhibitor against methicillin-resistant Staphylococcus aureus (MRSA) strains. p-Cymene downregulates EP gene mepA expression in MRSA that can lead to loss of virulence in resistant isolates. This property can be utilized for the efficient management of MRSA-related infections (Mouwakeh et al. 2019).

7  Hepatoprotective Activity Thymoquinone mechanism of the hepatoprotective action is not confirmed, but may be associated with the maintenance of intracellular glutathione, which is known to be depleted by oxidative stress, inducing the susceptibility of cells to irreversible injury (Saheb et al. 2016). El-Dakhakhny et al. (2000b) stated that the pre-treatment of rats for 4  weeks with Nigella sativa oil was effective in the defensive against D-galactosamine and CCl4-induced hepatic damage. Türkdoğan et  al. (2001) detected that N. sativa oil has a strong hepatoprotective effect on CCl4-administrated rabbits, wherein that hepatocellular degenerative and necrotic alterations are slight without advanced fibrosis and cirrhosis process in the group treated with the oil. Also, Al-Ghamdi (2003) studied the protective influence of Nigella sativa against CCl4-induced liver damage. Animals treated with CCl4 exhibited significant centrilobular fatty alterations and this influence was significantly declined in animals pretreated with N. sativa (Grypioti 2006). Ghadlinge et  al. (2014) confirmed that N. sativa oil has a hepatoprotective effect, and the administration of this oil can prevent the hepatotoxicity induced by paracetamol. Thymoquinone has a favorable prophylactic impact in a variety of situations, where cellular damage is a consequence of nitrosativa or/and oxidative stress (Nagi et al. 2010).

8  Antidiabetic Activity Nigella sativa oil keeps hypoglycemic effects in streptozotocin-induced diabetic rats and recommends that it may be a beneficial supplemental remedy in diabetes (Abdellatif 2013). Also, N. sativa and its active component, TQ, possess positive effects in controlling lipid profiles and glucose levels in diabetics (Heshmati and Namazi 2015). Although the molecular mechanism of TQ on the secretion of insulin has not been totally clarified, this component causes an increase in glucose use by boosting the serum concentration and diminishing blood glucose by preventing gluconeogenesis (Heshmati and Namazi 2015). Nigella sativa extract (20 mL/kg body weight) declined blood glucose from 340 to 194 mg/dl in alloxan-induced diabetic rabbits (Meral et al. 2001). Even though N. sativa oil is considered as an effective agent for glycemic control in type 2 diabetes mellitus (DM) patients, it was inferior

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Induction: Inhibition:

x

Free iron

Iron

Free radicals

Protein destruction

DNA damage

Bactericidal

x Aging& immune deficiency

x x

NS antioxidant effect

x

x

Carcinogenesis

Apoptosis

x

x

x

Lipid peroxidation

Ischemia reperfusion injury

x Cell death

Fig. 29.3  Mechanism of antidiabetic effect of N. sativa

to the activity of metformin in terms of diabetes management. Administration of N. sativa oil was also found to be tolerable and no side effects (Moustafa et al. 2019). Oral administration of N. sativa extract (300 mg/kg body weight/day) produced a significant hypoglycemic influenceand improved the antioxidant status of streptozotocin-­induced diabetic rats (Kaleem et al. 2006). N. sativa extract (2 mL/ kg body weight) induced significant hypoglycemic influence on the tenth day connected to improved antioxidant status in streptozotocin-induced diabetic rats (Abdelmeguid et al. 2010). The mechanism of the antidiabetic effect of N. sativa is summarized in Fig. 29.3.

9  Anti-inflammatory Activity Progression of an acute or chronic state of inflammation is mediated by a number of mediators, including enzymes, eicosanoids and cytokines secreted by the inflammatory cells neutrophils and macrophages (Lefkowitz et al. 1999). The inhibition of both cyclooxygenase and lipoxygenase pathways is a key factor mediating the anti-­ inflammatory impacts of N. sativa oil and its active ingredients (Salem 2005). Thymoquinone has shown potent anti-inflammatory influences on numerous inflammation-based models including experimental colitis, encephalomyelitis, oedama, arthritis and peritonitis through suppression of the inflammatory mediators’ leukotrienes and prostaglandins (Salem 2005).

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Thymoquinone protected against lung injury from exposure to sulfur mustard in Guinea pigs (Hossein et al. 2008), and affected the cyclooxygenase expression and prostaglandin E2 (PGE2) production of airway inflammation in mice (El mezayen et  al. 2006). Thymoquinone possesses anti-arthritic activity due to its anti-­ inflammatory and immunomodulatory effects. This is mediated by downregulating IL-1, TNF-α, TLR2, TLR4, and NFκB expression levels (Arjumand et al. 2019). The analgesic and anti-inflammatory activity exhibited by N. sativa is attributed to free radical scavenging activity and due to the interaction with several molecular targets that are involved in inflammation, mostly pro-inflammatory enzymes and cytokines (Amin and Hosseinzadeh 2016).

10  Antineurodegenerative Effects Nigella sativa is commonly used in traditional medicine due to its neuroprotective properties. Both hydroalcoholic extract of N. sativa seeds and the active constituent (TQ) was found to be beneficial in managing cerebrovascular insufficiency states and dementia (Fanoudi et al. 2019). Medicinal plants are preferred over synthetic drugs for the treatment of nervous diseases due to their easy availability, less chance of toxicity, and lower production cost (Samarghandian et al. 2018). The methanol extract of N. sativa has the ability to modify brain neurotransmitter amino acid levels. This property of the extract can be utilized for the treatment of certain neurodegenerative diseases that are related to the imbalance of the central nervous system amino acid levels (El-Naggar et  al. 2017). Nigella sativa oil provided neuro-­ safeguard from spinal cord injury and avoid damage to the cells of the brain from numerous nerve toxins in experimental animal models. Moreover, it showed promising therapeutic and prophylactic impacts on murine toxoplasmosis (Randhawa and Alenazi 2016). Treatment using anticholinesterase agents are considered as the best strategy for managing memory impairment. The extract of N. sativa exhibits dose-dependent inhibition of the acetylcholinesterase enzyme. Identification of this active ingredient can help to synthesize new anticholinesterase agents (Ansari et al. 2019). N. sativa and TQ have several properties like anticonvulsant, antidepressant, anxiolytic, antipsychotic, and memory enhancer that can be utilized in the treatment of neurodegenerative diseases such as Alzheimer, Parkinson and multiple sclerosis (Javidi et  al. 2016). They also exhibit well established protective effects against neurological affections like depression, encephalomyelitis, epilepsy, ischemia, and traumatic brain injury in various cell lines and experimental animal models (Samarghandian et al. 2018).

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11  Anti-Alzheimer Effects Alzheimer’s disease is a neurodegenerative disorder characterized by the accumulation of amyloid-beta in the hippocampus. Herbal plant oils have gained wide attention in their use in the treatment and management of psychiatric, degenerative and neurological diseases, due to their no or fewer side-effects (Yadav et  al. 2009; Kumar et al. 2012). Compelling evidence has reported that black seed oil (BSO) exhibits protective activities against many diseases depending on its high antioxidant and anti-inflammatory properties (Kanter et al. 2008; Majdalawieh et al. 2010). This could suggest its use as a potential therapy for cognitive impairments in Alzheimer’s disease. Earlier studies evidenced the use of BSO in neurodegenerative diseases like Alzheimer due to its antioxidant potential (Hajra 2011; Hobbenaghi et al. 2014), a property related to the presence of TQ, which has the potential to prevent neuronal cell death (Kim et al. 2013). BSO can be a probable complementary in the management of memory dysfunctions, due to its impactsonhippocampal dependent memory, working memory and cortico-hippocampal neuron, against scopolamine-induced memory dysfunctions in rats (Imam et al. 2016). The mechanistic action of N. sativa against Alzheimer’s disease is illustrated in Fig. 29.4.

Nigella sativa (Thymoquinone)

cell

ROS Oxidative stress Increased lipid peroxidation

Decreased oxidative stress

decreased antixidatant enzymes

decreased beta cell activity

Decreased lipid peroxidation

Increased antixidatant enzymes

Enhanced beta cell activity

decreased insulin production

Increased insulin production

Diabetes mellitus type 1

decrease incidence Diabetes mellitus type 1

Fig. 29.4  The mechanistic action of N. sativa against Alzheimer disease

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12  Fertility-enhancing Activity Thymoquinone is the most important phytochemical compound found in Nigella sativa that has antioxidant activity. This compound has the ability to improve fertility by increasing the number of healthy sperms and by preventing sperm anomalies (Tufek et al. 2015). The oil extracted from Nigella sativa has the ability to enhance fertility in male rats. This property has great potential and can be effectively utilized in the modulation of male fertility (Akour et  al. 2016). Oral supplementation of N. sativa oil in infertile men improves the semen quality by improving semen parameters like sperm count, motility, morphology, semen volume, and pH (Kolahdooz et al. 2014).

13  Antiparasitic Activity Nigella sativa was found to have an anthelmintic activity against tapeworm comparable to that of piperazine. The hydroalcoholic extract of N. sativa has scolicidal activity against the protoscolices of camel hydatid cysts. The scolicidal activity of the extract depended upon the exposure time and concentration (El-Bahy et  al. 2019). TQ has antibabesial activity against in vitro and in vivo growth of piroplasma parasites. The highest activity was reported against the in vitro growth B. divergens and B. bigemina which can be utilized for the treatment of animal piroplasmosis under field conditions (El-Sayed et  al. 2019). Plant-derived natural compounds obtained from N. sativa like TQ exhibit anthelmintic potential that can be used for countering the emerging resistance against available anthelminthic compounds. Thymoquinone exhibits anthelmintic activity against Fasciola gigantica by inhibits the worm motility and severely disrupting the tegumental surface. It also suppresses the detoxification and free radical scavenging ability as well as their invasive capacity which is mediated by inhibiting the Cathepsin L gene expression (Ullah et al. 2017).

14  Analgesic Activity The essential oil produced significant analgesic activity using chemical and thermal noxious stimuli methods such as acetic acid-induced writhing, hot plate, and tail-­ flick tests. Topical N. sativa seed oil was found to be safe and clinically effective in managing cyclic mastalgia in humans. The analgesic activity exhibited by N. sativa seed oil was comparable to topical diclofenac making it an efficient alternative (Huseini et al. 2016). The analgesic effect of N. sativa extract is due to the presence of the active compounds (i.e., TQ). The extracts produce analgesic activity by the inhibition of pain mediators. The mechanism of action that produces analgesia

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might be analogous to the action of acetylsalicylic acid (Zakaria et al. 2018). Nigella sativa seed oil was found to possess dose-dependent analgesic activity, which can be utilized for the treatment of arthritis (Nasuti et al. 2019).

15  Applications of Black Cumin Oil in Animals and Poultry Studies are being undertaken to explore the use of Nigella sativa seeds as an additive to improve the growth performance and immunity in animals and poultry. A study with the black seed (Nigella sativa L.) in broilers at Peshwar in 2005, revealed that the addition of these seeds at 40  g/kg improved the body weight gain, feed intake, feed efficiency, dressing percentage and immune response (Durrani et  al. 2007; Shewita and Taha 2011). The incorporation of Nigella sativa seeds into the ration of broilers resulted in higher antibody concentration against common viral infections (Newcastle disease, Infectious Bursal Disease and Infectious Bronchitis) during a 35 days study. The authors further assessed whether the addition of N. sativa is profitable or not, and they had more gross return as well as return per unit of feed cost at 40 g/kg feed. Black cumin seeds could be used digestive and appetite stimulant (Gilani et al. 2004; Arif et al. 2019), antidiarrheal (Gilani et al. 2001), anthelmintic (Agarwal et  al. 1979; Chowdhury et  al. 1998) and antibacterial agents (Ferdous et al. 1992; El-Kamali et al. 1998). A study was performed to evaluate the protective effects of N. sativa oil against oxytetracycline-induced hepatorenal toxicity in 40 white New Zealand male rabbits. Result conferred the preventive role of N. sativa oil upon oral administration. Owing to the antioxidant defense mechanism by scavenging the free radical to impart hepatoprotective effect herbal oil minimized the damage in liver and kidney tissue (Abdel-Daim and Ghazy 2015). TQ was administered in adult Wistar albino male rats via the intraperitoneal route to assess the cardioprotective effect of TQ against myocardial ischemia/Reperfusion (I/R) injury and ischemia and reperfusion-­ induced ventricular arrhythmias in anesthetized rats. Results showed a reduction in the size of the infarct, frequency of myocardial ischemia/reperfusion and lowers reperfusion-induced arrhythmias after treatment with TQ (Gonca and Kurt 2015). The seeds of N. sativa are a good source of protein as well as the unsaturated fatty acids, hence there were studies on animals/birds to explore this as a feed ingredient/feed additive. Supplementation of seeds of black cumin at a 1% level exhibited a positive effect on performance which may be due to its antibacterial activity against the pathogenic bacteria, fungi and parasites in the gut (Gilani et al. 2004). Various researchers have reported antimicrobial activities of black cumin against Streptococcus mutans (Namba et al. 1985); Escherichia coli (Ferdous et al. 1992); Staphylococcus aureus, Pseudomonas aeruginosa (Sokmen et al. 1999). In addition to its antimicrobial activities, the black cumin also exhibits anthelmintic activities (Agarwal et al. 1979). Guler et al. (2006) reported that the seeds of black cumin could be used as a natural growth promoter instead of antibiotics.

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Abu-Dieyeh and Abu-Darwish (2008) fed seed powder of Nigella sativa at 1.5% to broilers during the finisher stage and observed that the seed powder exhibited positive response in both the growth performance and as well as the livability of the birds. However, feeding up to 2% of black cumin seeds resulted in negative effects in broilers. Guler et al. (2006) reported that feeding of 1% black cumin feeds could be considered as a potential candidate for an alternative natural growth promoter instead of antibiotics in broilers. Akhtar et  al. (2003) observed that feeding of N. sativa seeds at a 1% level increased the egg production, egg mass, shell thickness and reduced the yolk cholesterol content. N. sativa exhibits potent antioxidant activity in the biological system, hence they could be used as heat stress alleviating agent in broilers. Hermes et al. (2009) fed black cumin seeds to heat-stressed broilers and found that the seeds exerted beneficial effects through manipulating the body’s metabolism. A study was carried out on the use of N. sativa seed extract in Japanese quail to study their effects on egg production as well as the egg quality (Denli et al. 2004). They reported that supplementation with black cumin seed extract did not have any effect on feed intake. The positive effect of dietary black cumin seeds on gain and feed conversion ratio could relate to increased efficiency of feed utilization and/or altered carcass composition. Similarly, recent studies reported that essential oils blocked the effect of pathogens in the digestive system and improved feed intake, feed conversion ratio and carcass yield (Tucker 2002; Giannenas et al. 2003). The reason for the lower performance of 2% and 3% black cumin groups compared with the 1% black cumin group may be due to negative effects of components such as alkaloids, saponin, volatile oils and other anti-nutritional factors found in black cumin. Saleh (2014) fed 0.001% Nigella seed oil to broilers and observed that the feeding of the oil leads to improved body weight gain and feed efficiency without affecting the feed consumption. The author further observed that the supplementation of oil at 1 mL/kg leads to a reduction in plasma cholesterol and triglycerides levels and increased the glutathione peroxidase activity. Siddiqui et al. (2015) studied the comparative effect of broilers fed with N. sativa seed powder (1.5, 2.5 and 3.0%) versus 0.2 and 0.4% acetone extracts. They did not find any effect of N. sativa (either as seed powder or acetone extract) on the growth performance. However, feeding of 3.0% of seed powder or 0.4% extract markedly reduced the serum cholesterol and triglycerides levels. Attia and Al-Harthi (2015) fed broilers with N. sativa seed oil (0.5 g/kg) and reported an improvement in the feed efficiency and body weight gain during the later stages of growth period, where the birds experienced moderate to severe heat stress.

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16  Conclusion and Future Prospects N. sativa is a storehouse of several compounds that have not yet been studied or even identified. Among the identified major constituents, some of them like TQ has been studied extensively for their biological activity. The literature published so far have shown several activities like antioxidant, antimicrobial, anti-inflammatory, anti-cancer, anti-parasitic, anti-neurodegenerative, antibabesial, diuretic, hepatoprotective, antidiabetic, antihypertensive agents, antidiarrheal, analgesic, and anthelmintic fertility-enhancing activities that can be utilized for the treatment of various diseases and disorders. Further evaluation of the herb can identify several biologically active compounds that have potential health applications. Currently studies are being conducted to explore the potential application of this herb as a feed additive to improve the growth performance, health and immunity in animals and poultry. Supplementation with Nigella seed oil or seed powder can improve the body weight gain and feed efficiency making it suitable to be used as a natural origin performance enhancer in the poultry industry. Further researches are necessary to identify newer compounds from N. sativa that has potential health benefits in both the human and animal health sector.

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Mahmoud, Y. K., & Abdelrazek, H. M. A. (2019). Cancer: Thymoquinone antioxidant/pro-oxidant effect as a potential anticancer remedy. Biomedicine & Pharmacotherapy, 115, 108783. https:// doi.org/10.1016/j.biopha.2019.108783. Majdalawieh, A. F., Hmaidan, R., & Carr, R. I. (2010). Nigella sativa modulates splenocyte proliferation, Th1/Th2 cytokine profile, macrophage function, and NK anti-tumor activity. Journal of Ethnopharmacology, 131, 268–275. Mansour, M.  A., Nagi, M.  N., El-Khatib, A.  S., & Al-Bekairi, A.  M. (2002). Effects of Thymoquinone on antioxidant enzyme activities, lipid peroxidation and DTDiaphorasein different tissues of mice: A possible mechanism of action. Cell Biochemistry and Function, 20, 143–151. Meral, I., Yener, Z., Kahraman, T., & Mert, N. (2001). Effect of Nigella sativa on glucose concentration, lipid peroxidation, antioxidant defense system and liver damage in experimentally-­ induced diabetic rabbits. Journal of Veterinary Medicine Series A, 48(10), 593–599. Moustafa, H. A. M., El Wakeel, L. M., Halawa, M. R., Sabri, N. A., El-Bahy, A. Z., & Singab, A.  N. (2019). Effect of Nigella sativa oil versus metformin on glycemic control and biochemical parameters of newly diagnosed type 2 diabetes mellitus patients. Endocrine, 65(2), 286–294. https://doi.org/10.1007/s12020-019-01963-4. Mouwakeh, A., Kincses, A., Nove, M., Mosolygo, T., Mohacsi-Farkas, C., Kisko, G., & Spengler, G. (2019). Nigella sativa essential oil and its bioactive compounds as resistance modifiers against Staphylococcus aureus. Phytotherapy Research, 33(4), 1010–1018. https://doi. org/10.1002/ptr.6294. Nagi, M. N., & Mansour, M. A. (2000). Protective effect of thymoquinone against doxorubicin-­ induced cardiotoxicity in rats: A possible mechanism of protection. Pharmacological Research, 41, 283–289. Nagi, M.  N., Alam, K., Badary, O.  A., Al-Shabanah, O.  A., Al-Sawaf, H.  A., & Al-Bekairi, A. M. (1999). Thymoquinone protects against carbon tetrachloride hepatotoxicity in mice via an antioxidant mechanism. Biochemistry and Molecular Biology International, 47, 153–159. Nagi, M. N., Almakki, H. A., Sayed-Ahmed, M. M., & Al-Bekairi, A. M. (2010). Thymoquinone supplementation reverse acetaminophen-induced oxidative stress, nitric oxide production and energy decline in mice liver. Food and Chemical Toxicology, 48(8–9), 2361–2365. Namba, T., Tsunezuka, M., Saito, K., Kakiuchi, N., Hattori, M., Dissanayake, D.  M. R.  B., & Pilapitiya, U. (1985). Studies on dental caries prevention by traditional medicines, screening of Ayurvedic medicines for anti-plaqueaction. Shoyakugaku Zasshi, 39, 146–153. Nasuti, C., Fedeli, D., Bordoni, L., Piangerelli, M., Servili, M., Selvaggini, R., & Gabbianelli, R. (2019). Anti-inflammatory, anti-arthritic and anti-nociceptive activities of Nigella sativa oil in a rat model of arthritis. Antioxidants, 8(9). https://doi.org/10.3390/antiox8090342. Nickavar, B., Mojab, F., Javidnia, K., & Amoli, M. A. (2003). Chemical composition of the fixed and volatile oils of Nigella sativa L. from Iran. Z Natutforsch C, 58(9–10), 629–631. Ramadan, M. F., Kroh, L. W., & Morsel, J. T. (2003). Radical scavenging activity of black cumin (Nigella sativa L.), coriander (Coriandrum sativum L.), and Niger (Guizotia abyssinica Cass.) crude seed oils and oil fractions. Journal of Agricultural and Food Chemistry, 51, 6961–6969. Randhawa, M. A., & Alenazi, S. A. (2016). Neuropsychiatric effects of Nigella sativa (black seed) A review. Alternative & Integrative Medicine, 5, 1. https://doi.org/10.4172/2327-5162.1000209. Randhawa, M.A., & Alghamdi M.S. (2002) A review of the pharmaco-therapeutic effects of Nigella sativa. Pakistan Journal of Medical Research, 41, 77–83. Rastogi, R.  P., & Mehrotra, B.  N. (1993). Compendium of Indian medicinal plants (Vol. 3, pp. 452–453). NewDelhi: CSIR. Saheb, S.  H., Desai, S.  D., Das, K.  K., & Haseena, S. (2016). Hepatoprotective effect of Nigella sativa seed in streptozotocin induced diabetic albino rats: Histological observations. International Journal of Anatomy and Research, 4(2), 2459–2463. Saleh, A. A. (2014). Nigella seed oil as alternative to avilamycin antibiotic in broiler chicken diets. South African Journal of Animal Science, 44(3), 254–261. Saleh, F. A., El-Darra, N., Raafat, K., & Iman El Ghazzawi, N. B. (2018). Phytochemical analysis of Nigella sativa L. utilizing GC-MS exploring its antimicrobial effects against multidrug-­ resistant bacteria. Pharmacognosy Journal, 10(1), 99–105.

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Salem, M. L. (2005). Immunomodulatory and therapeutic properties of the Nigella sativa L. seed. International Immunopharmacology, 5(13–14), 1749–1770. Salim, E. I., & Fukushima, S. (2003). Chemopreventive potential of volatile oil from black cumin (Nigella sativa L.) seeds against rat colon carcinogenesis. Nutrition and Cancer, 45, 195–202. Samarghandian, S., Farkhondeh, T., & Samini, F. (2018). A review on possible therapeutic effect of Nigella sativa and Thymoquinone in neurodegenerative diseases. CNS & Neurological Disorders Drug Targets, 17(6), 412–420. https://doi.org/10.2174/1871527317666180702101455. Shafiq, H., Ahmad, A., Masud, T., & Kaleem, M. (2014). Cardio-protective and anti-cancer therapeutic potential of Nigella sativa. Iranian Journal of Basic Medical Sciences, 17(12), 967. Shewita, R. S., & Taha, A. E. (2011). Effect of dietary supplementation of different levels of black seed (Nigella sativa L.) on growth performance, immunological, hematological and carcass parameters of broiler chicks. World Academy of Science, Engineering, and Technology, 77, 788–794. Siddiqui, M. N., Islam, M. T., Sayed, M. T., & Hossain, M. A. (2015). Effect of dietary supplementation of acetone extracts of Nigella sativa L. seeds on serum cholesterol and pathogenic intestinal bacterial count in broilers. The Journal of Animal and Plant Sciences, 25(2), 372–379. Sokmen, A., Jones, B. M., & Erturk, M. (1999). The effect of black seed oil from Nigella sativa against murine cytomegalovirus infection. International Journal of Immunopharmacology, 22, 729–740. Sultan, M. T., Butt, M. S., Anjum, F. M., Jamil, A., Akhtar, S., & Nasir, M. (2009). Nutritional profile of indigenous cultivar of black cumin seeds and antioxidant potential of its fixed and essential oil. Pakistan Journal of Botany, 41(3), 1321–1330. Tayman, C., Cekmez, F., Kafa, I.  M., Canpolat, F.  E., Cetinkaya, M., Tonbul, A., & Sarici, S. U. (2013). Protective effects of Nigella sativa oil in hyperoxia-induced lung injury. Archivos de Bronconeumología, 49(1), 15–21. Tucker, L. (2002). Botanical broilers: Plant extracts to maintain poultry performance. Feed International, 23, 26–29. Tufek, N. H., Altunkaynak, M. E., Altunkaynak, B. Z., & Kaplan, S. (2015). Effects of thymoquinone on testicular structure and sperm production in male obese rats. Systems Biology in Reproductive Medicine, 61(4), 194–204. https://doi.org/10.3109/19396368.2015.1044135. Türkdoğan, M. K., Ağaoğlu, Z., Yener, Z., Sekeroğlu, R., Akkan, H. A., & Avci, M. E. (2001). The role of antioxidant vitamins (C and E), selenium and Nigella sativa in the prevention of liver fibrosis and cirrhosis in rabbits: New hopes. Deutsche Tierärztliche Wochenschrift, 108(2), 71–73. Ullah, R., Rehman, A., Zafeer, M. F., Rehman, L., Khan, Y. A., Khan, M. A., Khan, S. N., Khan, A.  U., & Abidi, S.  M. (2017). Anthelmintic potential of Thymoquinone and curcumin on Fasciola gigantica. PLoS One, 12(2), e0171267. https://doi.org/10.1371/journal.pone.0171267. Varol Y. (2008). Investigation of the effects of Nigella sativa oil on cuta- neous wound healing in rats [Ph.D. Thesis], Marmara Universitesi Saglik Bilimleri Enstitusu, Istanbul, Turkey. Wajs, A., Bonikowski, R., & Kalemba, D. (2008). Composition of essential oil from seeds of Nigella sativa L. cultivated in Poland. Flavour and Fragrance Journal, 23(2), 126–132. Yadav, R. S., Sankhwar, M. L., Shukla, R. K., Chandra, R., Pant, A. B., Islam, F., et al. (2009). Attenuation of arsenic neurotoxicity by curcumin in rats. Toxicology and Applied Pharmacology, 240, 367–376. Yi, T., Cho, S. G., Yi, Z., Pang, X., Rodriguez, M., Wang, Y., Sethi, G., Aggarwal, B. B., & Liu, M. (2008). Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing AKT and extracellular signal-regulated kinase signaling pathways. Molecular Cancer Therapeutics, 7, 1789–1796. Zakaria, A., Jais, M. R., & Ishak, R. (2018). Analgesic properties of Nigella sativa and Eucheuma cottonii extracts. Journal of Natural Science Biology and Medicine, 9(1), 23–26. https://doi. org/10.4103/jnsbm.JNSBM_131_17. Zaoui, A., Cherrah, Y., Lacaille-Dubois, M.  A., Settaf, A., Amarouch, H., & Hasar, M. (2000). Diuretic and hypotensive effects of Nigella sativa in the spontaneously hypertensive rat. Thérapie, 55, 379–382.

Part IV

Nigella sativa Seed Extracts: Chemistry, Technology, Functionality and Applications

Chapter 30

Composition and Functionality of Nigella sativa Seed Extracts Songul Kesen

Abstract  In this chapter, the composition and functionality of Nigella sativa seed extracts were reported. Nigella sativa, known as a black seed, is an annual flowering plant that has been used for many years in traditional treatment in the Middle East regions, India, Africa and Asia. N. sativa can be reached to a height of 40–90 cm. Its fruit is similar to a large, swollen capsule containing 3–7 follicles, each with many seeds. When the capsule of a fruit matures, it opens and the seeds are fall out then turn black. The chemical composition of Nigella sativa consists of fixed oil (35–40%), proteins (23%), volatiles (0.05%), vitamins, alkaloids, sugars, phytosterols, resins, organic acids, and minerals. Black cumin seed has many therapeutic properties. It is known to have a curative effect especially in cardiovascular, diabetes, inflammatory, renal and liver diseases. The seeds, oils and extracts of Nigella sativa are used in traditional treatment all over the world to cure and protection of various diseases including cough, asthma, dizziness, bronchitis, fever, influenza, diarrhea, headache, eczema and dyslipidemia. It is the essence of black seed mentioned in many scientific sources. The black cumin seed extract is obtained by washing, drying, powdering the seeds and then extracting with alcohol. The seed extract contains certain compounds with potential biological activity such as flavonoids, phenolics, proteins, carbohydrates and alkaloids. Nigella sativa extract is also folksy used up in numerous cancer therapy in some countries. Keywords  Black seed · Seed extract · Functional properties · Health-promoting Antioxidants · Olfactometric

Abbreviations BCS Black cumin seed DPPH· 1,1-Diphenyl-2-picrylhydrazyl DTQ Ditimoquinone S. Kesen (*) Gaziantep University, Naci Topcuoglu Vocational High School, Gaziantep, Turkey © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_30

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EO Essential oil FD Flavor dilution GAE Gallic acid equivalent GC-MS-O Gas chromatography-mass spectrometry-olfactometry IC50 The half maximal inhibitory concentration NP Nanoparticle NS Nigella sativa NSE Nigella sativa extract QE Quercetin equivalent TEAC Trolox equivalent antioxidant capacity TPC Total phenolic content TQ Thymoquinone

1  Nigella sativa (Black Cumin Seed) Nigella sativa (family Ranunculaceae) is a green and blue flowering plant with small black seeds. Black cumin seeds are used as a flavoring substance in curry and a component in vinegar because of its aromatic flavor that resembles hot pepper taste, as well as they are substitutes for pepper in cooking and bakery foods (Mamun and Absar 2018). It has also a rich history and religious background due to its use in traditional medicine. It is important because of the potential source of biologically active phytochemicals (Anvari et al. 2012; Kadam et al. 2019). The NS seeds are the source of the active ingredients and include fixed oil (30–40%), essential oil (0.5–1.5%), sugars, proteins, and pharmacologically active ingredients like thymoquinone, ditimoquinone, and nigellin (Boskabady et al. 2007; Hosseini et al. 2015). The other ingredients extracted from seeds are vitamins (riboflavin, thiamin, pyridoxine, folic acid, and niacin), minerals, alkaloids (nigellicine, nigellimine and nigellidine) and proteins (Berkoz et  al. 2019). The plant has been the subject of many investigations due to certain biological activities and therapeutic potentials. In particular, studies have investigated the effects on renal, diabetes, cancer, microbial situation, cardiovascular diseases, immune gastrointestinal, respiratory systems, and nervous (Ramadan 2007; Norouzi et al. 2019).

2  Nigella sativa Seed Extracts By-products derived from NS such as oil and extract are used worldwide for the herbal treatment. Oil and extract obtained from NS are different from each other. Ksouda et al. (2018) stated that the black seed oil is obtained as follows; 100 mg dried plant seeds were grounded with 1 mL diethyl ether using a homogenizer for 5 min (4500 rpm at room temperature). The organic phase and the residue were then

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separated by centrifugation (3000 rpm for 3 min). From the collected organic phase, diethyl ether was evaporated by nitrogen flow to obtain the oil. In another research, extraction of seed oil was conducted as follows; 50 g dry seeds were milled and received with petroleum ether for 4 h in a Soxhlet apparatus. Then, the extract was condensed under reduced pressure (Hosseini et al. 2019). According to Soleimanifar et  al. (2019), to obtain seed oil, powdered black cumin seed was grounded with n-hexane at a ratio of 1:4 (w/v) at room temperature for 28 h, then the mixture was filtered and the n-hexane was evaporated under reduced pressure at 40 °C. On the other hand, NS extract is obtained by the hydroalcoholic extraction method using different solvents such as methanol or ethanol. The methodology for obtaining the black cumin seed extract is shown in Fig. 30.1. The methods used to obtain NS seed extract from different studies are summarized in Table 30.1. Many researchers have used the hydroethanolic extraction method to obtain seed extract. Norouzi et al. (2019) described how they obtained the seed extract by reference to Seghatoleslam et al. (2016). According to this process, 100 g the seeds were cleaned, dried, pulverized and extruded in a Soxhlet apparatus containing 70% ethanol. The ethanolic extract was then concentrated under vacuum to remove ethanol. According to Tappeh et al. (2019), to obtain seed extract, 200 g of the seed powder was soaked into 800 mL of 70% ethanol. The solution was then shaken once every 24 h for 3 Fig. 30.1 The methodology for obtaining black cumin seed extract

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Table 30.1  Extraction methods of Nigella sativa seeds Extraction method Amount Hydroethanolic 100 g Hydroethanolic 200 g

Hydroethanolic 500 g

Solvent Ethanol (70%) 800 mL ethanol (70%) 2 L ethanol (80%)

Hydroethanolic Unspecified

2 L ethanol (50%)

Hydroethanolic 200 g

800 mL ethanol (50%) Methanol

Methanolic

2.5 kg

Methanolic

Methanol (80%)

Methanolic

1:4 (w/v) ratio (seed: Methanol) 10 g seed oil

Methanolic

50 g

500 mL methanol (80%)

Methanolic

200 g

1 L methanol (70%)

100 mL methanol

Extraction condition Concentrating under reduced pressure After 3 days filtration and concentrating with rotary evaporator Refrigerating for a week, filtration and concentrating with rotary evaporator 48 h at room temperature, filtration, drying in an oven at 40 °C for 72 h 72 h at 40 °C, filtration, drying by rotary evaporation at 40 °C After 2 weeks filtration and concentrating with rotary evaporator 28 h at room temperature, filtration, evaporating under vacuum at 40 °C 100 min at ambient temperature, evaporating at 45 °C 24 h at room temperature, filtration, concentrating with rotary evaporator at 40 °C 72 h shaking, filtration, evaporating under reduced pressure at 40 °C

Reference Norouzi et al. (2019) Tappeh et al. (2019) Nourbar et al. (2019) Abbasnezhad et al. (2015) Gholamnezhad et al. (2019) Aftab et al. (2019) Soleimanifar et al. (2019) Ahmad and Beg. (2013) Kiari et al. (2018)

Tayel et al. (2018)

days and finally filtered through a Whatman filter paper number one. Alcohol was removed from the extract solution by concentrated in the vacuum using a rotary evaporator until a liquid gel was formed. Similarly, the hydroethanolic extraction method was used to gain seed extract by Nourbar et  al. (2019). To this purpose, 500  g seeds were pulverized and macerated with an adequate amount of ethanol (2 L) 80%, the solution was then refrigerated for a week. Finally, the solution was passed through a filter paper and concentrated using a rotary evaporator. Abbasnezhad et al. (2015) used the following method to extraction. The seeds were pulverized and soaked in 2 L of an ethanol/water (1:1, v/v) for 48 h at room temperature. The extraction solution was strained and dried in an oven at 40 °C for 72 h.

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3  Composition of Black Seed Extract Although there are many studies on the composition of black seed oil, studies on black seed extracts are limited. The chemical composition of NS seeds and oils was studied by some researchers. Ksouda et al. (2018) examined the total phenolic contents (TPC), antioxidant activity, oil contents and fatty acid profiles of NS seeds. The researchers found that the oil content was 42.3%, while palmitic, stearic, oleic, linoleic and linolenic acids were 12.3, 3.3, 24.0, 55.8 and 0.2%, respectively. At the same time, total phenolic content was determined as 750 mg GAE/100 g and antioxidant activity as 31 mg TEAC/100 g by DPPH· method and 38 mg TEAC/100 g by ABTS method. In another study, physicochemical properties and stability (Kiralan et  al. 2014), volatile compounds (Kiralan and Kiralan, 2015; Liu et  al. 2012) of the black seed oil were investigated. According to the literature research, there are limited number of studies about extract composition. The methanolic extract had a patty appearance, a strong and specific odour and characterized by a dark brown to black colour (Kiari et al. 2018). The obtained black seed extract (BSE) was subjected to some analysis and the determination of the secondary metabolites such as glycosides, alkaloids, anthraquinone glycosides, cardiac glycosides, steroids, tannins, flavonoids, and saponins (Mamun and Absar 2018). Results show that all chemical groups except anthraquinone glycosides were screened. Among them, alkaloids, flavonoids, saponins and tannins were detected in high quantity. Their amounts were determined as 10.1, 3.78, 7.58 and 2.21 mg % for alkaloids, flavonoids, saponins and tannins, respectively. It was reported that the seedcake extract contains high amount of phenolics, protein, carbohydrate, alkaloids and flavonoids with potential biological activity (Kadam and Lele 2017). In another study, seed extracts were prepared using diverse solvents, such as ethanol, water, ethanol: water (3:2, v/v) and methanol: hot water (3:2, v/v). TPC, antioxidant activity and reducing power were measured. Results indicated that NS extracts had a great amount of TPC (4.4–7.4 mg GAE/g) and DPPH· radical scavenging activities (33.9–44.2%), with water, extract, and ethanol: water extracts indicating high reducing power and encouraging antioxidant activity (Chauhan et  al. 2018). Mechraoui et al. (2018) investigated total flavonoid, TPC, and antioxidant activities of BSE extracted with different solvents (methanol: water, and acetone:water). According to results, important phenolic compounds were identified, wherein methanolic extract showed the highest value. On the other hand, the methanolic extracts showed moderate antiradical activity, whereas the acetone extract revealed a low antioxidant activity. Antioxidant activity results showed that NS extract contains a large number of active ingredients that can act simultaneously against pathological microorganisms. The results are summarized in Table 30.2. The extraction of NS seed was obtained by the percolation-assisted extraction method with ethanol (50%) and evaporated at 55 °C for 12 h. The extract was dried at 50 °C for 18 h (Petrujkic et al. 2018). The individual amino acid content and total protein were determined (Table 30.3). The glutamine amino acid had the highest

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Table 30.2  Phenolic profile, flavonoid content and antioxidant activities of methanolic and acetonic extracts of Nigella sativa seed meal. (Mechraoui et al. 2018) Property Total phenol content mg/g (mg GAE/g dw) Total flavonoid content (mg QE/g dw) DPPH· radical scavenging IC50 (mg/mL) Phenol compound Gallic acid Catechin Vanillic acid Epicatechin Coumaric acid Apigenin Naringenin Rutin Quercetin Kaempferol

Methanolic extract 1.3714 0.4418 0.0734 μg/g dw 69.64 139.2 – – – – – – – – 15.06 139.28 0.84 1.68 120.2 240.4 3.30 6.60 2.82 5.64

Acetonic extract 0.5962 0.2746 0.1202 11.9 – – – – 10.27 0.40 22.3 0.30 0.36

23.8 – – – – 20.4 0.80 44.7 0.60 0.76

Table 30.3  Amino acids for the Nigella sativa seed meal extract. (Petrujkic et al. 2018) Molar composition of total proteina (%) Amino acid Glutamine 32.7 Glycine 11.8 Asparagine 9.3 Arginine 6.3 Alanine 5.7 Serine 5.3 Proline 5.0 Valine 4.1 Leucine 4.0 Threonine 3.9 Lysine 2.8 Isoleucine 2.7 Phenylalanine 1.8 Methionine 1.6 Tyrosine 1.6 Histidine 1.5 Total

Weight composition of total proteina (g/kg) 50.2 7.97 12.7 11.7 4.92 5.58 5.72 4.92 5.45 4.65 4.25 3.59 3.19 2.39 3.06 2.39 132.7

Total protein in the NSE = 132.9 g/kg

a

ratio in the total protein of the BCE (32.7% molar composition and 50.24  g/kg weight composition). Mariod et al. (2009) studied the content of phenolics and antioxidant capacity of phenolic extract from BCS cake. The phenolic compounds obtained from methanol

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extracts of BCS (syringic, hydroxybenzoic and p-cumaric acids) have shown considerable antioxidant properties. Similarly, TPC and antioxidant activity were measured by Soleimanifar et  al. (2019). The TPC of the extract was determined as 955.7 mg/kg. Moreover, IC50 results were found as 104.7 mg/mL and 8.06 mg/mL for DPPH· and BHT, respectively. In a study conducted by Kiari et al. (2018) on TPC and flavonoid content of methanol extract and essential oil, they found higher amounts of these compounds in methanol extract. The TPC and flavonoid contents were determined as 28.1  mg GAE/g extract and 5.64  mg Catechin equivalent/g extract. In addition, thin layer chromatography analysis indicated that quercetin, gallic acid and catechin were most likely found in the extracts. Fourier transform infrared spectrometry analysis showed the existence of certain chemical groups such as aldehydes, acids, alcohols, ethers, and phenols. In general, these molecules are antimicrobial and shows therapeutic properties. As it is known, the aroma, aroma-active compounds and sensory profiles of the alcoholic black cumin seed extract were first identified (Kesen et al. 2018). Aroma compounds are one of the most important factors that affect consumer preferences and shape food quality. They determine the organoleptic properties of foods. However, only a small number of these compounds contribute to the whole aroma. These compounds are called as aroma-active compounds and identified by olfactometric technique. This technology serves to classify aroma compounds in the samples as aroma-active or non-aroma-active compounds according to their intensity. The aroma compounds of NS extract were obtained by using the purge and trap extraction technique. In this technique, a special cartridge (C18) is used in which aroma compounds are trapped. The flow of nitrogen carrier gas is controlled by a flow meter and it is connected to a separation system to split the flow. The needles of cartridge and nitrogen gas sources were placed in the septum of the glass bottle to retain the aroma compounds. After that, the aroma and aroma-active compounds of NSE were detected GC-MS-O. The analysis and GC conditions are described in detail (Kesen et al. 2018). According to results of analysis acids, alcohols, phenols, terpenes, esters, ketones, aldehydes, lactones and hydrocarbons (total 32) were determined. Table  30.4 shows the aroma compounds of BCS extract with high amounts. Aroma-active compounds consisting of alcohols, phenols carboxylic Table 30.4 Aroma compounds of BCS extract

Chemical class Ketone Acid Acid Alcohol Ketone Acid Alcohol Hydrocarbon

Aroma compound Acetoin Acetic acid Propanoic acid 2-Methyl-3-butanol Hydroxyacetone (E)-3-Hexenoic acid 3-Penten-2-ol m-Xylene

Concentration (μg/L) 9394 3088 1187 2627 808 516 394 357

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Table 30.5  Aroma-active compounds of BCS extract Chemical class Ketone Acid Terpene Hydrocarbon Acid Phenol Acid Phenol

Aroma-active compounds Acetoin Isobutanoic acid Limonene Styrene Propanoic acid Eugenol Hexanoic acid Guaiacol

Odor descriptionsa Buttery Cheesy Citrusy Balsamic Fatty Smoky Cheesy Smoky

FDb 1024 512 256 256 128 128 64 64

Odor description felt by panelists during olfactometric analysis FD factor is the maximum dilution of the extract at which an odorant compound detected by aroma extract dilution analysis a

b

acids, terpenes, ketones and hydrocarbons (total 13 compounds) were identified. Table 30.5 presents aroma-active compounds with the highest values of the FD factor. Among them, acetoin had the most intense aroma (FD = 1024) and ensured a buttery odor. This compound also displayed the most plentiful compound of the whole aroma profile. Isobutanoic acid (FD = 512) is followed by this compound and it gave a strong smell aroma of cheesy notes. Sensory defining notes were also detected in the study. A total of nine descriptive odors perceived by panelists in aromatic extracts and the reference sample (black seed extract) are shown in the spider graph (Fig. 30.2). As shown in the figure, the descriptive odors were citrus, buttery, oily and green, cheesy, balsamic, spicy-­ smoky, hard fruit, burnt plastic. Among these, buttery and cheesy odors were the most powerful and received maximum sensation points. Panelists interpreted the sensory characteristic of the aromatic extract in a similar manner to the original black seed extract. In the definitions, the most effective aroma-active compound was found to be acetoin. This compound was perceived as a buttery note.

4  Functionality of Seed Extracts 4.1  Health Effect The medical use of NS dates back to 2000  years. In addition to being used as a spice, it is also used to treat diseases such as asthma, diabetes, inflammation, cough, hypertension, bronchitis, eczema, dizziness, headache, fever and influenza. Interestingly, NS may exhibit antimicrobial, antitumor, anti-inflammatory, antioxidant, hypoglycemic, hypotensive, hypolipidemic and immune system-boosting effects (Nourbar et al. 2019). The health effects of black seed extract (BSE) have been more examined. According to literature studies, the effects of antitoxic, antimicrobial, neurological,

489

30  Composition and Functionality of Nigella sativa Seed Extracts

Buttery

8 7 6 5 4 3 2 1 0

Green

Fatty

Cheesy

Citrusy

Burnt plastic

Balsamic

Putrid fruit

Reference

Smoky

Extract

Fig. 30.2  Odors of BCS extracts. (Kesen et al. 2018)

protective, cardiovascular, wound healing and regulatory on some metabolic disorders were evaluated. In this context, the following studies indicate the effects of BSE on some diseases. 4.1.1  Antitoxic and Hepatoprotective Effect According to the literature, Nigella sativa extract showed antitoxic and hepatoprotective properties against some organophosphate pesticides such as diazinon, dimethoate, mancozeb, propoxur, and malathion. The memory booster effect of Nigella Sativa extract was investigated by Norouzi et al. (2019). In this work, the effects of the extract on lipopolysaccharide (LPS)-induced learning and memory impairments, brain tissues oxidative damage and hippocampal cytokine levels were studied in rats. Researchers stated that the BS hydroalcoholic extract improved the LPS-induced learning and memory impairments induced by LPS. In addition, extract effects were attended with progressing hippocampal cytokine levels and brain tissue oxidative damage criteria. Similarly, the impact of NSE on memory capacity and its likely mechanisms in scopolamine (Sco)-induced spatial memory impairment were evaluated. The hydroalcoholic extract of NS has shown to reduce the oxidative stress of brain tissue in rats. Results also supported the traditional

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belief about the beneficial effects of NS on the nervous system (Hosseini et  al. 2015). The effects of NS seed extract on oxidative stress were studied in the hippocampus of streptozotocin (STZ)-induced diabetic rats by Abbasnezhad et  al. (2015). Oxidative stress is known to play a key role in the etiology and complications of diabetes. On the other hand, NS has a strong antioxidant effect and therefore can protect the brain against oxidative stress following lipid peroxidation in transient global ischemia of the brain. The NS hydroethanolic extract showed antidiabetic and protective effects against the oxidative stress in the hippocampus of the STZ-induced diabetic rats. In another similar study, the inhibitory effect of NS extract on doxorubicin-induced oxidative stress in rat kidneys was examined. In this work, intravenous doxorubicin injection at a dose of 5 mg/kg resulted in an increase in the MDA and decreased activity of SOD, catalase enzymes and total thiol concentrations in renal tissues. In the treated groups, Nigella sativa was reported to provide renal protection against doxorubicin (Mohebbati et al. 2016). Methyl parathion (MP) is an organophosphorus pesticide that stimulates hepatotoxicity in living organisms. It is easily absorbed from the skin, mucous membranes and eyes and is potentially dangerous. To determine the hepatoprotective effect of NS extract in methyl parathion-exposed rats, the experimental procedure was planned. The aim of this study was to investigate the effect of BSE on oxidative stress and anti-inflammatory. To prepare methyl parathion and NSE group, methyl parathion (0.28 mg/kg) was first dissolved in maize oil and applied orally to rats. After two applications, rats were orally treated with 300  mg/kg NS dissolved in 1 mL of 5% Na-CMC for 28 days. It was found that methyl parathion application caused significant liver damage and oxidative stress in Wistar albino rats. In addition, the application of NSE markedly reversed these symptoms. The result showed that the NSE application commuted the possible liver damage of methyl parathion reducing the oxidative stress and inflammation. NSE significantly reduced the activities of liver enzymes such as ALT, AST, GGT, LDH and ALP compared to those in the methyl parathion treated group (Berkoz et al. 2019). 4.1.2  Antimicrobial Effect Kiari et  al. (2018) examined the antimicrobial activity of essential oil (EO) and methanol extract (ME) obtained from Algerian NS seeds against microbial strains isolated from the oral cavities of periodontal patients. For this purpose, 12 gram-­ positive, 11  gram-negative and 3 microscopic fungi species were separated. The activity of EO and ME against some microorganisms such as Staphylococcus aureus, Streptococcus pneumoniae and Staphylococcus epidermidis were tested. Results displayed that the perfect antimicrobial activity of EO (Minimum Bactericidal Concentration, MBC): 16,500 μg/mL and reasonable performance of ME (MBC: 125,000 μg/mL) against all the microorganisms tested. The fungi are mostly pathogens living in the host cell. They interact with the host and raiding species. The pathogens infect both plant and animal cells and make the survival of host cells quite difficult. The antifungal activity of vegetative parts of

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Nigella sativa against soil-borne fungal was conducted to evaluate species Fusarium oxysporum and Macrophomina phaseolina (Aftab et al. 2019). To this purpose, the black cumin seed was crushed, sieved and extracted by maceration in methanol, followed by removal of methanol on the rotary evaporator. Subsequently, partitions with different solvents such as chloroform, n-hexane, ethyl acetate, and n-butanol were formed. The formed fractions were tested for antifungal activity against two soil-borne fungal strains, F. oxysporum and M. phaseolina, collected from Fungal Culture Bank. As a result of the analysis, it was observed that the vegetative parts of NS had significant antifungal potential against F. oxysporum and M. phaseolina. Ethyl acetate extract completely inhibited fungal biomass production in both fungal strains, while chloroform, n-hexane and n-butanol extract inhibited biomass production against F. oxysporium fungal in 88, 78 and 76%, respectively (at 50 mg/ mL). Both chloroform and ethyl acetate extract fully inhibited M. phaseolina, while n-butanol and n-hexane extract showed 86% inhibition. The aqueous extract, however, worked very poorly (5%) to inhibit the biomass production against F. oxysporium fungal. Table 30.6 presents the reduction in fungal biomass production over control at different concentrations of bioassays. Staphylococcus aureus is a very hazardous pathogen and causes many health problems. Especially methicillin-resistant strains (MRSA) are very dangerous for health and it is responsible for some difficult to treat infections in humans. Staphylococcus aureus is a Gram-positive bacteria and is usually associated with Table 30.6  Fungal biomass reduction in different concentrations of fractions of methanolic extract of vegetative parts of Nigella sativa. (Aftab et al. 2019) Reduction in fungal biomass over control (%) Concentration (mg/mL) n-Hexane Chloroform Ethyl acetate n-Butanol Fusarium oxysporum f. spp. Cepae 1.562 43 45 62 65 3.125 42 43 58 65 6.25 49 47 66 67 12.5 58 50 73 70 25 62 76 82 74 50 78 88 100 76 100 100 100 100 100 200 100 100 100 100 Macrophomina phaseolina 1.562 60 48 67 59 3.125 63 59 72 63 6.25 63 65 74 64 12.5 66 77 79 72 25 71 82 89 78 50 86 100 100 86 100 100 100 100 89 200 100 100 100 100

Aqueous 12 15 13 8 2 5 10 22 35 38 38 39 43 57 57 61

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food poisoning, toxic shock, wound infections, scalded skin syndrome, endocarditis and osteomyelitis. In a study on this topic, the effects of different plant seeds including BSE as microbial inhibitory agents against S. aureus isolates were investigated. Antibacterial activity of BSE was evaluated both qualitatively and quantitatively using a well diffusion method and minimal inhibitory concentration (MIC). The results showed that BSE had antibacterial activity against normal S. aureus strains but not against MRSA isolates (Tayel et al. 2018). Similar results were found by Enomoto et al. (2001) and they stated that the methanolic extract of BSE has antibacterial activity against many bacterial strains except MRSA. Malaria in tropical countries is known as one of the most common invasive diseases. When drug resistance occurs, treatment becomes even more difficult. Therefore, they are in search of alternative effective and safe new drugs. Conventional medicine is seen as a significant source for new drugs, especially because of its low cost, low serious complication rate and higher tolerability. For this purpose, Tappeh et al. (2019) carried out a study examining the NSE effect on malaria. It was determined that the extract had a weak effect against the malaria parasite. 4.1.3  Cardiovascular Effect Shakeri et al. (2018) reported in their review article the cardiovascular effects of NS and its constituents. They summarized the effect of NS and its constituents on vascular smooth muscle, endothelial dysfunction, heart rate and heart contractility. It is stated that the hydroethanolic extract of NS showed the vasorelaxation effect by using a study model of rat aortic smooth muscle contracted by both KCl and phenylephrine (Niazmand et al. 2014). In addition, the inhibitory effect of aqueous and macerated extracts of NS on heart rate and heart contractility was displayed by applying a study model of the isolated heart in guinea (Boskabady et  al. 2005; Shafei et al. 2005). In the same study, the effect of NS and its constituents on the lipid profile was reported. According to this, methanol extract of NS increased high-­ density lipoprotein cholesterol (HDL-C) and reduced serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C) on hyperlipidemia rat (Ahmad and Beg 2013). Depending on the situation in the ethanol extract of NS seeds, TC, TG, and LDL-C were reduced and HDL-C was increased in rats by utilizing a study model of adrenaline-induced dyslipidemia and left ventricular hypertrophy (Ali et al. 2013). 4.1.4  Effect on Cancer The incidence of cancer is rapidly increasing worldwide. This brings efforts to seek effective natural anticancer treatments as an alternative to the chemotherapies used. The use of medicinal plants in cancer treatment has been increased in recent years. For 5 consecutive days, ethanol extract of BCS (250 mg/kg) was applied to diethyl

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nitrosamine-induced hepato carcinogen cancer model in Wistar rats. Analysis findings showed that liver weight, serum AFP and VEGF levels, hepato-somatic indices and hepatic HGF𝛽 protein expression were reversed by increased NS extract. Secondly, the histopathological change of the liver due to the bioactive chemicals in NS was reduced in mice receiving NS extract without any detrimental effect (Shahin et al. 2018). 4.1.5  Neurological Effect Epilepsy and epileptic syndromes are widespread neurological disorders in children. More than 25% of children with epileptic seizures are resistant to anti-seizure drugs and require different methods of treatment. Recently, studies have been conducted to determine whether herbal agents can be used for this purpose. Among these agents, NS and Thymus vulgaris extracts containing primary active chemical agents (i.e., TQ) are the most notable. Some studies have demonstrated the controlling effects of chemical agents in animal models of seizures. Momen et al. (2019) examined whether the mixture of black seed and Thymus vulgaris extracts had effects on epileptic seizures in children. As a result of the applications, it was found that the mixture of the extracts of NS and Thymus vulgaris did not affect the frequency and duration of seizures in children with refractory seizures. However, it has been noted that very few of the highly selected children with refractory seizures may benefit from the effects of these agents. 4.1.6  Wound Healing Effect The effect of hydroethanolic NSE on skin wound healing in diabetic male rats was investigated (Nourbar et al. 2019). Wound healing refers to a series of processes to multiply the lost tissue as much as possible. Healing of diabetic wounds is particularly important, because wound healing may be delayed in diabetic patients due to irritating inflammation. Shortening the wound healing time has long attracted the attention of medical researchers, and for this purpose plant-based drugs are increasingly used for different classes of pharmacological drugs. NS is one of the plants studied for the treatment of diseases. This study showed that NS extract may cause inflammation reduction and acceleration of wound healing after topical application to clinical and histological full-thickness skin wounds. 4.1.7  Triglyceride Lowering Effect Hypertriglyceridemia (HTg) is generally a part of metabolic disorders and is defined as a high amount of triglyceride (TG) that can cause serious complications in the blood over time. Different drugs are used in the treatment of HTg, and also, many plants have been tested experimentally for the treatment of HTg as an alternative

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therapy. Mollazadeh et al. (2019) researched on the herbs that have a lowering effect on TG. According to results, many plants showed positive effects but NS, Allium sativum, Curcuma longa, Commiphora mukul and Anethum graveolens had the best TG lowering effect with full action mechanism. It was stated that petroleum ether extract of NS is a strong extract for its TG-lowering effect. 4.1.8  Serologic and Hematologic Effect Since a sedentary lifestyle is an important risk factor for various chronic pathologies that increase mortality and morbidity of many chronic non-contagious diseases such as cancer, type 2 diabetes, neurodegenerative diseases and cardiovascular, studies on this subject were planned. For example, different exercise loads and the effect of NS seed extract on hematological and serological parameters in rats were performed by Gholamnezhad et al. (2019). Seven groups of animals consisting of control sedentary, moderate trained, overtrained, recovered overtrained, control sedentary + NS, moderate trained + NS and overtrained + NS were investigated. As a result of applications, overtraining induced chronic inflammatory symptoms, such as fatique, lethargy, catabolic/anabolic imbalance, stress hormone elevation, performance decline and WBC count reduction. Although prolonged administration of NS developed some changes such as metabolic condition and corticosterone elevation, NS did not show an important effect on hematological factors. 4.1.9  Effect on Infertility and Sexuality Application of alcoholic extract of NS in male rats resulted in marked increase in production of living and motile sperm cells, weight gain of reproductive organs, increased epididymal sperm reservation, gonadotropin content, blood testosterone concentration, quantity of mature Leydig cells and fertility indices according to control group (Parandin et al. 2012; Yimer et al. 2019). Although several studies have been conducted on the utility of Nigella sativa seeds in male infertility, a direct effect on penile erection was first demonstrated by Aminyoto and Ismail (2018). NS has been reported to increase reproductive parameters. In the study, the response of ethanolic NS seed extract on penile erection was examined in vitro. The results obtained confirmed that the NSE directly influenced the penis erection response by relaxation of blood vessels in the corpus cavernosum of male rats. This data shows that NS seed can be used as sex-enhancing drugs.

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4.2  Effects on the Synthesis of the Metal Nanoparticle (NPs) In recent years, the aim of researchers is to focus on the synthesis of metal that differs significantly from the properties of bulk materials. It differs especially due to its mechanical optical, magnetic, electronic and chemical properties. These properties may be due to their small sizes and large surface areas. Therefore, metal NPs have found applications in different fields (Alaghemand et al. 2018). Green-nanotechnology is a new trend and is concerned with the method of NPs and green synthesis of nanomaterials. Metal NP synthesis is generally done by physical, chemical and biological methods. Among these methods, the biological synthesis of metal NPs is non-toxic, less expensive, and does not carry significant risk and does not involve the use of hazardous chemicals to the biological system. It has been found that metal NPs such as gold, copper and silver are active against the fungal strains, gram-positive and gram-negative bacteria with their characteristic effect and specificity for multifaceted targets. Recently, black seed extract has been used in the synthesis of metal NPs. Biogenic silver NPs (Kadam et al. 2019) and zinc oxide NPs (Alaghemand et al. 2018) were synthesized using NSE with potential antioxidant activity. Silver NPs are reported to exhibit a unique biological property, such as antimicrobial and healing stability. These NPs are resistant to microorganisms such as fungi, bacteria, and viruses. Since silver NPs are used in human and environmental contact practice, they develop environmentally friendly and non-toxic green synthesis approaches. Since films and coatings containing chitosan polymer matrix are known to be resistant to oxygen, moisture and microorganisms, they are estimated to increase food quality and shelf life when used as packaging. However, the practical applications of pure CS films are difficult due to their fragility and low mechanical strength. Therefore, in order to improve the physical, mechanical and biological properties of these films, Kadam et al. (2019) examined the effect of incorporating silver NPs into the CS polymer. Experimental design including analysis of antioxidant property, physical and mechanical properties (film thickness, moisture content, and water vapor permeability), tensile strength, extensibility, colour, opacity, thermal stability, and antimicrobial properties of chitosan/NSE-AgNPs composite film was planned. According to the data obtained from the study, the researchers reported that the addition of AgNPs to CS polymer matrix showed a general change in the chemical interaction between CS-CS and CS-water. In addition, various degrees of AgNP exhibited antibacterial activities have been reported depending on the inoculated AgNP concentration. In light of these data, possible applications of AgNPs in the development of active biodegradable packaging have been proposed. Similarly, Alaghemand et al. (2018) synthesized zinc oxide NPs using Nigella sativa extract. Zinc NPs have wide interest since they have numerous applications such as nonlinear optics, spectrally selective coating for solar energy absorption, bio-labeling, coupling materials for electrical cells as optical receptors, the catalyst for chemical reactions and antibacterial capacities. In the study, NPs were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM).

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During the experiment, the height of the plant and the number of branches were monitored. It was emphasized that the formulation in this study could be used to increase yield, improve products and eliminate food deficiencies.

4.3  Effects on Foodstuffs and Other Applications The application of black seed extract in food or other fields and their effects on these applications have been investigated by some researchers. For example, NSE was added to sunflower oil and palm olein and then, its effect on frying performance was monitored. Two frying systems containing 0% NSE (Control) and 0.02% butylated hydroxytoluene (BHT) were used and compared with each other. To see the effect of NSE, fatty acid composition, anisidine value, peroxide value, TPC, Totox value (TV), C18:2/C16:0 ratio and oil viscosities were measured. The results showed that NS extract improved the oxidative stability of frying oils throughout the frying process. In addition, palm olein oil was determined as more consistent than sunflower oil, depending on the fatty acid composition and the ratio of linoleic acid to palmitic acid. In both oil, the value of anisidine, known as the decomposition product, was significantly lower in the NSE-containing system compared to the control (Solati and Baharin 2015). Similarly, Bassim and Imaizumi (1998) investigated the antioxidant activity of BCS and found that BCS ethanolic extracts applied to the corn oil prevented oxidative damage of triglycerides. To determine the effect of adding extracts to oil on oven stability, BCS extract and BHT (100 mg/mL) were separately added to 1000 mL of soybean oil without antioxidant and placed in an oven at 60 °C. Then, 10 mL of samples were taken at 24-hour intervals for 14 days to determine peroxide value (PV) and thiobarbituric acid (TBA) levels. PV showed no significant difference between samples. On the other hand, TBA measurement results showed significant differences between control and antioxidant-containing samples. The highest TBA value was found in the control and then in soybean oil samples loaded with BHT and BCS. As a result, it was emphasized that the increase in TBA value in oil samples may be due to the formation of secondary oxidation products (Soleimanifar et al. 2019). Livestock producers are using antibiotics to control disease and increase production, but they are under pressure to reduce it. This pressure arises from the concern that such medical procedures may contribute to the emergence and proliferation of antibiotic-resistant microbial populations used to treat major diseases. To this end, Nigella sativa was used as an alternative antibiotic feed supplement and monitored growth performance in weanling pigs. The results of the study showed that the application of NS extract as oral feeding to weanling pigs improved feed conversion by 63.7% in 4.5 g/kg NS extract (Petrujkic et al. 2018). Chauhan et al. (2018) investigated that the antioxidant effect of BCS extracts to suppress lipid and protein oxidation in raw ground pork meat. Seed extracts were prepared using various solvents [ethanol, water, ethanol: water (60:40), and methanol: hot water (60:40)]. Water and ethanol: water extract was chosen and added

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(1.5%) into just minced pork meat and compared with BHT (100  ppm), on the restraining performance of lipid and protein oxidation during storage. Compared with BHT, the samples treated with BCE had lower thiobarbituric acid reactants, PV, free fatty acids, the formation of protein carbonyls, and the development of offodor or rancid odour. In meat samples treated with BCE, the color was not adversely affected.

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Momen, A. A., Hemati, A. A., Houshmand, G., Azadzadeh, M. H., & Malamiri, R. A. (2019). The effect of a mixture of Nigella sativa and Thymus vulgaris extracts in children with refractory epilepsies: A randomized trial. Journal of Herbal Medicine, 15, 100242. Niazmand, S., Fereidouni, E., Mahmoudabady, M., & Mousavi, S.  M. (2014). Endothelium-­ independent vasorelaxant effects of hydroalcoholic extract from Nigella sativa seed in rat aorta: The roles of Ca2. BioMed Research International, 2014, 1–7. Norouzi, F., Hosseini, M., Abareshi, A., Beheshti, F., Khazaei, M., Shafei, M. N., Soukhtanloo, M., Gholamnezhad, Z., & Anaeigoudari, A. (2019). Memory enhancing effect of Nigella Sativa hydro-alcoholic extract on lipopolysaccaride-induced memeory impairment in rats. Drug and Chemical Toxicology, 42(3), 270–279. Nourbar, E., Mirazi, N., Yari, S., Rafieian-Kopaei, M., & Nasri, H. (2019). Effect of Hydroethanolic extract of Nigella sativa L. on skin wound healing process in diabetic male rats. International Journal of Preventive Medicine, 10(18), 1–8. Parandin, R., Yousofvand, N., & Ghorbani, R. (2012). The enhancing effects of alcoholic extract of Nigella sativa seed on fertility potential, plasma gonadotropins and testosterone in male rats. Iranian Journal of Reproductive Medicine, 10(4), 355–362. Petrujkic, B. T., Beier, R. C., He, H., Genovese, K. J., Swaggerty, C. L., Hume, M. E., Crippen, T. L., Harvey, R. B., Anderson, R. C., & Nisbet, D. J. (2018). Nigella sativa L. as an alternative antibiotic feed supplement and effect on growth performance in weanling pigs. Journal of the Science of Food and Agriculture, 98(8), 3175–3181. Ramadan, M.  F. (2007). Nutritional value, functional properties and nutraceutical applications of black cumin (Nigella sativa L.): An overview. International Journal of Food Science & Technology, 42(10), 1208–1218. Seghatoleslam, M., Fatemeh, A., Shafieian, R., Hassanzadeh, Z., Edalatmanesh, M. A., Sadeghnia, H. R., & Hosseini, M. (2016). The effects of Nigella sativa on neural damage after pentylenetetrazole induced seizures in rats. Journal of Traditional and Complementary Medicine, 6(3), 262–268. Shafei, M. N., Boskabady, M. H., & Parsaee, H. (2005). Effect of aqueous extract from Nigella sativa L. on Guinea pig isolated heart. Indian Journal of Experimental Biology, 43(07), 635–639. Shahin, Y. R., Elguindy, N. M., Abdel Bary, A., & Balbaa, M. (2018). The protective mechanism of Nigella sativa against diethylnitrosamine-induced hepatocellular carcinoma through its antioxidant effect and EGFR/ERK1/2 signaling. Environmental Toxicology, 33(8), 885–898. Shakeri, F., Khazei, M., & Boskabady, M.  H. (2018). Cardiovascular effects of Nigella Sativa L. and its constituents. Indian Journal of Pharmaceutical Sciences, 80(6), 971–983. Solati, Z., & Baharin, B. S. (2015). Antioxidant effect of supercritical CO2 extracted Nigella sativa L. seed extract on deep fried oil quality parameters. Journal of Food Science and Technology, 52(56), 3475–3484. Soleimanifar, M., Niazmand, R., & Jafari, S.  M. (2019). Evaluation of oxidative stability, fatty acid profile, and antioxidant properties of black cumin seed oil and extract. Journal of Food Measurement and Characterization, 13(1), 383–389. Tappeh, K. H., Ghaderi, M., Seyedi, S., Mikaili, P., Aminpour, A., & Khademvatan, S. (2019). The effect of hydroalcoholic extract of Nigella sativa on plasmodium berghei-infected mice: An evaluation of ımmune deviation and serum levels of interferon gamma (IFN-Ɣ) and interleukin 4 (IL-4). Journal of Clinical and Diagnostic Research, 13(3), 12–15. Tayel, A. A., Shaban, S. M., Moussa, S. H., Elguindy, N. M., Diab, A. M., Mazrou, K. E., Ghanem, R. A., & El-Sabbagh, S. M. (2018). Bioactivity and application of plant seeds’ extracts to fight resistant strains of Staphylococcus aureus. Annals of Agricultural Sciences, 63(1), 47–53. Yimer, E.  M., Tuem, K.  B., Karim, A., Ur-Rehman, N., & Anwar, F. (2019). Nigella sativa L. (black cumin): A promising natural remedy for wide range of illnesses. Evidence-based Complementary and Alternative Medicine, 2019, 1–16.

Chapter 31

Nigella sativa Seed Extracts in Functional Foods and Nutraceutical Applications Ranga Rao Ambati and Mohamed Fawzy Ramadan

Abstract Plants are used as traditional medicine for curing various diseases. Medicinal plants are used in formulations of herbal drugs due to their safety than allopathic medicines. Medicinal herbs are one of plant species with several medicinal and culinary applications. These herbs are exhibited several biological roles in various applications. Nigella sativa is a medicinal plant extensively used in various medicinal uses globally. N. sativa extracts and seed oils contain bioactive ingredients such as essential oils, fatty acids, sterols, and vitamins. The plant is used as antioxidant, antimicrobial, antiviral, antifungal, immunomodulatory, antidiabetic, anti-inflammatory, anticancer, improve the skin pigmentation, and wound healing. It has been confirmed that the therapeutic traits of N. sativa is due to the thymoquinone (TQ), the main bioactive compound in the seed extracts and also in the seed oils. The present book chapter is focused on bioactive compounds, as well as the therapeutic and nutritional aspects of seed extracts and seed oil of N. sativa with exploring their properties for the development of novel foods and pharmaceuticals. Keywords  N. sativa · Seed extracts · Seed oil · Bioactive molecules · Health benefits

R. R. Ambati (*) Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research University (VFSTRU) (Deemed to be University), Guntur, Andhra Pradesh, India M. F. Ramadan Deanship of Scientific Research, Umm Al-Qura University, Makkah, KSA Agricultural Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_31

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1  Introduction Medical plants are used for various diseases in the indigenous medicinal systems. These medical plants are used in herbal medicine and considered safe when compared with allopathic medicine. Researchers are focusing on various medical plants, however few plant species were examined for their potential traits, mechanisms and safety. Among medicinal plants, Nigella sativa (known as black seed, family Ranunculaceae) showed a wide spectrum of biological activities (Gholamnezhad et al. 2016). It is a native to North Africa, Southern Europe, and Southwest Asia. The plant cultivated in countries such as Saudi Arabia, Turkey, Syria, Pakistan, India, Europe, and Middle Eastern (Khare 2004). N. sativa seed extracts and oils are extensively used in the treatment of ailments in globally. It is considered as one of healing medicine for several diseases and recommended to be used in Prophetic Medicine (Al-Bukhari 1976). Both medicines Unani and Ayurveda are prepared from N. sativa seeds (Sharma et al. 2005; Goreja 2003). N. sativa is well studied by various researchers (Table 31.1) who reported on N. sativa therapeutic aspects such as antioxidant, antidiabetic, antihypertensive, anticancer, diuretic, analgesic, antimicrobial, immunomodulatory, hepatoprotective, renal protective bronchodilator, spasmolytic, gastroprotective, anti-inflammatory, analgesics, and anthelmintics (Kazmi et al. 2019, Yimer et al. 2019; Ramadan 2007). N. sativa seeds are commonly used in the treatment of diarrhea, skin disorders, rheumatism, asthma, and bronchitis (Perveen 2019). N. sativa seeds are also used in the digestive, liver tonic, appetite stimulant, anti-diarrheal, and also in nursing mothers to enhance milk production (Abel-Salam 2012; Khaled 2009, Assayed 2010; Abdel-Zaher et al. 2011; Boskabady et al. 2010; Goreja 2003). Seeds of N. sativa contains various bioactive compounds such as carvacrol, thymohydroquinone, dithymoquinone, p-cymene, 4-terpineol, t-anethol, alkaloids, and α-hederin (Abdelmeguid et al. 2010; Pari and Sankaranarayanan 2009). Thymoquinone (TQ), the major bioactive compound in N. sativa, showed most of the therapeutic activity. Black cumin seeds are also used as a food additive in the bread and pickles (Al-Ali et al. 2008). The current book chapter is focusing on extracts of Nigella sativa seeds and their effects on functional food applications.

2  Geographical Distribution of N. sativa for Traditional Use N. sativa is known as black caraway, black seeds, black cumin and it is belonging to the family Ranunculaceae and class Magnoliophyta. It is recognized as an annual flowering plant and cultivated in various countries India, Turkey, Pakistan, Iran, Saudi Arabia, Middle East, and South Europe (Rajeskhar and Kuldeep 2011). N. sativa seeds are black and having dimensions between 2 and 3.5 mm. The powder of N. sativa seed was observed under a microscope wherein brown-black parenchymatous cells and oil globules were detected (Mukhtar et al. 2019). The N. sativa

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Table 31.1  Biological activities of N. sativa seeds or seed oil N. sativa seed or seed oil Activity Seed extract, seed oil Antioxidant

Bioactive compound –

Seed, seed oil

Antischistosomiasis

–

Ethanol extract Hexane, ethanol extract Seed

Anti-stress Contraceptive and antifertility Curative for rheumatoid arthritis Gastroprotective Human neutrophil elastase inhibitory Immuno-modulatory Nephroprotective Therapeutic in sickle cell anemia Neuropharmacological Antimicrobial

– –

Seed Essential oil Volatile oil Seed Oil extract

Seed extract Methanol, hexane, chloroform, acetone, ethylacetate, ethanol-water extract, seed oil Hexane, chloroform, Antifungal acetone, ethyl acetate, ethanol-­ water extract Seed extract, seed Antidiabetic oil, ethanol extract Volatile oil Antiepileptic

Seed, boiled extract Aqueous extract

Antiasthmatic Pulmonary protective Anti-inflammatory

– Carvacrol

References Abbasnezhad et al. (2015) and Beheshti et al. (2017) Mohamed et al. (2005) and Mahmoud et al. (2002) Roshan et al. (2010) Keshri et al. (1995) and Agarwal et al. (1990) Umar et al. (2012) and Mariod et al. (2009) Al Mofleh et al. (2008) Kacem and Merihi (2006)

– – –

Torres et al. (2010) Saleem et al. (2012) Ibraheem et al. (2010)

Thymoquinone Thymoquinone, melanin

Kanter (2008a, b) Gerige et al. (2009), Bita et al. (2012), Alshareef (2019), Mohammed et al. (2019), and Al-Ameedy and Omran (2019) Al-Ameedy and Omran (2019) and Suthar et al. (2010)

Thymoquinone

Thymoquinone

Thymoquinone Thymoquinone р-cymene, α-pinene Dithymoquinone Thymoquinone, р-cymene,

Abdelmeguid et al. (2010) and Kapoor (2009) Biswas and Guha et al. (2007) and Raza et al. (2008) Begrow et al. (2010) and Boskabady et al. (2010) Al-Ghamdi (2011) and Mutabagani and EI-Mahdy (1997)

Adopted from Mukhtar et al. (2019)

seeds and seed oils contain various constituents having high nutritional values. The production of constituents in N. sativa varies upon geographic distribution and cultivation methods (Datta et al. 2012). N. sativa seeds and seed oils are considered valuable drugs in the conventional Indian medical system (Ayurveda and Unani). N. sativa seeds are used for the treatment of asthma, cough, dizziness, diabetes,

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abdominal diseases, hemorrhage, jaundice, and hydrophobia (Srinivasan 2018). It is a remedy for fatigue, headaches, cold, fever, piles, diarrhea, and paralysis (Shakeri et al. 2016). Seed oil applied to anesthetic and analgesic, while roasted seeds are used to stop vomiting (Yarnall and Abascal 2011). Based on the various conventioanl uses of N. sativa seed extracts and seed oils, the seeds could be exploited as a bioactive agent in the rapidly growing nutraceutical and functional food industries.

3  High-value Compounds in N. sativa Seed and Seed Oils High-value compounds such as minerals, vitamins, fatty acids, and sterols in N. sativa seed and seed oil were reported in the literature (Tables 31.2 and 31.3). The most important bioactive compounds are thymoquinone (TQ), dithymoquinone, thymohydroquinone, p-cymene, t-anethol, sesquiterpene longifolene, carvacrol, 4-terpineol, α-pinene and thymol (Ahmad et  al. 2013). Black seeds contain alkaloids such as nigellicimine, isoquinoline, and nigellicimine-N-oxide, as well as pyrazol alkaloids or indazole ring bearing alkaloids (nigellidine and nigellicine). N. sativa seed contains other compounds such as α-hederin, pentacyclic triterpene and saponin which showed potent anticancer activities (Al-Jassir 1992; Atta-Ur-­ Rahman 1995). Other compounds such as limonene, carvone, and citronellol are  reported in traces. Most of the biological activities of N. sativa are showed because of quinine constituents such as TQ. The seeds of N. sativa contain lipids, carbohydrates, proteins, crude fiber and total ash. N. sativa seeds oil also contain amount of minerals such as Ca, Mg, K, Na, Fe, M, Cu, P, Zn, Fe, and vitamins like α-tocopherol, β-tocopherol, γ-tocopherol, β-tocotrienol, thiamin (B1), phyridoxin (B6), niacin, folic acid riboflavin (B2). N. sativa seeds also contain carotenoids that are converted by the liver into vitamin A. Root and shoots of N. sativa were reported to accumulate vanillic acid (Nickavar et al. 2003). The seeds of N. sativa contain fatty acids such as linoleic, oleic, eicodadienoic, dihomolinoleic, palmitic, and stearic (Cheikh-Rouhou et  al. 2008; Mehta et  al. 2008; Bourgou et  al. 2008). Other chemicals such as campesterol, avenasterol-5-ene, avenasterol-7-ene, nigellone, cholesterol, cycloeucalenol, obtusifoliol, gramisterol, citrostadienol, lophenol, stigmasterol-­7-ene, stigmastanol, butryro-speermol, β-amyrin, taraxerol, tirucallol, 24-methylene-cycloartanol, volatile oil, dehydrostearic acid, aliphatic alcohol, hederagenin glycoside, β-unsaturated hydroxyl ketone, melanthigenin, melanthin, tannin, resin, reducing sugar, and saponin were also detected (Morikawa et al. 2004a, b; Ali et al. 2008; Mehta et al. 2009). Chemical constitutes such as thymol, thymoquinone, α-, β-pinene, р-cymene and thymohydroquinone were reported in essential oils of N. sativa seeds (Fig.  31.1 and Table  31.3). These important nutrients and phytochemicals can be used as functional ingredients in the foodstuffs.

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Table 31.2  Bioactive compounds in N. sativa seeds and seed oil Chemical composition Fixed oil

Seed Vitamin (%) 22–38 α-Tocopherol

Volatile oil

0.40– 1.6 8.4

β-Tocopherol

2.30

Myristoleic acid

γ-Tocopherol

1.90

Palmitic acid

4.8 35.6– 41.6 1–4

β-Tocotrienol γ-Tocotrienol

14.2 1.00

Palmitoleic acid Stearic acid

Thiamin (B1)

0.83

Oleic acid

20.8– 31.2 24.9– 40 3.7– 7.0

Pyridoxin (B6)

0.78

Linoleic acid

Niacin

6.3

Linolenic acid

Folic acid

0.042

Arachidic acid

riboflavin(B2)

0.063

Eicosadienoic acid Lignoceric acid

Fibre Total ash Fatty oils Vitamins Protein Carbohydrate Minerals

Mineral Calcium (Ca)

Oil (g/ Phytosterol mL) 1.54 Cholesterol

Oil Fatty acid (mg/100 mg) 4.80 Myristic acid

Oil (%) 0.16– 1.2 0.18– 11.2 11.0– 13.1 0.1–0.3 2.2–3.7 3.16– 25.6 47.5– 60.8 0.30– 2.10 0.14– 0.25 2.3–3.0 0.2– 1.08 10.0 Seed (%) 30– 48% 7–15%

Oil (%)

Dihomolinoleic acid Chemical constituent

0.9–2.0

Thymoquinone

Thymohydroquinone, dithymoquinone, p-cymene Carvacrol 6–12%

Phosphorus (P) 4.5

Campesterol

10.4–13.1

Magnesium (Mg) Potassium (K) Sodium (Na) Zinc (Zn) Copper (Cu) Iron (Fe) Manganese (M)

1.25

Stigmasterol

6.57–20.9

7.7 0.118 0.015 0.013 0.016 0.011

β-Sitosterol Trimethylsilyl Δ7-Stigmasterol Δ7-Avenasterol Δ5-Avenasterol Lanosterol

49.0–60.2 9.33 0.6–1.60 1.17–2.1 2.4–12.4 3.4

4-Terpineol t-Anethol Sesquiterpene longifolene

2–7% 1–4% 1–8%

Matthaus and Ozcan (2011), Takruri and Dameh (1998), Nergiz and Otleș (1993), Kiralan et al. (2014), Ramadan and Morsel (2002), Ramadan et  al. (2003), Mcrtz and Rogimsky (1971), Medeiros (1985), Al-Saleh et al. (2006), Ansari et al. (1998), EI-Tahir Kamal and Bakeet (2006), Al-Jassir (1992), Cheikh-Rouhou et  al. (2008), Gharby et  al. (2015), Hamrouni-Sellami et  al. (2008), Khan (1999), Houghton et al. (1995), Nickavar et al. (2003), Babayan et al. (1978), Ahmad et al. (2013), Mukhtar et al. (2019), and Atta (2003)

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Table 31.3  Essential oil composition (%) of N. sativa seeds Thymoquinone α-Pinene β-Pinene р-Cymene Carvacrol Carvone Sabinene Thymol α-Thujene γ-Terpinene Linalool cis-Thujone Camphor 4-Terpineol α-Terpineol α-Longipinene

13.7–24.0 1.2–15.04 1.3–7.7 14.8–46.8 1.6 0.9–21.1 1.4–5.5 0.4 2.4–9.8 0.5–2.0 9.9 0.2 0.6 1.0 2.2 2.1

Citronellyl acetate Longifolene Davanone Fenchone Dihydrocarvone р-Cymene-8-ol α-Longipinene Longifolene Estragole Anisaldehyde trans-Anethole Mysristich Dill apiole Myrcene α-Phellandrene Limonene

0.4 6.4 0.1 1.1 0.3 0.4 0.3 0.7 1.9 1.7 38.3 1.4 1.8 0.4 0.6 4.3

Rajeskhar and Kuldeep (2011), Al Mofleh et al. (2008), Nickavar et al. (2003), Tekeoglu (2007), Ashraf et al. (2006), and Mukhtar et al. (2019)

Fig. 31.1  Bioactive compound in N. sativa seed and essential oil (Mukhtar et  al. 2019). (a) р-Cymenem, (b) Thymol, (c) Thymoquinone, (d) Thymohydroquinone, and (e) Dithymoquinone

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3.1  Antioxidant Activity Synthetic antioxidants are used in food and medical industries for the preservation process. Currently, synthetic antioxidants are forbidden to be used in various industries due to their side effects (Kahl and Kappus 1993). Many consumers are looking for natural antioxidants from various medicinal plants due to their potential health benefits (Bhattacharjee et  al. 2020). The antioxidant activity of N. sativa seeds extract was tested against STZ-induced diabetic rats and the results indicated an increase in the levels of malondialdehyde in the rat hippocampus (Abbasnezhad et al. 2015). Another study conducted that N. sativa also showed antioxidant traits against oxidative damage induced in the brain tissue of animals by propylthiouracil. Increased serum thyroxine was found in rats by the combination of vitamin C and N. sativa as compared to the group with propylthiouracil (Beheshti et  al. 2017). Several studies were conducted on antioxidant properties of ethanol and methanol extracts of N. sativa seed and also oils. The results indicated that N. sativa had potential antioxidant property due to antioxidant compounds (i.e, phenolics and tocopherols) present in the seed extracts and also the oils (Burits and Bucar 2000; Mohammed et al. 2016; Houcher et al. 2007, Awan et al. 2018).

3.2  Antimicrobial Activity The major bioactive compound (thymoquinone) found in the black seed extract of N. sativa showed potential antibacterial activity (Emeka et  al. 2015). It showed bactericidal activity against gram-positive bacteria species such as Staphylococcus epidermidis and Staphylococcus aureus with minimum inhibition concentration (MIC) in the range of 8–32 μg/mL. However, there was no effect on Enterobacter bacteria and E. coli (Chaieb et al. 2011). Antibacterial activity of methanol, chloroform, and water extracts of N. sativa was reported by Gerige et al. (2009), and Bita et al. (2012). Among the extracts, methanol extracts showed antimicrobial activity against Candida albicans followed by chloroform extract (Gerige et  al. 2009). Black seed extract showed several multidrug-resistant clinical bacterial activities (Islam et al. 2013) and also effectively work against H. pylori (Salem et al. 2010). Antibacterial activity was exhibited by N. sativa due to thymoquinone while melanin was the major compound in the extracts (Bita et al. 2012). Some of the authors were also reported antimicrobial activity of N. sativa seed extracts and seed oils (Mohammed et al. 2019; Al-Ameedy and Omran 2019; Alshareef 2019).

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3.3  Antiviral Activity N. sativa extracts showed antiviral activity against the hepatitis C virus (HCV), a study was reported by Barakat et  al. (2013). Patients with HCV received 450 mg N. sativa oil in the form of capsules, wherein a decrease in the viral count was reported after 3 months of treatment. Furthermore, increased antioxidant activity in the cells indicates a reduction in the red blood cells hemolysis and platelet count. The results indicated that extracts of N. sativa oil decreased the viral load in the HCV patients, and enhanced the clinical condition, oxidative stress and glycemic in the diabetic patients.

3.4  Antifungal Activity N. sativa extract contains a rich source of thymoquinone that showed potential antifungal activity against fungal strains (Gupta et al. 2012; Rogozhin et al. 2011). The antifungal activities were compared with standard drug Amphotericin-B. Thymoquinone showed antifungal activity against Cryptococcus albidus Candida albicans, Isatchenkia orientalis, and Aspergillus fumigatus. Another study reported that essential oil of N. sativa has antifungal potential on T. mentagrophytes, M. canis, and M. gypseium (Mahmoudvand et  al. 2014). Khosravi et al. (2011) reported that N. sativa oils inhibited the growth of A. flavus and A. fumigatus due to volatile molecules. Some studies reported the control of fungal infections by natural products from N. sativa (Rogozhin et  al. 2011; Bita et  al. 2012; Asdadi et  al. 2014). Other studies showed effective antifungal traits against Aspergillus niger, Aspergillus flavus, Candida tropicalis, C. albicans, and C. krusei (Piras et al. 2013). The antifungal activity was observed against fungal culture-Fusarium Solani, while hexane and ethanol fractions of black seed showed higher antifungal activity when compared to chloroform, methanol, acetone, ethyl acetate and water factions (Al-Ameedy and Omran 2019). Bioactive compounds in N. sativa seed extracts and seed oils and their mechanisms of action make it a useful candidate as an antifungal agent.

3.5  Anti-diabetic Effects Diabetes is the most prevalent civilization disease globally. The natural extracts of N. sativa seed or seed oil have been reported a good agent for the development of anti-diabetic drugs (Table  31.4). N. sativa oil decreased blood glucose levels in STZ-diabetic rats after feeding for 2–6 weeks (El-Dakhakhny et al. 2002). Fararh et al. (2004) reported the impact of N. sativa oil on hypoglycemia. The extract of N. sativa increased the glucose consumption by hepatocytes via the activated

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Table 31.4  Anti-diabetic potential of N. sativa seeds N. sativa Seed oil

Observation Lowered blood glucose in STZ diabetic rats

Seed oil

Decreased blood glucose and increased serum insulin in STZ/NA-diabetic hamsters Decrease in hepatic gluconeogenesis in STZ-diabetic hamsters Increased glucose consumption by hepatocytes

Seed oil Defatted extract Seed extract Ethanol extract Seed oil Seed extract Seed oil

N. sativa

Decreased lipid peroxidation and increased antioxidant defense system in diabetic rabbits Reduced hyperglycemia and improved antioxidant enzymes in live and kidney in STZ-diabetic rats Decreased hyperglycemia and restored lowered serum insulin in STZ-diabetic rats Significantly increased serum insulin and SOD activity in tissues in STZ-diabetic rats Improved clinical and biochemical composition of the insulin resistance syndrome in patients with diabetes Reductions in fasting blood glucose, postprandially, and glycosylated hemoglobin

Period of study 2–6 weeks

–

References El-Dakhakhny et al. (2002) Fararh et al. (2002) Fararh et al. (2004) Yuan et al. (2014)

2 months

Meral et al. (2001)

1 month

Kaleem et al. (2006)

1 month

Kanter et al. (2003) Abdelmeguid et al. (2010) Najmi et al. (2008)

4 weeks –

– –

–

Bamosa et al. (2010)

Source from Srinivasan (2018)

protein kinase (Yuan et al. 2014). N. sativa extracts fed to induced-diabetic rabbits for 2 months showed potential antioxidant activity and prevented lipid peroxidation in the liver (Meral et al. 2001). Oral administration of ethanolic N. sativa extract to STZ-diabetic animals improved altered lipid peroxidation and antioxidant enzymes levels in the kidney and liver of rats (Kaleem et al. 2006). Black cumin seeds control diabetic complications through antioxidant activity, decreased serum glucose and restored lowered insulin proliferation of β-cells in STZ-diabetic animals after feeding of N. sativa oil for 30 days (Kanter et al. 2003). Feeding of N. sativa oil to STZ-­diabetic animals preserved pancreatic -β-cell integrity and increased insulin levels (Najmi et al. 2008; Abdelmeguid et al. 2010). Bamosa et al. (2010) reported that N. sativa seeds used for the adjuvant therapy purpose to control glycemic levels in type-2 diabetes mellitus. Anti-diabetic activity of ethanol extract of black seeds was studied in Meriones shawi after the onset of diabetes. The results indicated that ACC phosphorylation and levels of muscle Glut 4 protein increased by ethanol extracts of N. sativa seed which in turn showed an insulin-sensitizing impact (Benhaddou-Andaloussi et al. 2011). The results indicated that TQ as well as various extracts of black seeds and seed oil could be applied in the treatment of diabetes.

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3.6  Anti-inflammatory Activity The anti-inflammatory potential of bioactive compounds in N. sativa seed extracts and seed oils controlled common health issues such as inflammation and rheumatoid arthritis (Babar et al. 2019; Bordoni et al. 2019). Many biological activities are attributed to the antiradical effect as well as interaction with molecular targets linked with inflammation (Bordoni et  al. 2019). N. sativa essential oil, contains p-cymene (37.3%) and TQ (13.7%), was tested for its anti-inflammatory effect in rats (Hajhashemi et  al. 2004). The water extract of N. sativa showed anti-­ inflammatory effect in animal models, while the anti-osteoporotic impact of N. sativa and TQ was evidenced by monitoring the inhibition of inflammatory cytokines and the transcription factor (Shuid et al. 2012). Al-Ghamdi (2011) reported the anti-inflammatory activity of N. sativa aqueous extract against carrageenan-­ induced right hind paw edema. The presence of bioactive molecules such as phenolics and TQ supports N. sativa extracts and seed oils for the development of anti-inflammatory drugs (Table 31.5).

3.7  Immuno-modulatory Activity N. sativa extracts and its bioactive constituents showed immuno-modulatory activities in vitro and in vivo (Koshak et al. 2017, 2018; Othman et al. 2019). The immunomodulatory effects of N. sativa water extract investigated in BALB/c mice and C57/BL6 primary cells, showed that N. sativa extract improved splenocyte proliferation. Methanol extract of Nigella sativa increased the total white blood cells count and the spleen weight in BALB/c mice, suggesting the immunomodulatory effect of black seeds (Ghonime et  al. 2011). Duncker et  al. (2012) reported that black seed extract enhanced the immune and symptoms parameters in murine ovalbumin-­induced allergic diarrhea in rats. N. sativa essential oil was tested for Table 31.5  Anti-inflammatory potential of N. sativa seeds N. sativa Essential oil N. sativa oil N. sativa Aqueous extract Seed extract

Observation Significant analgesic effect on acetic acid-induced writhing, formalin, and light tail-flick tests in rats model Significantly inhibited carrageenan-induced paw edema in rats Observed inhibition of inflammatory cytokines and the transcription factor in human trails Enhanced total while blood cells count and increased spleen weight in BALB/c mice; enhanced splenocyte proliferation in C57/BL6 primary cells Improved symptoms and immune parameters in murine OVA-induced allergic diarrhea in mice

Source from Srinivasan (2018)

References Hajhashemi et al. (2004) Hajhashemi et al. (2004) Shuid et al. (2012) Ghonime et al. (2011) Duncker et al. (2012)

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immunomodulatory effect in a Long-Evans rats, wherein the results indicated a two-­ fold decrease in the production of antibodies in response to the vaccination of typhoid when compared to the control rats. Additionally, neutrophils count was decreased while those of monocytes and lymphocytes were increased (Torres et al. 2010). The results indicated that TQ can be used as an immunosuppressive agent in Type-1 diabetes mellitus.

3.8  Anti-cancer Activity As per the reports in the literature (Table  31.6), N. sativa controlled the tumor growth, initiation of tumor and metastasis with chemotherapeutic drugs (Aisa et al. 2019; Butt et al. 2019; Tiwari et al. 2019). The anti-cancer potential of N. sativa was tested in different in vitro and in vivo models. Black cumin might be used as an anti-­ proliferative and anti-cancer agent in animal and cell culture models (Majdalawieh and Fayyad 2016). Black cumin extract enhanced the benz(α-)pyrene-induced carcinogenesis in the forestomach of animals (Aruna and Sivaramakrishnan 1990). Salim and Fukushmia (2003) reported that colon cancer-induced to rats with 1,2-dimethylhydrazine and prevented the same after administration of N. sativa oil, this may be suppression of cell proliferation in the colonic mucosa. Aqueous extracts Table 31.6  Anticancer activity of N. sativa N. sativa Seed extract

Observation Improved benz(a-)pyrene-induced carcinogenesis in the forestomach in mice model

N. sativa oil

Reduced the induction and development of 1,2-dimethylhydrazine-induced aberrant crypt foci, putative preneoplastic lesions for colon cancer in a rat model Improved the ulcer severity and basal gastric acid secretion. The anti-ulcer effect is possibly prostaglandin mediated through it antioxidant and anti-secretory activities in a rat model Reduced malignant and benign colon tumor sizes, incidences and multiplicities in Waster rats Prevented inflammatory in the blood; protected colonic tissue against ulcerative colitis in the rat model Protected against DMBA induced breast cancer Exhibited cytotoxicity to MCF-7 Cells

Aqueous extract

Volatile oil Seed oil Seed oil Aqueous extract Seed extract

Reduced the carcinogenic effects of DMBA in mammary carcinoma

Source from Srinivasan (2018)

References Aruna and Sivaramakrishnan (1990) Salim and Fukushima (2003)

Al Mofleh et al. (2008)

Salim (2010) Isik et al. (2011) Linjawi et al. (2015) Mahmoud and Torchilin (2012) Shafi et al. (2008)

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of N. sativa prevented formation of gastric ulcer by necrotizing agents in Shay rats (Al Mofleh et  al. 2008). N. sativa oil showed chemoprotective activity on multi-­ organ tumor formation in rat models (Salim 2010), wherein the results showed significant decreased in tumor size, multiplicities and incidences in lungs and alimentary canal. Administration of N. sativa (Isik et al. 2011) resulted in a decrease in the pro-inflammatory cytokines in the blood of animals with experimental colitis induced by trinitrobenzene sulfonic acid. Black cumin seed oil prevented breast cancer induced by 7,12-dimethylbenz [α] anthracene in rat models, and the results indicated controlled tumour markers, regulation of genes, and histopathological studies related to breast cancer (Linjawi et al. 2015). Aqueous extracts of N. sativa exhibited cytotoxicity in MCF-7 cells at 50 mg/mL (Mahmoud and Torchilin 2012). Administration of black cumin reduced the carcinogenic impacts of DMBA in mammary carcinoma, which revealed its protective effect in mammary carcinoma (Shafi et al. 2008). Thymoquinone (TQ) effects on pancreatic cancer in vitro and in vivo model studies revealed a reduction in tumour metastasis and expression of NF-kB and MMP-9 protein in tumour tissues upon treatment with TQ (Wu et al. 2011).

3.9  Improve the Skin Pigmentation N. sativa is used for the dermatological disorder, treatment of skin conditions, wounds healing, burn, injury treatment, skin-inflammatory and skin pigmentation (Yaman et al. 2010; Abu-Zinadah 2009; Ali and Meitei 2011; Chaieb et al. 2011). Extract of N. sativa oil has shown effective protection against skin diseases (vitiligo), that is a hypopigmentation disorder causing psychological morbidity. N. sativa rich in thymoquinone has the ability to increase melanin within the skin using the active ingredient of N. sativa oil (Ghorbanibirgani et al. 2014). A possible mechanism of this action was that it increased the melanin intensity by increasing the sensitivity of cholinergic receptors inside the melanopsin. N. sativa oil used for the treatment of skin diseases as therapy, wherein a decrease in the size of the lesion on the skin was noted (Ghorbanibirgani et al. 2014).

3.10  Wound Healing Property Thymoquinone extracted from N. sativa was reported as an antioxidant, prevent lipid membrane in tissue and wound healing property (Yaman et  al. 2010). TQ increased the wounding process compared with silver sulfadiazine due to immunomodulatory and anti-inflammatory activities. Extracts of N. sativa showed high activity in the improvement of collagen formation and epithelialization rate, and these results indicated that extracts of N. sativa are an excellent candidate for the treatment of wound healing property (Sarkhail et al. 2011). Abu-Al-Basal (2011)

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reported the effect of N. sativa ether extract applied on the skin, wherein the results indicated a decrease in white blood cells, reduction in tissue damage, and a decrease in bacterial expansion. Another study conducted by Ab Rahman et al. (2014), indicated an increase proliferation rate. Based on these observations of extracts of N. sativa can be used in the treatment of the wound healing process.

4  Conclusion Herbal products are derived from plants and used globally for medicinal applications. N. sativa is one of the best herb and extensively used for therapeutic due to it offers various beneficial effects and also available commercial products (hair oil, cream, shampoo, Hair mask, capsule…etc) in the market. N. sativa seed extracts and oils contain various bioactive compounds showed potential health benefits in vitro and in vivo. The health benefits of N. sativa seeds and seed oils associated with TQ and unsaturated fatty acids. The availability of practical applications is to be standardized for providing good therapeutic use. Studies conducted on N. sativa recommended N. sativa in the manufacture of natural drug products for food, nutraceutical and pharmaceutical applications. Acknowledgments  The authors acknowledge Vignan’s Foundation for Science, Technology and Research (Deemed to be University) for providing the facility for this work.

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Chapter 32

Health Promoting Activities of Nigella sativa Seed Extracts Arzu Kart and Ebru Aydın

Abstract  Many medical plant have recently attracted attention due to their important role as dietary supplements with health-promotive effects amd minimal side impacts. Nigella sativa, also known as black seed, belongs to Ranunculaceae family is cultivated in various areas of the world mostly in Middle Eastern, Far Eastern, Asian and East Mediterranean countries. Valuable seeds, extracts and oils of Nigella sativa, which contain vitamins, minerals, essential fatty acids and many other useful components, have been used since ancient times. Today, Nigella sativa seeds and seed extracts have been used for healing several disorders due to their antioxidant properties. Studies have shown that Nigella sativa is very effective in terms of health promotion and disease prevention. Nigella sativa seeds have useful effects such as neuroprotective, antihypertensive, anticancer, antidiabetic, anti-inflammatory, analgesic, antibacterial, antifungal, antiviral and antiparasitic activities. Nigella sativa seed extracts might be a potential new health promotive agent due to the proven beneficial activities. Nigella sativa seed extracts might be used as essential nutrients sources or as a nutritional supplement. This chapter provide detailed information about the botanical properties of Nigella sativa, functionalities of phytochemicals, therapeutic effects of Nigella sativa extracts, protective effects against certain diseases to reveal the potential of the use of Nigella sativa seed extracts and to shed light on the future Nigella sativa-related studies. Keywords  Pharmacological · Antimicrobial · Anti-inflammatory · Respiratory Neuroprotective

A. Kart (*) · E. Aydın Department of Food Engineering, Faculty of Engineering, Suleyman Demirel University, Isparta, Turkey e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0_32

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Abbreviations EO Essential oil IDF International Diabetes Federation MBC Minimal bactericidal concentration MIC Minimum inhibitory concentration TQ Thymoquinone WHO World Health Organization

1  Introduction Nigella sativa L., commonly known as black seed, belongs to the Ranunculaceae family. It is cultivated in various areas of the world, mostly in Middle Eastern, Far Eastern, Asian and East Mediterranean countries. N. sativa has been used for thousands of years as a spice and food preservative. It has been utilized as a protective and curative remedy for several diseases. Seeds, seed extracts and oils of N. sativa, which contain vitamins, minerals, essential fatty acids and many other useful components, have been used for various reasons. Today, N. sativa seed and seed extracts have been used traditionally for several diseases due to their high antioxidant properties. Recently, beneficial effects of N. sativa seed extracts have been demonstrated in many in vitro or in vivo. Scientists have shown that N. sativa is very effective in terms of health promotion and disease protection. Many remarkable studies have approved that its effective pharmacological actions. It has been revealed that N. sativa seed extracts has many pharmacological effects such as neuroprotective, antihypertensive, antianticancer, nephroprotective, antidiabetic, anti cardiovascular, anti-inflammatory and antibacterial, antifungal antiviral and antiparasitic activities (Mbarek et al. 2007; Hannan et al. 2008; Okeola et al. 2011; Dilshad et al. 2012; Bita et al. 2012; Abbasnezhad et al. 2015; Enayatfard et al. 2019). N. sativa seed extracts might be a potential new health promotive agent due to the proven beneficial activities. Hence, N. sativa seed extracts might be used as essential nutrients sources, as a nutritional supplement or as a natural drug for health promotive effect and diseases prevention.

2  Health Promoting Activities of N. sativa Seed Extracts 2.1  Antioxidant Effects of N. sativa Seed Extracts Phenolic compounds such as flavonoids, phenolic acids, and tannins have received attention for their high antioxidative activity. It is a known fact that these synthetic antioxidants cause health problems. Therefore, in recent years there has been an increasing interest in the use of natural antioxidants, such as tocopherols, flavonoids

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and plant extracts for the preservation of food materials. Many plants have phytochemicals as unartificial antioxidants, one of these plants is N. sativa (Tubesha et al. 2011; Ahmed et al. 2015). Some studies have evaluated the antioxidant activity of Nigella sativa seed extracts. N. sativa has been used traditionally for the treatment of many disease, such as influenza, headache, eczema, rheumatism, ischemia, asthma, cough, bronchitis, cancer and cardiovascular. The antioxidant activity of N. sativa has been demonstrated in the several studies and it is suggested that the positive and curative effects of N. sativa seeds on some diseases are due to its antioxidant property (Hosseinzadeh et al. 2007; Tubesha et al. 2011; Meziti et al. 2012; Ahmed et al. 2015). The toxic oxygen metabolites are removed by the antioxidant compounds and excessive amounts of oxygen free radical production may cause ischemia and reperfusion (I/R) during an oxidant injury. Hind limb ischemia was induced to rats and analyzed with the administration of N. sativa seed extracts to see the extract’s effect on I/R injury. N. sativa seed extract had shown a protective effect on I/R injury-­ induced oxidative stress in rat muscle (Hosseinzadeh et al. 2007). Tubesha et al. (2011) evaluated the antioxidant character of N. sativa seeds methanol extracts. The highest total phenolic and flavonoid contents values was detected in N. sativa hydrolyzates. On the other hand, non-hydrolyzed seed extracts have shown the lowest total phenolic and flavonoid contents. The method used for extraction has provided different results in terms of obtaining antioxidants. The hydrolysis process is important to recover the antioxidants from seeds (Tubesha et al. 2011). Meziti et al. (2012) reported similar results in their study. They evaluated the antioxidant activity of various extracts prepared from N. sativa seeds including methanolic extract (ME), chloroform extract (CE), hexane extract (HE: fixed oil), ethyl acetate extract (EAE), and water extract (WE). According to the Folin-­ Ciocalteu assay CE and EAE have contained a high level of phenolic compounds (81.3 and 72.4 μg GAE/mg extract, respectively). It has been observed that CE and EAE exhibited the highest DPPH· radical scavenging activity, with IC50 values of 106.5  μg/mL and 121.6  μg/mL, respectively. The antioxidant activities of the methanol extract and the fixed oil have been proven by an in vivo animal study. Significant enhancement of the blood total antioxidant capacity and the plasmatic antioxidant capacity towards DPPH· radical has been determined after daily oral administration of methanol extract (500 and 800  mg/kg/day) during 21  days in mice (Meziti et al. 2012). The shoots, roots, and seeds methanol extracts of N. sativa was studied for its antioxidant activity. The seeds hexane fraction of the methanol extract inhibited reactive oxygen species (ROS) production and protected A-549 lung carcinoma cells from oxidative stress (Bourgou et al. 2012). Abbasnezhad et  al. (2016) observed the antioxidant effect of N. sativa seeds hydro alcoholic extracts on STZ-induced diabetic rats. The level of thiol and malondialdehyde in the hippocampus was higher in the rat treated with the seed (200 mg/ kg) compare to the untreated group. Recently, scientists indicated that N. sativa seed hydro alcoholic extracts have antioxidant activity contrary to oxidative damage induced in the brain tissue of rats during their neonatal and juvenile growth. Rats were administered with vitamin C

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(100 mg/kg) and N. sativa extract (100, 200, and 400 mg/kg). It was reported that N. sativa had a protective effect on oxidative damage of brain tissues (Beheshti et al. 2017). Sen et  al. (2018) studied the antioxidant activity of methanolic extract of N. sativa seeds obtained from six different cities in Turkey. It was reported that the amount of the total phenolic content had a positive effect on their antioxidant activity. Pranav (2018) analyzed the oxidative deterioration of ground pork in the presence of N. sativa seed extract and a synthetic antioxidant; butylated hydroxytoluene (BHT) as control. Water and ethanolic extract of the seeds did not change the meats’ color and also its free fatty acids, peroxide formation of protein carbonyls and rancid odour development were lower than control due to the phenolic content of the black cumin seed extract.

2.2  Anti-inflammatory Effects of N. sativa Seed Extracts Pharmacological effects have been described for N. sativa and its constituents, including an anti-inflammatory effect. There are several studies about the anti-­ inflammatory feature of N. sativa plants. These studies focused on thymoquinone (one of the main compounds of N. sativa fixed and essential oils), oil and aqueous extracts of the N. sativa seeds (Dirjomuljono et al. 2008; Boskabady et al. 2011a). Extracts obtained from the seeds of N. sativa are used as a spice or remedy for the treatment of various inflammatory diseases (Ghannadi et al. 2005). The aim of Ghanadi group’s study was to investigate the analgesic and anti-inflammatory effects of N. sativa phenolics. In their study, N. sativa seed polyphenols (NSP) were prepared and its anti-inflammatory effects on mice and rats were examined using acetic acid-bound wrinkles, formalin, slight tail movement, carrageenan-induced paw edema, and croton oil-induced ear edema tests. It was observed that intraperitoneal and oral administration of NSP suppressed in a dose-dependent manner the nociceptive response in the formalin test. It was observed that NSP did not produce significant analgesia in the light tail-flick test in mice. Oral administration of NSP had not produced a significant reduction in carrageenan-induced paw edema. However, it was emphasized that NSP which injected via intraperitoneally, inhibited paw edema in a dose-dependent manner. NSP when applied topically failed to reduce croton oil-induced ear edema. Briefly, the study has revealed that seed extract has potential anti-inflammatory and analgesic features (Ghannadi et al. 2005). In a similar research study, a placebo-controlled study, the anti-inflammatory effect of extract mix obtained from N. sativa and Phyllanthus niruri (NS-PN extracts) in patients with acute tonsillopharyngitis were tested. During supplementation, patients received a capsule days (360 mg N. sativa extract and 50 mg P. niruri extract) for 7 days. Positive results were obtained from the first day of this study. A reduction was observed in swallowing, inflammation and pain in patients compared with the placebo group (Dirjomuljono et al. 2008).

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Another clinic experiment demonstrated the anti-inflammatory effects of N. sativa seed extract (Boskabady et al. 2011b). It was concluded that the protective feature of the extract of N. sativa on pathological disorders of the lungs in test animals is probably due to its alleviating characteristic on inflammation. Since N. sativa is widely used in the treatment of respiratory diseases such as asthma, researchers have investigated the potential for use in the treatment of pulmonary inflammation. Çelik Altunoğlu et  al. (2017) analyzed the effect of N. sativa seed methanol extract on the immune response and growth performance of rainbow trout for 30 days. It was reported that N. sativa consumption did not affect the fish’s growth. Also, cytokine-mediated immune responses were obtained ineffective in rainbow trout and it was shown that N. sativa seed may only have an effect on higher animals. Ikhsan et al. (2018) studied the effects of N. sativa ethanol extract as an anti-­ inflammatory agent in asthmatic patients. The results showed that N. sativa ethanol extract had anti-inflammatory features and no toxic effects. Therefore, they emphasized that the anti-inflammatory feature of N. sativa extracts might be potential as an agent for use to the treatment of asthma (Ikhsan et al. 2018).

2.3  Respiratory Effects of N. sativa Seed Extracts Boskabady and Farhadi (2008) studied the prophylactic effect of an aqueous seed extract on chemical war victims exposing to sulfur mustard (SM). The results showed that the prophylactic effect of seed extracts on respiratory problems might be due to its alleviate feature on inflammation.

2.4  Neuphroprotective effects of N. sativa seed extracts Nephrotic syndrome is considered a secondary disorder of diseases such as diabetes and cancer. There are various factors related to kidney diseases. Oxidative stress and Inflammatory processes and have a crucial role in nephrotic diseases. It is well established by scientific results that oxygen free radicals have mediated in nephrosis-­ induced damage in animal models. Adriamycin (ADR, doxorubicin) is an antibiotic that is used in treatment due to its anticancer activity. However, it is a known fact that this antibiotic may have several side effects. Especially nephrotoxic effects were reported after treatment with ADR. Acute and chronic side effects of ADR on kidneys have been emphasized. Also, it is suggested that the use of ADR is limited in clinical practice because it might lead to oxidative damage and nephrosis progression. Nowadays, there is various studies that indicated Curcuma longa and Nigella sativa might be used for treating renal disorders (Mohebbati et al. 2016, 2017a). According to this literature, seed extracts might be used as an antioxidant source for

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adriamycin-induced oxidative stress in renal illnesses. Mohebbati et al. (2016) had done such study since there was not any study showing the effects of using the combined of both plant extracts (C. longa ve N. sativa) on kidney disease. They had studied theneuroprotective effects of hydro-alcoholic seed extract in animal trials. At the end of the study, it had been concluded that the administration of hydro-­ alcoholic extracts of N. sativa and C. longa might be used for the treatment of kidney disorders. In a recent study, similar results were obtained. It was demonstrated that the mixed extracts of N. sativa and C. longa have positive synergistic effects on ADR-induced nephropathy in kidney damage in rats (Mohebbati et al. 2017a). As a result of both studies, it was demonstrated that C. longa and N. sativa might be used together to enhance the antioxidant potential in the animal kidney. Nigella sativa seed extract could be used in the treatment of kidney diseases. Urinary calculi are the third prevalent disorder in the urinary system. Urinary calculi may cause obstruction, hydronephrosis, infection, and hemorrhage in the urinary tract system. Hadjzadeh et al. (2007) studied the effects of the ethanol seed extract on kidney calculi. At the end of the study, it was observed that ethanolic seed extract has a potential preventive character on calculus formation in the kidney.

2.5  Neuroprotective effects of N. sativa seed extracts Several in vitro studies have been conducted to investiagate the neuroprotective activity of N. sativa seed extracts. These studies confirmed that seed extracts have a modulatory feature on the nervous system and brain neurotransmitter amino acid levels. Al-Naggar et al. (2003) suggested that aqueous, and methanolic seed extracts have a potential sedative and depressive characters and inductive effect on analgesia. It had been indicated that neuroprotective features of seed extract on the central nervous system resulted in a reduction in normal body temperature. Furthermore, it was suggested that the sedative and depressive effects of N. sativa could be based on changes in inhibitory/excitatory amino acid levels (Arafa et al. 2013). El-Naggar et al. (2010) conducted a similar study and at the end of this study, considerable results had been shown that exposure of the cultured neurons has a modulatory effect on the release and contents of amino acids. El-Naggar et  al. (2010) demonstrated that pretreatment with methanol extracts of N. sativa seeds modulates the neuronal release of several amino acid neurotransmitters, such as glycine, aspartate and gamma-aminobutyric acid (GABA). A similar study had been conducted by El-Naggar et  al. (2017). They investigated the effects of N. sativa methanol extract on brain neurotransmitter amino acid levels. The changes were measured in aspartate, glutamate, glycine and c-aminobutyric acid in five brain regions of rats after N. sativa methanol extract treatment. The results had shown notable variations in amino acids. It had been concluded that injected N. sativa methanol extract had modified amino acid levels. Also, it had been suggested that

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the results might be considered because of neurodegenerative disorders are associated with the instability of amino acid dose. There is another striking study revealing the neuroprotective effect of N. sativa seed extracts. Ciprofloxacin is a drug commonly used for the remedy of infections. Also, it is accepted to has a probable epileptogenic effect. Arafa et  al. (2013) ­investigated the neuroprotective effect of Nigella sativa (NS) extract on amino acid neurotransmitter alteration in pentylenetetrazole (PTZ) and ciprofloxacin (CFX) treated rats in different brain regions. In the study, researchers had concluded that the administration with seed extract is notably effective on side effects due to PTZ and ciprofloxacin. It was suggested that N. sativa seed extracts might be used as a natural agent to remediate neurotoxicity arising from ciprofloxacin administration. Some studies indicated that seed extracts might be used for the treatment of epilepsy. There is a high prevalence of mental and neurological disorders worldwide that account for 13% of total disability-adjusted life years lost due to all diseases and injuries in the World. Around 50 million people worldwide are afflicted with epilepsy (Arafa et al. 2013; Bhandari 2014). There are drugs, like benzodiazepines and barbiturates, that have used as effective antiepileptic products. It is stated that in spite of using the antiepileptic drugs, approximately 15% of epilepsy patients in childhood are resistant to the drug treatments. There is a need for screening the medicinal plants or constituents for use as alternative natural products. There are several remarkable in vivo and in vitro research. These studies have investigated the antiepileptic and adverse effects of N. sativa and TQ against several animal models of epilepsy. According to conducted trials, more information has been generated from this research. Proven data, providing a better understanding of the antiepileptic activity of N. sativa and constituents. One of the studies related to the antiepileptic activity of N. sativa seed extracts, conducted by Arafa et al. (2013). The results of their study indicated that N. sativa seed extracts regulated the balance of some amino acids and antioxidant capacity induced with the drugs. Also, it was mentioned that seed extract has similar activity to an antiepileptic medicine such as sodium valproate. Some of the studies to determine the neuroprotective effects of N. sativa and its constituents have focused on hypothyroidism. Thyroxine (T4) and Triiodothyronine (T3) hormones are secreted by the thyroid gland. These hormones have crucial effects on the modulation of some metabolic reactions and mental evolution (Wagner et al. 2008). Thyroid hormones are significant in neural production and evaluation of the central neural system during the fetal cycle (Shin et al. 2013). One of the severe chronic disorders is hypothyroidism. Hypothyroidism disease is related to the inadequate secretion of thyroid hormones. Late recognition of hypothyroidism might be lead to crucial some health problems. It is also stated that hypothyroidism might cause learning or thought problems (Asiaei et al. 2017). The studies which are related to the activity of N. sativa seed extracts on hypothyroidism are remarkable. Another study related to hypothyroidism was conducted by Beheshti et al. (2014). The aim of their research was to evaluate the activity of hydroalcoholic seed extract on hypothyroidism-related problems. In the trials, a part of test rats

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received PTU (propylthiouracil) during lactation. Also, a part of the test rats received seed extract at different doses. After the lactation stage, test rats evaluated the learning and thought abilities. According to the findings of this study, it was emphasized that treatment with hydroalcoholic seed extract might improved pernicious impacts of hypothyroidism on learning or thought stages (Beheshti et al. 2014). Similarly, Mohebbati et  al. (2017b) investigated the effects of hydroalcoholic extract of Nigella sativa on renal tissue oxidative damage associated with propylthiouracil (PTU)-induced hypothyroidism during neonatal and juvenile growth in rats. In this study, they had shown that N. sativa hydroalcoholic extract can improve oxidative damage parameters (Mohebbati et al. 2017b). In order to reveal the possible neuroprotective act mechanism of N. sativa extract, results were compared to previous studies. Researchers reported various mechanisms. However, the mechanism of action could not be clearly demonstrated. Asiaei et al. (2017) investigated the neuroprotective properties of N. sativa seed extracts. In the experiments, rats had administrated by mixtures of PTU and hydroethanolic seed extract at various concentrations and combinations. Hydroethanolic seed extract reduced the release of neurons compared to the test group administrated with PTU. The study also stated that seed extract might be inhibited neural damage after hypothyroidism.

2.6  Neurotoxicity Effects of N. sativa Seed Extracts Butt et al. (2018) investigated the protective effects of Nigella sativa seed extract on lead (Pb)- induced neurotoxicity during different life stages in the mouse. Lead might produce adverse effects on the brain via increased production of reactive oxygen species (ROS) and causes oxidative stress. Almost every organ system, especially the evolving brain and renal systems, might be affected by Pb. In related articles, it was stated that Pb can indirectly lead to Alzheimer’s disease (AD). Scientists had tried to demonstrate the neuroprotective feature of ethanolic seed extract on oxidative stress due to Pb in animals. In the clinical study, rats were exposed to low (0.1%) and high (0.2%) doses of Pb. Pb exposure doses were administered via drinking water. Seed extracts were given at different levels in a normal diet. From the results of this study, researchers had supported that damaging property of Pb might be crucially reduced by administrated seed extract, emphasizing its antioxidant and neuroprotective features (Butt et al. 2018). Some studies that determined the neurotoxic effects of N. sativa and its constituents focused on Alzheimer’s disorder. It was indicated that antioxidants have a potential preventive feature on memory impairments in some studies (Hosseini et al. 2015). It had used tests to evaluate the effect of seed extract on memory performance and its possible mechanisms in the scopolamine-induced memory test model. Beneficial aspects of seed extracts have been exhibited on the nervous system in rats (Hosseini et al. 2015).

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2.7  Anticancer Effects of N. sativa Seed Extracts Herbal treatments are generally used for the prevention and treatment of cancer. Therefore, pharmacological investigations of the N. sativa seed extracts were performed in order to reveal its anticancer activities (Kumara and Kwong Huat 2001; Chehl et al. 2009; Dilshad et al. 2012). Dilshad et al. (2012) assessed the inhibitory activity of N. sativa methanol extracts on human breast cancer MDA-MB-231 cell proliferation and reported that N. sativa methanol extracts were able to alleviate the proliferation of cancer cells through induction of apoptosis. In another study, Nigella sativa seed extract was used to possess anti-carcinogenic activity on pancreatic ductal adenocarcinoma (PDA). PDA is known as one of the most lethal cancers of the gastrointestinal tract. It had been demonstrated that thymoquinone (TQ) which had been dissolved in methanol has cytoprotective effects that are mediated through its antioxidant and anti-inflammatory activities. Chehl et al. (2009) indicated that TQ reduces PDA cell growth and promotes apoptosis. They had suggested that surgical resection of PDA combined with a TQ treatment represents a promising novel therapeutic alternative treatment. Kumara and Kwong Huat (2001) studied in vitro cytotoxic activity of column fraction of an ethanolic extract of N. sativa seeds. This fraction and α-hederin had been evaluated for its in vivo antitumor activity. α-Hederin and saponin obtained from the NS seeds extracts exhibited antitumoral activity. It had been also suggested that apoptosis is a major mechanism by which α-hederin in preventing tumor growth. In Swamy and Tan research, α-hederin was isolated from black seed and dose-dependent tumor inhibition had been shown when given intraperitoneally for 7 days at 5–10 mg/kg to mice with formed tumors (Swamy and Tan 2001). In another study, the essential oil, ethyl acetate fractions and the ethyl acetate and butanol extracts of N. sativa seeds possessed a strong cytotoxic effect against tumor cells. The results demonstrated that the cytotoxic activity of black seed extracts is a complex phenomenon depending not only on the nature of the extract and its components but also on the tumor cell type (Mbarek et al. 2007).

2.8  Anticardiovascular Effects of N. sativa Seed Extracts In the past years, remarkable in vitro and in vivo animal studies were published that showing the cardioprotective effect of N. sativa plant, its seed, seed extract and its constituents. The explorations indicated cardioprotective characteristics of N. sativa seed extracts on blood pressure disorder, hypertension, lipid profile disturbance, hyperlipidemia, endothelial dysfunction, endothelium-independent vasorelaxant, heart rate, and atherogenesis. Therefore, it is suggested that N. sativa acts as a natural drug could be a preventive and curative product in cardiovascular diseases (Dehkordi and Kamkhah 2008; Haas et al. 2014; Niazmand et al. 2014; Hebi et al. 2016; Al-Naggar et al. 2017; Enayatfard et al. 2019).

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Dehkordi and Kamkhah (2008) conducted a study on the cardioprotective effect of Nigella sativa seed extract. It was observed that N. sativa seed extract caused a considerable decline in total and low-density lipoprotein (LDL) cholesterol. Dehkordi and Kamkhah (2008) recruited people with mild hypertension diseases with a randomized, double-blind, placebo-controlled trial to assess the effect of N. sativa seed extract supplement. After 8 weeks it was found that the supplement was able to lower blood pressure significantly. The mechanisms responsible for the vasorelaxant effect of Nigella sativa were suggested (Niazmand et al. 2014). The effects of hydroalcoholic extract of N. sativa seed on the vasomotor tone of the aortic rings and its possible mechanism(s) of action have been investigated. It has been observed that the inhibitory effect of N. sativa seed extract on the contraction induced by phenylephrine and KCl was endothelium-independent. The relaxation might be mediated through the inhibition of Ca2+ and KATP channels and also intracellular calcium release. In another study, Boskabady et al. (2005) have shown that N. sativa seed extracts have an effect on cardiovascular diseases. It has been demonstrated that seed extracts might be diminished cardiac contractility and heart rate. Hebi et al. (2016) published another article that demonstrates that Nigella sativa seed extracts has a play role in cardioprotective effects. They studied the cardiovascular effect of aqueous extract of Nigella sativa seeds (NSAE) in rats. Aqueous seed extract has traditionally been used in the treatment of hypertension and cardiovascular diseases. Seed extracts had a potential reductive effect on arterial blood pressure and heart rate. The study also indicated the possible use of seed extract in the control of hypertension. Enayatfard et  al. (2019) published another article that demonstrated that Nigella sativa seed extracts have a significant role in hypertension. The study demonstrated that hydroalcoholic seed extract notably reduces the cardiovascular problems caused by hypertension. Rizka et al. (2017) evaluated the effect of N. sativa seed extract for hypertension in the elderly. It is known that N. sativa seed extracts have diuretic character, and also might be increased formation of nitric oxide. Nigella sativa seed extract can be used as a natural antihypertensive product for the elderly population. It was found that Nigella sativa seed extract can be used as a natural antihypertensive product for the elderly population. In this clinical study, the effect of Nigella sativa seed extract on two different blood pressure (SBP, DBP) of elderly sicks with hypertension have been measured. As a result of trials, no negative effects on hypotension, liver, and kidney has been observed. However, the researcher’s final interpretation of the study was striking. They concluded that N. sativa has not shown itself to be effective enough for decreasing blood pressure in sicks with hypertension. In a recent study, hydroalcoholic extract of N. sativa seed was assessed for its activity on the vasoreactivity of aortic rings to acetylcholine (Ach) in STZ-induced diabetic Wistar rats. The rats were administered with different concentrations of N. sativa (100, 200, and 400 mg/kg daily) for 6 weeks. It was found that treatment with N. sativa seed improved aorta’s of diabetic rats through its effect on vascular inflammation (Abbasnezhad et al. 2019).

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2.9  Antidiabetic Effects of N. sativa Seed Extracts Diabetes mellitus is known as one of the common metabolic disease worldwide and is a metabolic syndrome which is generally formed by high blood glucose concentration as a result of insulin deficiency. Many plants possess an antihyperglycemic character. Nigella sativa is one such plant. Abbasnezhad et al. (2015) studied possible antihyperglycemic characteristic of N. sativa seed hydroalcoholic extract on oxidative stress caused by induced diabetes. It has been observed that glucose level decreased in diabetic rats given seed extract. Hydroalcoholic extract of Nigella sativa had a possible antidiabetic characteristic and decreased oxidative stress in the hippocampus of diabetic rats. Nehar et  al. (2015) published another article that demonstrated Nigella sativa seed ethanolic extracts role as antidiabetic effects. At the end of the trials, a significant decrease in blood glucose levels and considerable improvement in glucose tolerance was reported. Researchers have also concluded that the higher doses of seed extracts were more effective than lower doses. The aim of another study related to antidiabetic research was to evaluate the hypoglycemic and hypolipidemic properties of hydroalcoholic seed extract in diabetic animals (Asaduzzaman et al. 2015). Diabetes mellitus induced by the treatment with streptozotocin and hyperlipidemic characteristic induced by treatment with a mixture including cholesterol and cholic acid. At the end of the experiment, serum glucose level was evaluated and it has been observed that hydroalcoholic seed extract has a potential hypoglycemic character. At the same time, antilipidemic effect was also observed. In the experiment, HDL-cholesterol levels showed an increase, while total cholesterol, LDL-cholesterol, and VLDL concentrations decreased. Another study related to antidiabetic researches has been conducted by Le et al. (2004). The research group investigated the effect of a four-week intragastric gavage with a petroleum ether extract of Nigella sativa seeds on blood glucose, insulin and lipids in the normal rats. At the end of the treatment, extract of Nigella sativa significantly reduced plasma triglycerides, increased HDL-cholesterol and showed anti-­ diabetic potential. Petroleum ether extract of Nigella sativa seeds exerted a beneficial action on serum lipids and insulin, and especially rendered liver cell intracellular signaling pathways. Authors suggested that Nigella sativa seeds extracts might be used in the treatment of type 2 diabetes. Alimohammadi et al. (2013) investigated antidiabetic characteristics of hydroalcoholic seed extract in diabetic rats. The experimental study was conducted to investigate the mechanism of the hypoglycaemic effect of N. sativa hydroalcoholic extract, with respect to hepatic gluconeogenesis. Diabetes was induced in all groups by injection of STZ (60  mg/kg). STZ-diabetic groups have been treated with a hydroalcoholic extract of N. sativa (5, 10, and 20  mg/kg). Treatments have been evaluated to assess its effect on fasting blood glucose, and body weight. The administration of N. sativa suppresses STZ-induced diabetes in the rat. It was suggested that the hydroalcoholic extract of N. sativa at low doses has a beneficial effect on fasting blood glucose level and ameliorative effect on the regeneration of pancreatic

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islets. According to histopathological examination the N. sativa (at low doses) partially recovered hepatic glycogen content and protected a great deal of the pancreatic islet cells. El-Rabey et al. (2017) analyzed the effect of N. sativa seed and propolis methanol extract on STZ-induced male rats. The hyperglycemia and oxidative stress resulting from hyperglycemia were significantly reduced with both treatments. Besides, they healed the adverse biochemical and histopathological changes of hyperglycemia.

2.10  Antimicrobial Effects of N. sativa Seed Extracts It is a known fact that N. sativa is used for alternative medicine as an antimicrobial agent. Several studies demonstrated that N. sativa seeds have antimicrobial effects against different pathogens, including bacteria, viruses, schistosome and fungus. It has been taught that N. sativa and its extracts could provide a valuable product for microbial diseases. In this section, we reviewed the studies that showed the antimicrobial effects of N. sativa seed extracts on various microorganisms in vivo and in vitro. 2.10.1  Antibacterial Effects of N. sativa Seed Extracts Pathogenic infections have become an important health problem all over the worlds. Novel antimicrobial sources to overcome this problem are needed. Based on pharmacological properties exhibited by N. sativa seeds, scientists demonstrated the ethanolic seed extracts efficacy against pathogenic bacteria that are resistant to antibiotics. It is a known fact that methicillin-resistant infections Staphylococcus aureus (MRSA) are difficult to treat because of their resistance to the commonly used anti-­ staphylococcal antibiotics. WHO and scientists have overemphasized that find some new antimicrobial agents or new approaches are urgently needed to overcome this problem. Laboratory research demonstrated that various extracts of N. sativa seed have potential antimicrobial activity against different bacterial strains. Hannan et al. (2008) investigated the antibacterial activity of ethanolic seed extracts against clinical isolate strains of MRSA. It has been observed that all tested strains of methicillin-­ resistant Staphylococcus aureus were sensitive to the ethanolic extract of N. sativa (4 mg/disc). In another antibacterial research that has been conducted by an animal experiment, it has been observed that the methanol and chloroform seed extracts have a dose-dependent antibacterial activity. Staphylococcus aureus or Escherichia coli have been injected into mice. The inhibitory effect of the methanol extract has been observed in mice infected with S. aureus at approximately 88%. At the end of the

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study, it has been observed that the seed extract effective against Gram-positive and Gram-negative bacteria (Hosseinzadeh et al. 2007). In another study, the antibacterial activity of the aqueous extract of N. sativa seed against several pathogenic bacteria has been evaluated (Hasan et al. 2013). Aqueous extract of N. sativa seed demonstrated less antibacterial effect compared to the methanol extract. Methanol extract at the concentration of 100 mg/mL has shown a considerable sensitivity towards all tested pathogenic bacteria. Methanol extracts of N. sativa exhibited a higher antibacterial effect compared with aqueous extracts. At a concentration of 100 mg/mL, the highest antibacterial activity had been observed in Streptococcus pyogene. The same antimicrobial activity has been measured in Klebseilla pneumonia, Pseudomonas aeruginosa and Proteus vulgaris. Aqueous seed extract at a concentration of 100  mg/mL, had a considerable effect against Pseudomonas aeruginosa and Streptococcus pyogenes. Also, the aqueous seed extract at a concentration of 50  mg/mL showed inhibition zone (10, 12, 12 and 11  mm) for Streptococcus pyogenes, Pseudomonas aeruginosa, Proteus vulgaris and Klebseilla pneumonia, respectively. 2.10.2  Antifungal Effects of N. sativa Seed Extracts There are few studies demonstrated the antifungal effects of N. sativa seed extracts. Bita et al. (2012) compared the antifungal activity of aqueous, methanol and chloroform extracts obtained from the N. sativa seeds with the effect of traditional antifungals. Candida albicans strains have been used as test microorganism. Methanol extracts of Nigella sativa exhibited the strongest antifungal effect followed by the chloroform extracts against the tested candida strains. It was observed the aqueous extracts did not show antifungal activity. In another study related to antifungal effects of N. sativa seed extracts, antidermatophyte feature of ether seed extract has been analyzed against several dermatophyte species (Trichophyton rubrum, Trichophyton interdigitale, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum canis). The ether extract of N. sativa showed inhibitory features against tested fungal strains. The results suggested that N. sativa seed extracts might be used as a source of antidermatophyte medicament (Aljabre et. 2005). 2.10.3  Antiparasitic Effects of N. sativa Seed Extracts A few studies have been also demonstrated that N. sativa seed extracts have an antiparasitic activity. The antiparasitic (anticestod) effect of N. sativa seeds extracts has been studied in children infected naturally with the respective cestod worms (Akhtar and Riffat 1991). The activities have been judged on the basis of percentage reductions in the fecal eggs per gram counts. It has observed that a single oral administration of 40 mg/kg of N. sativa reduced the percentage of fecal eggs per gram counts.

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Okeola et  al. (2011) researched on antiparasitic effects of Nigella sativa seeds extracts. Antimalarial activities of methanol seed extract in mice have been investigated. It has been observed that methanol seed extract leads to suppression of a kind of a parasitic organism (plasmodium yoelli nigeriensis). Therefore, methanol N. sativa seeds extract has been suggested to be more effective than the chemical compounds used for parasitic organism treatment.

3  Conclusion Nigella sativa, which has been used in alternative medicine with its traditional applications, has drawn attention in the world of science today. N. sativa provides an alternative traditional medical treatment. N. sativa has many pharmacological properties and scientific evidence provide more opportunities for people to benefit from these properties. Most of the studies to date, have focused on the essential oil, fix oil and thymoquinone of N. sativa plant. Studies on N. sativa seed extracts have been observed to be increased. Therefore, in this book section, scientific studies on N. sativa seed extracts have been compiled separately.

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Chehl, N., Chipitsyna, G., Gong, Q., Yeo, C. J., & Arafat, H. A. (2009). Anti-inflammatory effects of the Nigella sativa seed extract, thymoquinone, in pancreatic cancer cells. HPB, 11(5), 373–381. Dehkordi, F. R., & Kamkhah, A. F. (2008). Antihypertensive effect of Nigella sativa seed extract in patients with mild hypertension. Fundamental & Clinical Pharmacology, 22(4), 447–452. https://doi.org/10.1111/j.1472-8206.2008.00607.x. Dilshad, A., Abulkhair, O., Nemenqani, D., & Tamimi, W. (2012). Antiproliferative properties of methanolic extract of Nigella sativa against the MDA-MB-231 cancer cell line. Asian Pacific Journal of Cancer Prevention, 13(11), 5839–5842. Dirjomuljono, M., Kristyono, I., Tjandrawinata, R.  R., & Nofiarny, D. (2008). Symptomatic treatment of acute tonsillo-pharyngitis patients with a combination of Nigella sativa and Phyllanthus niruri extract. International Journal of Clinical Pharmacology and Therapeutics, 46(6), 295–306. El Rabey, H.  A., Al-Seeni, M.  N., & Bakhashwain, A.  S. (2017). The Antidiabetic activity of Nigella sativa and propolis on streptozotocin-induced diabetes and diabetic nephropathy in male rats. Evidence-Based Complementary and Alternative Medicine : ECAM, 2017, 5439645. https://doi.org/10.1155/2017/5439645. El-Naggar, T., Gómez-Serranillos, M. P., Palomino, O. M., Arce, C., & Carretero, M. E. (2010). Nigella sativa L. seed extract modulates the neurotransmitter amino acids release in cultured neurons in vitro. Journal of Biomedicine and Biotechnology, 2010. El-Naggar, T., Carretero, M. E., Arce, C., & Gómez-Serranillos, M. P. (2017). Methanol extract of Nigella sativa seed induces changes in the levels of neurotransmitter amino acids in male rat brain regions. Pharmaceutical Biology, 55(1), 1415–1422. Enayatfard, L., Mohebbati, R., Niazmand, S., Hosseini, M., & Shafei, M. N. (2019). The standardized extract of Nigella sativa and its major ingredient, thymoquinone, ameliorates angiotensin II-induced hypertension in rats. Journal of Basic and Clinical Physiology and Pharmacology, 30(1), 51–58. Ghannadi, A., Hajhashemi, V., & Jafarabadi, H. (2005). An investigation of the analgesic and anti-­ inflammatory effects of Nigella sativa seed polyphenols. Journal of Medicinal Food, 8(4), 488–493. Haas, M. J., Onstead-Haas, L., Naem, E., Arnold, A., Rohrbaugh, N., Flowers, M., & Mooradian, A. D. (2014). The effect of black seed (Nigella sativa) extract on FOXO3 expression in HepG2 cells. Phytotherapy Research, 28(6), 873–879. Hadjzadeh, M. A. R., Khoei, A., Hadjzadeh, Z., & Parizady, M. (2007). Ethanolic extract of Nigella sativa L. seeds on ethylene glycol-induced kidney calculi in rats. Urology Journal, 4(2), 86–90. Hannan, A., Saleem, S., Chaudhary, S., Barkaat, M., & Arshad, M. U. (2008). Anti bacterial activity of Nigella sativa against clinical isolates of methicillin resistant Staphylococcus aureus. Journal of Ayub Medical College, Abbottabad : JAMC, 20(3), 72–74. Hasan, N. A., Nawahwi, M. Z., & Malek, H. A. (2013). Antimicrobial activity of Nigella sativa seed extract. Sains Malaysiana, 42(2), 143–147. Hebi, M., Zeggwagh, N., Hajj, L., El Bouhali, B., & Eddouks, M. (2016). Cardiovascular effect of Nigella sativa L. aqueous extract in normal rats. Cardiovascular & Haematological DisordersDrug Targets., 16(1), 47–55(9). Hosseini, M., Mohammadpour, T., Karami, R., Rajaei, Z., Reza Sadeghnia, H., & Soukhtanloo, M. (2015). Effects of the hydro-alcoholic extract of Nigella sativa on scopolamine-induced spatial memory impairment in rats and its possible mechanism. Chinese Journal of Integrative Medicine, 21(6), 438–444. Hosseinzadeh, H., Fazly Bazzaz, B.  S., & Haghi, M.  M. (2007). Antibacterial activity of total extracts and essential oil of Nigella sativa L. seeds in mice. Pharmacology, 2, 429–435. Ikhsan, M., Hiedayati, N., Maeyama, K., & Nurwidya, F. (2018). Nigella sativa as an anti-­ inflammatory agent in asthma. BMC Research Notes, 11(1), 1–5. Kumara, S.  S. M., & Kwong Huat, B.  T. (2001). Extraction, isolation and characterisation of antitumor principle, αhederin, from the seeds of Nigella sativa. Planta Medica, 67(1), 29–32.

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Le, P. M., Benhaddou-Andaloussi, A., Elimadi, A., Settaf, A., Cherrah, Y., & Haddad, P. S. (2004). The petroleum ether extract of Nigella sativa exerts lipid-lowering and insulin-sensitizing actions in the rat. Journal of Ethnopharmacology, 94, 251–259. Mbarek, L. A., Mouse, H. A., Elabbadi, N., Bensalah, M., Gamouh, A., Aboufatima, R., Benharref, A., Chait, A., Kamal, M., Dalal, A., & Zyad, A. (2007). Anti-tumor properties of blackseed (Nigella sativa L.) extracts. Brazilian Journal of Medical and Biological Research, 40(6), 839–847. Meziti, A., Meziti, H., Boudiaf, K., & Benboubetra, M. H. B. (2012). Polyphenolic profile and antioxidant activities of Nigella sativa seed extracts in vitro and in vivo. World Academy of Science, 6(4), 26–32. Mohebbati, R., Shafei, M.  N., Soukhtanloo, M., Mohammadian Roshan, N., Khajavi Rad, A., Anaeigoudari, A., Hosseinian, S., Karimi, S., & Beheshti, F. (2016). Adriamycin-induced oxidative stress is prevented by mixed hydro-alcoholic extract of Nigella sativa and Curcuma longa in rat kidney. Avicenna Journal of Phytomedicine, 6(1), 86–94. Mohebbati, R., Shafei, M. N., Beheshti, F., Soukhtanloo, M., Roshan, N. M., Anaeigoudari, A., Parhizgar, S., Hosseinian, S., Khazdeir, M. R., & Rad, A. K. (2017a). Mixed hydroalcoholic extracts of Nigella sativa and Curcuma longa improves adriamycin-induced renal injury in rat. Saudi Journal of Kidney Diseases and Transplantation: An Official Publication of the Saudi Center for Organ Transplantation, Saudi Arabia, 28(6), 1270–1281. Mohebbati, R., Hosseini, M., Haghshenas, M., Nazariborun, A., & Beheshti, F. (2017b). The effects of Nigella sativa extract on renal tissue oxidative damage during neonatal and juvenile growth in propylthiouracil-induced hypothyroid rats. Endocrine Regulations, 51(2), 105–113. Nehar, S., Kauser, H., Rani, P., & Alam, I. (2015). Effects of Nigella sativa seed extract on insulin resistant non-insulin-dependent diabetic guinea pigs. American Journal of Ethnomedicine, 2(1), 58–67. Niazmand, S., Fereidouni, E., Mahmoudabady, M., & Mousavi, S.  M. (2014). Endothelium-­ independent vasorelaxant effects of hydroalcoholic extract from Nigella sativa seed in rat aorta: The roles of Ca2+ and K+ channels. BioMed Research International, 2014. Okeola, V. O., Adaramoye, O. A., Nneji, C. M., Falade, C. O., Farombi, E. O., & Ademowo, O. G. (2011). Antimalarial and antioxidant activities of methanolic extract of Nigella sativa seeds (black cumin) in mice infected with Plasmodium yoelli nigeriensis. Parasitology Research, 108, 1507–1512. Pranav, C. (2018). Effect of Nigella sativa seed extract on lipid and protein oxidation in raw ground pork during refrigerated storage. Nutrition & Food Science, 48(1), 2–15. https://doi. org/10.1108/NFS-02-2017-0031. Rizka, A., Setiati, S., Lydia, A., & Dewiasty, E. (2017). Effect of Nigella sativa seed extract for hypertension in elderly: A double-blind, randomized controlled trial. Acta Medica Indonesiana, 49(4), 307–313. Sen, A., Choudhuri, P., & Chatterjee, R. (2018). Influence of inorganic nutrient, organic nutrient and bio-fertilizer on growth, yield and quality of cumin black (Nigella sativa L.) in eastern Himalayan region of West Bengal. Journal of Pharmacognosy and Phytochemistry, 7(2), 2571–2575. Shin, M.-S., Ko, I.-G., Kim, S.-E., Kim, B.-K., Kim, T.-S., Lee, S.-H., et  al. (2013). Treadmill exercise ameliorates symptoms of methimazole-induced hypothyroidism through enhancing neurogenesis and suppressing apoptosis in the hippocampus of rat pups. International Journal of Developmental Neuroscience, 31(3), 214–223. Swamy, S. M. K., & Tan, B. K. H. (2001). Extraction, isolation and characterization of anti-tumor principle, alpha-Hederin, from the seeds of Nigella sativa. Planta Medica, 67, 29–32. Tubesha, Z., Iqbal, S., & Ismail, M. (2011). Effects of hydrolysis conditions on recovery of antioxidants from methanolic extracts of Nigella sativa seeds. Journal of Medicinal Plant Research, 5(22), 5393–5399. Wagner, M. S., Wajner, S. M., & Maia, A. L. (2008). Theroleofthyroidhormonein testicular development and function. Journal Endocrinology, 199(3), 351–365.

Index

A Accelerated solvent extraction (ASE), 110 Acetylcholine esterase enzyme, 372 Acid-catalyzed process, 398 Acid secretion, 166 Acne vulgaris, 118, 237 Actual evapotranspiration (AET), 15 Acute lymphoblastic leukemia (ALL), 215 Adenosine triphosphate (ATP), 284 Adriamycin, 525 ADR-induced nephropathy, 526 Aeromonas hydrophila, 310 Affinity chromatography, 62 Aging-related disorders, 75 Agroecology, 25 Air-water interface, 74 AKT signaling, 370 Alanine aminotransferase (ALT), 166 Alcohol, 484 Alcoholysis, 399 Alkaline catalyzed transesterification, 398, 399 Alkaloids, 3, 47, 52, 132, 138, 195 Allergic rhinitis, 238 α/β-Thionins, 60, 61 α-glucosidase enzymes, 162 α-hederin, 116, 365, 463 α-longipinene, 414 α-pinene, 411 α-thujene, 411, 414 α-tocopherol, 325, 342 Alzheimer’s disease, 468, 528 Amino acids, 48, 49, 192, 336, 486, 526 AMPK phosphorylation, 371 Amyloidogenic pathway, 372 Anaerobic human pathogenic bacteria, 160

Analgesic activity, 469 Analgesic effects, 158 Analgesic efficacy, 237 Androecium, 27 Anisidine, 496 Anti-bacterial action, NSO, 372–373 Antibacterial activity, 507 Antibacterial agent, 465 Antibacterial effects, 532, 533 Antibacterial protein, 62, 63 Antibiotic activity, 186 Antibiotic growth promoters, 255 Antibiotic-resistant bacteria, 160, 298 Anticancer activities, 40, 116, 159, 160 effect, 417, 462, 463, 511, 512, 529 TQ, 86, 87 Anticancer role, NSO, 365 antioxidant effects, 366, 367 cell signaling effect, 367–370 cytotoxic activity, 365 TQ, 365 Anticarcinogens, 68 Anticardiovascular effects, 529, 530 Anticholinesterase agents, 467 Anticlastogenic and antioxidants activities, 310 Anti-dandruff agents, 234 Antidiabetic activities, 39, 40, 68, 161, 162 Anti-diabetic effect, 115, 162, 465, 466, 508, 509 Anti-diabetic effect role, NSO AMPK phosphorylation, 371 DM, 371 hyperlipidemia, 371

© Springer Nature Switzerland AG 2021 M. F. Ramadan (ed.), Black cumin (Nigella sativa) seeds: Chemistry, Technology, Functionality, and Applications, Food Bioactive Ingredients, https://doi.org/10.1007/978-3-030-48798-0

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Index

540 Anti-diabetic effect role, NSO (cont.) insulin-degrading enzyme, 371 LDL-c level, 371 NADH and NAD+, 371, 372 sodium-glucose co-transporter, 371 Anti-dyspeptic agent, 166 Antifungal activity, 161 Antifungal effects, 508, 533 Anti-Helicobacter pylori effect, 166 Antihyperlipidemic activity, 164–166 Antihypertension effects, 162–164 Antihypertensive product, 530 Anti-inflammatory, 68, 237, 466, 467, 490, 510, 524, 525 activities, 39 cytokine, 309 effect, EO, 114, 158, 417 Anti-inflammatory role, NSO COX pathway, 370 DNA binding proteins, 370 inflammation, 370 5-lipoxygenase, 370 Antimicrobial activities, 40, 68, 160, 161, 490–492 Antimicrobial agents, 60, 182, 464, 465, 532 Antimicrobial chitosan, 446 Antimicrobial peptides (AMPs), 59–61 Antimicrobial potential, 61 Antimicrobial properties, 273 Antioxidant, 68, 197–199, 251, 257 activities, 40, 70, 116, 117, 156, 157, 427, 443 enzymes, 461 free radicals, 460 hepatonephrotoxicity, 462 hepatotoxicity, 461 N. sativa oil, 460 oxidative changes, 462 oxidative damage, 461 TQ, 461, 462 Antioxidant effects, 485, 487, 495, 496, 507, 509, 522–524 Antioxidant enzymes, 463 Antioxidative status, 283, 284 Antiparasitic effects, 469, 533, 534 Antiradical activity, 75 Antitumoral, 68 Antiviral activity, 508 Anti-virus, 161 Aquaculture, 298 Arachidonic acid, 258 Aroma-active compounds, 487, 488 Aroma compounds, 487

Aromatic plants, 2 Arrhythmia, 166 Artificial fertilizers, 404 Ascaris lumbricoides, 216 Asian Seabass diet, 301 Aspartate aminotransferase (AST), 166 Asthma, 12, 33, 132, 199 Atherosclerosis, 164 Atomic force microscopy, 61 Auto-oxidation, 445 Avenasterol, 195 Ayurveda, 32 Azotobacter chroococcum, 15 B Back pain, 199 Bacterial cell wall, 62, 63 Bacterial enzymatic activity, 306 Barbiturates, 527 Barbus grypus, 307 BCO food applications blended vegetable oils antiradical activity, 352 bioactive compounds, 351 conjugated diene and triene values, 353 CO oxidation, 352 ESR, 353 food product, 352 healthier traits, 351 inhibition, 352 natural antioxidant, 353 oxidative stability, 351, 354 PUFA, 352 radical scavenging activity, 351, 352 tocopherols/tocotrienols, 352, 353 volatile oxidation compounds, 354 cheese antibacterial activity, 354 moisture contents, 355 natural antimicrobial, 355 physicochemical properties, 355 sensory properties, 356 cookies functional and physical properties, 356 sensory analysis, 356 TQ content, 356 mayonnaise conventional, 358 O/W emulsion, 358 PV and conjugated diene, 358 meat and marine products chemical and sensory quality, 357

Index MUFA, 357 sensory analysis, 357 TAMB, 357 TBARS, 357 TVB-N, 357 BCSO, functional properties antibacterial features antimicrobial activity, 416 bioactive constituents, 415 carvacrol, 415 ciproflaxin effects, 415 disc infusion method, 415 Listeria strains, 416 maximum inhibition effect, 415 permeability cell membranes, 415 Staphylococcus strains, 415 TQ and longifolene, 416 zone inhibition effect, 415 antifungal features, 416 antioxidant features, 416 functional foods and nutraceuticals, 414 health-promoting effects, 417 BCSO production applied and ecological factors, 412 chemical composition/bioactive components chemotypes, 414 oxygenated monoterpenes, 414 oxygenated sesquiterpenes, 413 quercetin, 413 sesquiterpene hydrocarbon, 414 Sesquiterpenes, 414 terpenic hydrocarbon, 413 TQ and p-cymene, 413, 414 clevenger apparatus, 411 cultivation and agricultural conditions, 412 extraction methods, 412 HD, 411 molecular weight, 411 SCE, 411 steam distillation, 411 water salinity, 412 yield and composition, 411 Benzene, 84 Benzodiazepines, 527 Benzylisoquinoline alkaloids (BIAs), 138, 139 β-carotene bleaching test, 76 β-catenin, 369 Betamethasone, 239 β-sitosterol, 326, 341, 351 Bioactive components, 46, 192, 336, 351 Bioactive compounds, 32, 36, 39, 69, 156, 253, 337, 502, 504–506

541 Bioactive constituents, 300 Bioactive molecules, 510 Bioactive phenolic compound, 358 Bioactive phytochemicals, 459 Biochemical traits, 15 Biodegradable polymeric nanoparticles, 384 Biodiesel, 186 advantage, 391 biodegradable alternative fuel, 391 blends, 391, 393, 394 chemical formula, 390 diesel engines, 391 expensive, 395 long-chain alkyl esters, 390 National Biodiesel Board, 390 Nigella oil, 391 producing countries, 391 production process dilution, 397 micro-emulsification, 397 pyrolysis, 396 transesterification, 397–399 transformation, 396 reaction, 398 transport system, 396 Biofertilizer, 15 Biofuels analysis, 396 crop improvement programs, 403 definition, 390 engine development, 404 oxidized, 395 Biogenic silver NPs, 495 Biological activities TQ, 86, 87 Black cumin/black seed characterization, 410 primary farming, 410 Black cumin (Nigella sativa) applications, 117 aromatic plants, 2 BCSPs (see Black cumin seeds polysaccharides (BCSPs)) bioactive compounds, 3 bioactive phytochemicals, 3 biological impacts, 61 botanical classification, 154 botanical description, 192, 193 characterization, 69 chemical composition, 155, 194, 195 composition, 5 COVID-19, 2 cultivation, 5, 12, 13, 191

542 Black cumin (Nigella sativa) (cont.) drought stress, 13, 14 essential oil, 4 extraction, 69 fertilization, 14–16 food applications, 199–202 foodstuffs, 2 functionality, 5 genotypes, 12 germination, 16, 17 green synthesis (see Green synthesis) growth parameters, 16 health promoting activities (see Health promoting activities) history, 192, 193 ingredients, 3 irrigation regime, 17–19 market, 3, 4 medical plants, 2 metabolomics, 134–135 MS, 135–141 NMR (see Nuclear magnetic resonance (NMR)) nutritional profile, 192 nutritional value, 155 physical properties, 16 phytochemicals, 2, 4 production, 12, 192, 193 properties, 12 publications, 4 in PubMed database, 4 QC, 133–134 remedy, 12 salinity, 13, 14 Scopus database, 4, 5 seed extract, 100 seed oil, 3 soil characteristics, 16 therapeutic agent, 199 in therapeutic applications, 3 thionins (see Thionins) TQ, 3 traditional uses, 99 treatment, several diseases, 2 TRP-gene expression, 3 WHO, 2 Black cumin seed (BCS), 216 Black cumin seed oil (BCSO) chlorophyll and carotenoids, 327 fatty acid composition, 323–325 minerals, 336 NL, 321, 322 oil content, 320

Index oil functionality antimicrobial activity, 330 antioxidant activity, 329 oxidative stability, 329, 330 phenolic compounds, 326, 327 phospholipid composition, 323 physicochemical properties, 320, 321, 340 polar lipids, 322 sterols, 326, 327 tocochromanols, 325, 326 tocopherols, 326 volatile compounds, 328 Black cumin seeds polysaccharides (BCSPs), 75–76 antioxidant activities, 75–76 color, 72 extraction, 70 functional properties, 73–75 metabolism, 69 molecular weight distribution, 72 monosaccharide composition, 72 physicochemical characteristics, 71, 72 starch and protein, 70 structural properties, 72, 73 surface properties, 74 thermal properties, 72 Black seed, 2, 3, 24, 32, 46, 47, 132 flavoring substance, 482 source, 482 vitamins, 482 Black seed oil (BSO), 237, 468 Blending cold-pressed black seed oil, 338 Blood biochemical characters, 308 Blood glucose, 217 Blood hematology and variables, 299 Body weight gain (BWG), 269–271 Botanical classification, 154 Breast cancer, 86 Bronchitis, 33, 132, 199 Buckwheat seed (BWS), 216 C Campesterol, 195 Cancer, 75, 492, 493 Capsule-like structure, 192 Carbohydrate content, 214 Carbohydrate mechanism, 162 Carbohydrates, 76, 225 Cardioprotective effects, 115, 116, 162–164 Cardiovascular diseases (CVD), 75, 115, 162–164 Cardiovascular effects, 492

Index Carotenoids and chlorophyll content, 338, 343, 426 Carvacrol, 90, 415 Catalase (CAT), 301, 366 Catalytic transesterification method, 398 acid-catalyzed process, 398 alcoholysis, 399 alkaline catalyzed, 398 biodiesel production, 400 description, 397 glycerin, 397 lipase-catalyzed, 399 sodium methoxide, 400 triglycerides, 399 variables, 400 Ceftriaxone, 441 Cell hemoglobin concentration (MCHC), 307 Cell signaling effect β-catenin, 369, 370 Bcl-xL expression, 367 cancer cell proliferation, 369 cell activity, 368 DENA-induced hepatocellular carcinoma, 369 down-regulation XIAP, 369 E-cadherin expression, 370 expressions, 367 hepatocellular carcinoma, 368 LNCaP prostate cancer cells, 368 MAPK signaling pathway, 368 MCF-7/DOX, 367 MM, 369 MUC4, 368 NF-κB, 368 PANC-1, 370 PI3K/AKT signaling pathway, 369 TQ, 367 types, 367 XIAP, 368 Cell vacuoles, 60 Cell wall lignocellulose easy processing, 403 Chelating power, 76 Chemical analysis, 85 Chemical composition, 155, 194, 195 Chemically-induced toxicities, 417 Chemical synthetic additives, 437 Chemometrics, 145, 146 Chemotherapeutic agents, 159, 267 Chest congestion, 199 Chlorophyll and carotenoid, 342 Chromatographic and identification techniques GC-MS, 111 GC-MS-O, 111

543 HPLC, 111 HPTLC, 111 NMR, 112 TLC, 110 Chromatographic fingerprints, 137 Chromatography analysis, 85, 487 Chromium Cr (VI) toxicity, 310 Chronic disease, 237 Chronic headache, 2, 199 Chronic illness, 166 Chronic inflammatory disorder, 238 Ciprofloxacin, 441, 527 cis and trans-4-methoxythujane, 411 Cisplatin, 86 c-Jun NH (2)-terminal kinase (JNK), 368 Classical grinding (CLG), 426 Clevenger apparatus, 411 Clinical efficiency, 165 Cohen’s Kappa analysis, 145 Cold press extraction method, 197, 320, 337, 364 Cold-pressed oil, 427 Colon cancer, 116 Common names, 24, 25 Composition, 47, 68 Contingency matrix, 145 Conventional medicine, 492 Conventional methods, 424 Cookies, 356 Cooled acetone, 61 Corn oil (CO), 338, 352 Coronavirus disease (COVID-19), 2 Cosmetic industry, 195 Cosmetic products cosmeceutical preparations, 232, 233 herbal cosmetics, 233, 234 natural beauty care ingredient, 236–240 Nigella sativa, 234, 235 Cosmetics, 227 acne vulgaris, 118 hair loss, 118 skin infections, 118 skin pigmentation, 119 sun protection, 119 tooth caring, 119 wound healing, 119 Cough, 199 Crude oil, 84 Crude protein (CP), 248, 273 Crunchy texture, 336 Culinary uses, 113 Cultivation methods, 13, 155 Cultured fish, 298, 311

Index

544 Cuminaldehyde, 212 Curcuma longa, 161 Curries, 32 Cyclin-dependent kinase-4 (CDK-4), 367 Cyclooxygenase (COX), 158 Cyclophosphamide-induced cardiotoxicity, 115 Cymene, 212 Cysteine-rich peptides, 60 Cytokine-mediated immune responses, 525 Cytoprotective effects, 166 Cytotoxic effects, 444 D Daily feed intake (DFI), 271 Daily weight gain (DWG), 248 Danio rerio, 311 DA roasting, 339 Defensins, 61 δ-tocopherol, 342 DENA-induced hepatocellular carcinoma, 369 Deproteinization method, 72 Derivative thermograms analysis (DTG), 428 Derivatization/methylation, GLC, 402 Dermatol Life Quality Index (DLQI), 239 Diabetes, 12, 132, 154, 161–164, 199, 508, 531 Diabetes mellitus (DM), 115, 371 Diabetic nephropathy (DN), 115 Diabetic wounds, 493 Diacylglycerols (DAGs), 321 Diastolic blood pressure, 163 Dietary supplementation, 154, 250, 252, 258, 275, 279, 281, 283, 285–287 Diethylnitrosamine (DENA), 366 Differential scanning calorimetry (DSC), 427 Digalactosyldiacylglycerol, 322 Digestible crude protein (DCP), 248 Dihydrothymoquinone, 87, 363 Dilution, 397 Dimethyl ether (DME) extraction, 196, 197 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 329 2,2-Diphenyl-1-picrylhydrazil assay (DPPH), 40 Disc infusion method, 415 Disease-preventing effects, 132 Dithymoquinone (DTQ), 3, 101, 192, 194 Diuretic and antihypertensive activity, 100 Dizziness, 132, 199 DNA damage, 310 1 D-NMR metabolites fingerprinting, 141

D-NMR metabolites fingerprinting, 142 Domiati cheese, 354 Doxorubicin, 86 Doxorubicin-induced cardiotoxicity, 116 Doxorubicin-resistant human breast cancer (MCF-7/DOX), 367 DPPH radical scavenging test, 443 Drainable lysimeter, 18, 19 Drought stress, 12–14 Dry air (DA), 339 Dry eyes, 117 Drying technology, 382 Dry matter (DM), 248 Dry matter intake (DMI), 248 Dyslipidemia, 164 Dysmenorrheal, 199 2

E Economic efficiency, 225, 249 Eczema, 132 Edman degradation method, 61 Egyptian NSEO, 442 Electrical mobility, 73 Electromagnetic waves, 339 Electron donors, 76 Electron spin resonance (ESR), 353 Electrospray ionisation (ESI) mass spectrometry, 135, 137 Emulsifying activity index (EAI), 74 Emulsion stability index (ESI), 74 Encapsulation, NS crude oil biodegradable polymeric nanoparticles, 384 drying conditions, 383 drying technique, 383 maltodextrin, 383 microencapsulated oil, 383 phospholipid vesicles, 384 Encapsulation, NS volatile oil expression/solvent extraction, 384 indomethacin, 384 micellar encapsulation, 384, 385 microemulsion, 384 nanoemulsion-based encapsulation, 385 surfactant micelles, 384 TQ, 384 Endothelial dysfunction, 116 Enterobacteriaceae, 307 Enzymatic oxidation, 444 Epidermal growth factor receptor (EGFR), 369 Epidermis and inflammatory cells, 237

Index Epilepsy, 493, 527 Epileptic syndromes, 493 Epithelial to mesenchymal transition (EMT), 370 Escherichia coli, 441, 532 Essential fatty acids, 3, 238, 350 Essential oil (EO), 3, 4, 13, 14, 17, 51, 52, 132, 192, 195, 382 agricultural practice, 101 Alzheimer’s disease, 468 analgesic activity, 469, 470 animals, 470 anti-cancer activity, 116, 462, 463 antidiabetic activity, 465, 466 anti-diabetic effect, 115 anti-inflammatory activity, 466, 467 anti-inflammatory effect, 114 antimicrobial activities, 470 antimicrobial agents, 464, 465 antineurodegenerative effects, 467 anti-oxidant activity, 116, 117 antioxidant effects, 460–462 antiparasitic, 469 ASE, 110 bioactive phytochemicals, 459 biological activities, 460 cardio-protective effects, 115, 116 chemical composition, 102–106 chromatographic and identification techniques, 110–112 composition, 101 and cosmetics, 117–119 culinary uses, 113 dry eyes, 117 DTQ, 101 egg production, 471 fertility-enhancing activity, 469 food flavor, 113 growth performance, 471 hepatoprotective, 465 MAE, 108, 109 nano-formulation, 112, 113 oxytetracycline-induced hepatorenal toxicity, 470 pathogens, 471 pharmaceutical activities, 460 pharmacological activities, 100 pharmacological effects, 113 protein, 470 pyrazine, 101 quinones, 101 SFE, 106, 109, 110 SPME, 108 TFAs, 101

545 TQ, 101 traditional extraction methods, 106 traditional volatile oil extraction methods, 107, 108 Ethanolic extract, 483 Ether extract (EE), 273 Ethyl acetate extract, 491 Experimental dry eye (EDE), 117 Extraction method, 155 chlorophyll and carotenoid, 342 microwave/soxhlet, 341 n-hexane and methanol/chloroform, 341 phenolics and carotenoids, 342 polysaccharides, 69, 70 predominant fatty acids, 341 PSs, 341 SFAs, 341 sterol composition, 341 tocols, 341 tocopherol, 342 γ-tocopherol, 342 F FAME quantification, 402, 403 Fat constituents, 393 Fat-soluble vitamins, 48 Fatty acid methyl ester, 186 Fatty acids, 17, 50, 51, 155, 192, 338, 504 Febrile neutropenia (FN), 159 Feed conversion ratio (FCR), 301 Feed-to-gain ratio (FCR), 272 Fenugreek, 72 Fermented milk and cheeses, 354 Ferric reducing ability, 427 Ferric reducing antioxidant power (FRAP) activity, 276 Fertility, 168, 169, 469, 494 Fertilization, 14–16 Fever, 132 Fibres, 194 Final body weight (FBW), 248 Fingerprinting techniques, 133 Fish (Barbus grypus), 357 Fish feed derivatives ameliorating agent, 309, 310 blood biochemical characters, 308 blood picture, 307, 308 growth promoting activities, 301, 306 immune response improvement, 308, 309 meat quality characteristics, 306, 307 N. sativa impact, 301–305 protection against fish pathogens, 310, 311

Index

546 Fish immune response, 308 Fish immunostimulants, 309 Fixed oil, 5 Flame ionization detector (FID), 322, 402 Flavonoids, 3 Flavoring agent, 156 Flaxseed kernel, 72 Flax seed oil (FO), 352 Flowers, 26 Fluorescence analysis, 33, 38 Fluorescent spectroscopy, 61 Foaming capacity (FC), 75 Foam stability (FS), 75 Foliar epidermal anatomy, 33, 36, 37 Folk medicine, 24 Follicle-stimulating hormone (FSH), 168 Food additive, 434 Food applications, 199–202 Food-borne pathogens, 354 Food flavor, 113, 117 Food industry, 336 Food oxidation, 437 Food safety, 145, 434 Fourier-transformed infrared spectroscopy (FTIR), 72, 73 Free fatty acids (FFA), 321, 342, 426 Free radicals, 460 Freeze-drying, 445 Fruits, 27, 46 FTIR spectroscopy, 142 Full factorial design (FFD), 424 Functional foods, 201 Functional properties, 5 BCSPs, 73–75 Functional properties, seed extract anisidine, 496 antimicrobial activity, 490–492 antioxidant effect, 496 antitoxic properties, 489 BHT, 496 cancer, 492, 493 cardiovascular effects, 492 fertility, 494 food, 496 health effects, 488 hematologic effect, 494 hepatoprotective properties, 489, 490 livestock producers, 496 metal NPs, 495, 496 neurological effect, 493 serologic effect, 494 sexuality, 494 skin wound healing, 493

TG, 493, 494 treatment, 488 Fungal species, 160 Fungi, 490 G Galactinol, 69 Galactolipids, 322 Galvinoxyl radicals, 329 γ- and δ- tocopherols, 352 Gamma-knife, 86 γ-terpinene, 414 γ-thionins, 60, 61 γ-tocopherol, 325, 351, 353 Gas chromatography (GC), 82–85, 133, 134, 322 Gas chromatography mass spectrometry (GC-MS), 111, 139–141, 435 Gas chromatography-mass spectrometry-­ olfactometry (GC-MS-O), 111 Gasoline, 391 Gastric mucosal glutathione (GSH), 117 Gastric ulcers, 69, 158, 166, 167 Gastrointestinal disturbances, 12, 132 Gastrointestinal (GI) motility, 162 Gastro-protective activities, 41 Gastro-protective effects, 166, 167 GATA transcription factors, 370 G × E interaction, 13 Genotoxicity, 87 Geographical location, 82, 83 Geographic distribution, 155 Germination, 12, 16, 17 Gibberellic acid (GA3), 14, 16 GL subclasses, 322 Glucocerebroside, 322 Gluconeogenesis, 371 Glucose, 256 Glucotoxicity, 115 Glutamine amino acid, 485 Glutathione depletion, 87 Glutathione peroxidase (GPx), 301, 366 Glutathione reductase (GR), 366 Glutathione-S-transferase (GST), 366 Glycemic control, 165 Glycolipids (GL), 50, 322 Gold nanoparticles (AuNPs), 183, 184 “Good Health and Well-Being”, 2 Gram-negative and gram-positive bacteria, 160 Gram-negative bacteria cell wall, 62 Gram-positive bacteria cell wall, 62

Index Gram-positive pathogenic bacterial strains, 449 Graphene oxide (GO), 182, 183 Green chemistry, 183, 184 Greenhouse gas emissions, 403 Green-nanotechnology, 495 Green synthesis AgNPs, 181 AuNPs, 183, 184 biodiesel, 186 iron corrosion inhibitor, 186, 187 Pt NPs, 182 RGO, 182, 183 ZnONPs, 184 Growth performance, 301, 306 Growth promoters, 246 Gut-associated lymphatic order, 269 Gynecological disorders, 12 Gynoecium, 27, 192 Gypsiferous soils, 18 H Hair loss, 118 Hand Eczema Severity Index (HECSI), 239 HbA1c, 217 Headspace-gas chromatography-mass spectrometry (HS-GC-MS), 140 Headspace/solid phase microextraction-gas chromatography/mass spectrometry (HS/SPME-GC/MS), 354 Health authorities, 133 Health benefits, 507, 513 Health-promoting activities analgesic effects, 158 anti-cancer, 159, 160 antidiabetic, 161, 162 antihyperlipidemic, 164–166 anti-inflammatory, 158 antimicrobial, 160, 161 antioxidant, 156, 157 cardioprotective and antihypertension effects, 162–164 fertility, 168 gastro-protective effects, 166, 167 neuro-protective effects, 167 Nigella sativa seeds, 156, 157 toxicological properties, 168 Health promoting activities, seed extracts antibacterial, 532, 533 anticancer effects, 529 anticardiovascular effects, 529, 530 antidiabetic effects, 531, 532 antifungal, 533

547 anti-inflammatory, 524, 525 antimicrobial agent pathogens, 532 antioxidant effects, 522–524 antiparasitic, 533, 534 neuroprotective, 525–528 neurotoxicity, 528 respiratory, 525 Health-promoting agent, 460 Heart diseases, 164 Heavy metals toxicity, 309 Hematocrit concentration, 307 Hematological characteristics, 301 Hematological factors, 494 Hematological variables, 307 Hemiplegia, 199 Hemoglobin (Hb), 307 Heparin, 62 Hepatitis C (HPC), 160 Hepatitis C virus (HCV), 508 Hepatocarcinogenesis, 160 Hepatocellular carcinoma (HCC), 160 Hepato-protective activities, 41, 465 Hepatoprotective properties, 489, 490 Herbal cosmetics, 233, 234 Herbal medicines, 23, 458 Herbal plant oils, 468 Herbal products, 513 Herb are black caraway seeds, 32 Hexanal, 343 High blood pressure, 162 High-density lipoprotein cholesterol (HDL-C), 163, 280 High-energy methods, 445 High linoleic corn oil (HLCO), 353 High performance liquid chromatography coupled to UV sperctrophotometry (HPLC-UV), 133, 137 High performance liquid chromatography (HPLC), 72, 85, 111, 137, 141 High performance thin layer chromatography (HPTLC), 85, 111, 133 High-pressure homogenisation, 112 High-value compounds, 504 Hind limb ischemia, 157 Histomorphology, 282 Histopathological analysis, 162 H9N2 avian influenza virus, 161 1 H NMR spectra, 141 HOMA-IR, 217 Hot water extraction, 70 HS-SPME method, 145, 328 Human fibrosarcoma cell line (HT1080) in vitro, 366

Index

548 Human telomerase reverse transcriptase (hTERT), 367 Hydroalcoholic extraction method, 483 Hydroalcoholic seed extract, 490, 526, 527 Hydrodistillated-black cumin essential oil, 414 Hydrodistillation (HD), 85, 106, 410–412, 414, 424 Hydroethanolic seed extract, 528 Hydro-/steam-distillation, 84 Hypercholesterolemia, 164, 217, 434 Hypercholesterolemic effect, 116 Hypercholesterolemic male white (HC) rabbits, 163 Hypercholesterolemic subjects, 163 Hyperglycemia, 115, 434, 532 Hyperhomocysteinemia (HHcy), 157 Hyperlipidaemic albino rats, 162 Hyperlipidemia, 164, 217, 218, 371 Hypersensitivity, 166 Hypertension, 12, 132, 163, 530 Hypolipidemic effects, 165 Hypothyroidism, 527 I Ictalurus punctatus, 311 Imiquimod application, 238 Immune-modulatory properties, 256 Immune response, 285, 286, 301 Immune systems, 59 Immunity, 301 Immunoglobulin, 308 Immunomodulators, 68 Immuno-modulatory activities, 510, 511 Immunostimulant, 310 Impotence, 166 Infectious bursal disease (IBDV), 285 Infectious Laryngotrachietis Virus (ILTV), 161 Inflammation, 12, 132, 370 Inflated capsule, 46 Influenza virus (H9N2), 160 Innate immune system, 309 Interleukins (IL), 309 Intestinal morphology, 282 In vivo and in vitro researches, 366 Iranian N. sativa seeds, 424 Iron corrosion inhibitor, 186, 187 Irradiation roasting (IR), 339 Irrigation regime, 17–19 IR roasting, 339 IRS/AKT/GSK-3β pathway, 372 Ischemia and reperfusion (I/R), 157

J Japanese quail, 227 K Kaempferol-3-O-sophorotrioside-7-Orhamnoside, 143 Kidney diseases, 526 Kinase signaling pathways, 463 Kinetin (KIN) spray, 15, 16 L Lactobacillus, 282 Laryngotrachietis virus, 160 L-asparaginase, 215 LC-ESI-Q-TOF-MS/MS, 137 LD50, 88 Leaf arrangement, 25 Limonene, 411 Linoleic acid, 50, 194, 257, 267, 435 Lipase-catalyzed transesterification process, 399 Lipid-lowering effects, 165 Lipid oxidation, 3, 76, 252, 277, 336, 352, 357, 443, 449 Lipids, 50, 51, 213 Lipid-soluble bioactive compounds, 428 Lipolytic enzymes, 340, 342 Lipopolysaccharide (LPS), 158, 489 Liposomes, 240 Lipoxygenase (LO), 158 Liquid chromatography (LC), 61, 134 Liquid chromatography-mass spectrometry (LS-MS), 135, 137 Liquid chromatography-multiple reaction monitoring (LC-MRM), 137 Listeria monocytogenes, 330 Listeria monocytogenes strains, 416 Live body weight (LBW), 269–271 Liver damage, 87 Livestock producers, 496 Low-density lipoprotein (LDL), 164, 165, 530 Low-density lipoprotein cholesterol (LDL-C), 163 LPS-induced pro-inflammatory cytokine, 368 Luteinizing hormone (LH) levels, 168 Lymphocyte proliferation, 309 Lysozyme, 308

Index M Macromolecules, 69, 73 MAE/HD and steam distillation bioactive compounds, 424 CLG essential oils, 426 fluctuation, 425 GC-MS, 425 microwave irradiation, 425 monoterpene hydrocarbons, 425 oxygenated monoterpene content, 425 p-cymene/TQ chemotype, 425 sesquiterpenes hydrocarbons, 425 Maillard reaction, 339 Malaria, 492 Malondialdehyde (MDA), 88, 115, 217, 276 Maltodextrin, 383 MAPK signaling pathway, 368–369 Mass analyser (MS), 134 Mass spectrometry (MS), 72 GC-MS, 139–141 LC-MS, 135, 137 UPLC-MS, 137–139 Mass spectrophotometer detector (GC-MS), 423 MDA-MB-468 breast cancer cells, 369 Mean arterial pressure, 163 Mean corpuscular hemoglobin (MCH), 307 Mean corpuscular volume (MCV), 307 Mean droplet diameter (MDD), 112 Meat quality, 251–253, 301 Medical plants, 2, 154 Medical purposes ailments treatments, 422 antioxidant activities, 422 beneficial effects, 422 choleretic and uricosuric activities, 422 history, 422 natural remedy, 422 notable pharmacological properties, 422 Medicament, 68 Medicinal plants, 32, 169, 308, 458, 467, 502 Melanthin, 53 Metabolic disorders, 218 Metabolites fingerprinting 1 D-NMR, 141 2 D-NMR, 142 Metabolomics, 134–136 Metal nanoparticle (NPs), 495, 496 Methane emissions, 247, 258 Methanol extract, 485, 492, 523, 533 Methicillin-resistant infections Staphylococcus aureus (MRSA), 465, 491, 532 Methyl esters, 391 Methyl jasmonate (MeJA), 138

549 Methyl parathion (MP), 490 Micellar encapsulation, 384, 385 Micro and macroscopic characterization fluorescence analysis and solubility test, 33, 38 foliar epidermal anatomy, 33, 36, 37 morphological characters, 33–35 palynology, 33, 35 physicochemical parameters, 36, 39 trichomes, 33 Microbial spoilage, 306, 437 Micro-electrical mechanical systems (MEMS), 181 Micro-emulsification, 397 Microemulsion, 90, 384 Microencapsulation definition, 382 drying technology, 382 N. sativa crude oil, 383 spray drying, 383, 385 Microfluidisation, 112 Microwave-assisted extraction (MAE), 70, 106, 108, 109, 198, 327, 338, 424 Microwave irradiation, 425 Microwave pre-treatment, 343 Microwave radiation, 339 Microwave roasting, 101 Microwave steam distillation (MSD), 425 Middle Eastern Mediterranean region, 336 Migraine, 199 Minerals, 47, 48, 132, 156, 194, 504 Minimal bactericidal concentration (MBC), 160 Minimal inhibitory concentration (MIC), 160, 492 MnO2/BC, 185, 186 Modern and green extraction method, 337 Moisture content, 339 Molecular pathways, 366 Molecular weight distribution, 72 Monoacylglycerols (MAGs), 321 Monosaccharides, 72, 435 Monoterpene hydrocarbons, 425 Monoterpenes, 3 Monounsaturated fatty acids (MUFA), 17, 256, 267, 323, 341, 357 Morphology, 33–35, 46 androecium, 27 flowers, 26 fruit, 27 gynoecium, 27 leaves, 25 root, 25 seeds, 28 stem, 25

550 Mucilage, 336 Mucin 4 (MUC4), 368 Mugil cephalus, 301 Multidrug resistance, 160 Multifunctional additives, 322 Multiple inflammatory diseases, 370 Multiple myeloma (MM), 369 Multivariate data analysis PCA, 143–145 phytomedicines QC research, 143 PLS, 144 PLS-DA, 144, 145 VIP analysis, 144 Myeloperoxidase (MPO), 309 N Nanocomposite MnO2/BC, 185, 186 Nanoemulsion-based encapsulation, 385 Nanoemulsion-based formulation, 112 Nanoemulsion-based system, 385 Nanoencapsulation, 385 nanoemulsion-based encapsulation, 385 nanotechnology, 382 N. sativa crude oil, 384 nutraceuticals, 382 solid-lipid, 385 TQ, 384 volatile oil formulation, 384 Nano-formulation, N. sativa volatile oil, 112, 113 Nano-medical field, 182 Nanoparticles (NPs) AgNPs, 181 AuNPs, 183, 184 poly (ε-caprolactone), 184, 185 Pt NPs, 182 ZnONPs, 184 Nanoprecipitation, 237, 445, 446 Nanostructured lipid carriers, 87 Nanotechnology, 90 Nasal mucositis, 239 National Biodiesel Board (USA), 390 Natural additives, 306 Natural antioxidants, 336 Natural beauty care ingredient, 236–240 Natural food additives, 434 Nephroprotective activities, 41 Nephrotic syndrome, 525 Net dietary protein energy percent (NDPE), 225 Net protein utilization (NPU), 225 Neurodegenerative diseases, 467

Index Neurodegenerative disorders, 167 Neurological effect, 493 Neuroprotective activities, 41 Neuroprotective effects, 167, 525–528 Neuroprotective function, NSO, 372 Neurotoxicity, 528 Neutral lipids (NL), 321, 322 Newcastle disease virus (NDV), 285 NF-κB signaling pathway, 368 n-Hexane extraction, 108, 341 Nigella herbs and spices, 421 Nigella sativa allopathic medicines, 211 antioxidative properties, 211 applications antiatherogenic effects, 215 anticholesterolemic effect, 215 avilamycin, 217 biochemical parameters, 216 butter and semi-skimmed flour, 214 efforts, 215 egg production, 216 feed conversion, 217 flavouring agent, 214 functional foods, 214 hypoglycaemic agents, 217 inflammatory and auto-immune disorders, 218 insulin resistance and β-cell function indicators, 217 layers and cholesterol content, 216 medicinal ingredient, 214 powder and oil, 214 prevention of disease, 218 protection mechanism, 215 serum concentration, 216 thyroid functions, 215 T-lymphocytes, 216 bioactivity, 427 biological activity, 212 biological properties, 211 compositional analysis, 212–214 diuretic and antihypertensive, 211 flowering plant, 211 medicinal seeds, 210 morphology, 422 principles, 211 protective effects, 299 Ranunculaceae family, 211 seed extract (see Seed extracts) taxonomic classification, 422 Nigella sativa essential oil (NSEO) analysis, 423

551

Index biological properties, 437 (see also Black cumin seed oil (BCSO)) chemical composition, 425 description and chemical composition, 435 functional benefits, 434 functional properties, 434 monoterpenes, 423 Nigella sativa oil (NSO), 444, 445 active constituents, 363 antidiabetic and neuroprotective effects, 363 complex mixture, 363 p-cymene, 363 Nigella sativa seeds (NSS), 310 Nigella sativa seeds essential oil (NSSEO), 183–185 Nigellone (dithymoquinone), 18 Nile tilapia, 301 Nitrogen (N), 13 Non-alcoholic fatty liver disease (NAFLD), 164, 166 Non-antibiotic feed supplementations, 268 Non-enzymatic oxidation, 445 Non-essential amino acids, 48, 156 Non-hydrolyzed seed extracts, 523 Non-specific cellular response, 309 Non-specific defense mechanisms, 308 Non-thermal pre-treatments, 344 4-nonylphenol-induced haematotoxicity, 310 Novel edible oils, 350 Novel foods, 195 N. sativa essential oil (NSEO), 112 N. sativa essential oil nanoemulsion (NSEO-NE), 112, 113 N. sativa seed polyphenols (NSP), 524 NS biodiesel advantages ASTM standard, 393 best lubrication, 393 conventional diesel, 393 cost-effectiveness, 394 EN standard values, 394 flash point, 393 less toxic pollutant/greenhouse gas, 391 low emission, 391 moderate prices, 393 oil content, 393, 394 renewable and clean-burning fuel, 391 smoke point, 394 NS biodiesel challenges anthropogenic carbon dioxide emissions, 395 demands, 394 disadvantage, 395 ethical concerns, 396 initiatives and political decisions, 395 non-edible feedstock, 395

non-OECD countries, 395 N. sativa oil consideration, 396 replacing fossil fuel oil, 395 NS biological activities, 362 NS crude oil extraction, 382 fatty acids, 382 herbal nutraceuticals, 382 nutraceuticals, 383 nutritive and health maintaining properties, 383 volatile oil fraction, 382, 385 NSEO encapsulation homogenization, 445 laboratory scale, 445 microcapsules, 449 nanoemulsion, 446 novel food products, 446 RAM, 446 systems, 445 TAMC, 446 volatile oil fraction, 446 volatilization, 445 NSEO extraction MAE/HD and steam distillation effects, 424–426 screw and cold pressed effects, 426–428 SFE and solvent effects, 423, 424 temperature and pressure effects, 424 NSEO food applications action mechanism, 437 advantageous properties antibacterial activity, 441, 442 antioxidant activity, 443 microbial growth and delay oxidation, 437 nutritional value, 443, 444 beauty and skin diseases, 437 edible food preservative and antioxidant, 437 food additives, 434 functional food products, 437 potential therapeutics, 437 TQ, 437 NS genetic resources, 403 NSO active ingredients, 365 NSO stability, 444, 445 NsW1, 61 NsW2, 61 Nuclear magnetic resonance (NMR), 72, 73, 112 1 D-NMR, 141 2 D-NMR, 142

552 Nutraceuticals, 69, 135, 382, 383Nigella sativa applications, 227 characterization, 225, 226 commercial cultivation, 224 Mediterranean regions, 224 Middle East and Asia, 224 nutrients, 225 Ranunculaceae family, 224 seed oil, 224 treatment of diseases, 224 See also Nutraceuticals market, 4 Nutrient digestibility, 249, 258 Nutrient-rich herbs, 24, 154, 192 Nutrients, 154 Nutrient utilization, 273, 274 Nutrition, 50, 68 Nutritional composition, 336 O Obesity, 162, 199 Oil blending, 337, 338 Oil components, 462 Oil composition, 336 Oil extraction method, 84, 85, 145, 196 blending, 338 efficiency, 339 nutritional and health properties, 337 physicochemical properties, 336, 342, 343 roasting, 342 SFE, 337 thermal pre-treatments, 339 thermal treatments, 337 ultrasound, 340 yields, 337–339 Oil-in-water (O/W), 358 Oil quality properties, 343 Oil retention capacity (OHC), 74 Oilseeds, 68 Olfactory epithelium (OE), 167 Oncoproteins (c-Myc), 367 Organic matter (OM), 248 Orthogonal projection to latent structures-­ discriminate analysis (OPLS), 145 Oxidation, 444 Oxidative stability, 329, 330, 338, 351 Oxidative stability index (OSI), 329 Oxidative stability test, 352 Oxidative stress, 87, 164, 465, 490, 525, 532 P Packed cell volume (PCV), 307 Palynology, 33, 35

Index Panacea, 99 Pancreatic carcinoma cell line (PANC-1), 370 Pancreatic ductal adenocarcinoma (PDA), 529 Paralysis, 199 Partial least square (PLS), 144 Pathogenic bacteria, 355 Pathogenic infections, 532 Pathogenic microorganisms, 268 p-cymene/TQ chemotype, 155, 365, 411, 414, 425, 435 PER antioxidants, 225 Percolation-assisted extraction method, 485 Peroxide value (PV), 352, 354, 356, 358 Pharmaceutical industry, 133, 195 Pharmacological activities, 132, 199–201 Pharmacological effects, 522 Pharmacological impacts, 310 Pharmacological potentials anticancer activities, 40 antidiabetic activities, 39, 40 anti-inflammatory activities, 39 antimicrobial activities, 40 antioxidant activities, 40 gastro-protective activities, 41 hepato-protective activities, 41 nephroprotective activities, 41 neuroprotective activities, 41 Pharmacological properties, 460 Pharmacological trials, 434 Pharmacology, 156 Phase inversion, 112 Phenolic compounds, 326, 327, 427, 485, 486, 522 Phenolics, 53, 59 Phenolics and carotenoids, 342 Phenolic substances, 441 Phenylpropanes, 411 Phosphatidylcholine (PC), 322 Phosphatidylethanolamine (PE), 322 Phosphoenolpyruvate carboxylase, 14 Phospholipids (PL), 50, 322 Phosphorus (P) fertilization, 15 Phosphorylated glycogen synthase kinase 3β (p-GSK-3β), 369 Physical properties TQ, 88, 89 Physicochemical characteristics BCSPs, 71, 72 Physicochemical parameters, 36, 39 Phyto-additives, 268, 277 Phytobiotics, 299, 310 Phytochemical analyses, 36, 194 Phytochemical composition, 133, 135 Phytochemical profile, 154, 195 Phytochemicals, 2, 4, 449

Index Phytogenic feed supplementations, 268, 269 Phytosterols (PSs), 195, 341 PI3K/AKT signaling pathway, 369 Plant-based extracts, 238 Plant defensins, 60 Plant growth characters (PGC), 13 Plant-mediated nanoparticles, 181 Plant peptides, 60 Plant phenolics, 252 Plasma lipid concentration, 162 Platelets (PL), 307 Platinum nanoparticles (Pt NPs), 182 Poly (ε-caprolactone), 184, 185, 384 Polyphenol-rich fraction, 240 Polysaccharides antioxidant activities, 75–76 application, 69 and BCSPs (see Black cumin seeds polysaccharides (BCSPs)) classification, 68 composition, 68, 69 extraction, 69, 70 functional properties, 69 nutraceuticals, 69 polyfunctional, 68 structure, 68, 69, 72 Polyunsaturated fatty acids (PUFA), 17, 267, 323, 341, 352, 357 Potassium ferricyanide reduction method, 76 Poultry feed active materials, 267 antioxidant properties of meat, 276, 277 antioxidant status, 283, 284 black cumin seeds, 267 blood biochemistry, 279–281 carcass traits, 274, 275 challenges and stress factors, 267 chemical composition of meat, 276, 277 chemotherapeutic agents, 267 egg production, 278, 279 green feed additives, 267 health disorders, 267 immune response, 285, 286 intestinal bacterial enumeration, 282, 283 intestinal morphology, 282 meat quality, 274, 275 medicinal herbs, 267 minerals, 267 MUFA, 267 phytogenic additives, 268, 269 productive performance BWG, 269–271 feed utilization, 271–273

553 LBW, 269–271 nutrient utilization, 273, 274 PUFA, 267 quality, 278, 279 reproductive performance, 277 vitamins, 267 Powdered N. sativa seeds (PBC), 164 Predominant compounds, 342 Predominant fatty acids, 341 Principal component analysis (PCA), 143–145 Pro-inflammatory cytokine, 309 Proprotein, 60 Propylthiouracil, 507 Prostaglandin E2 (PGE2), 368 Protein efficiency rate (PER), 225 Proteins, 48, 49, 74, 194, 225 Pseudomonas fluorescens infection, 310 Psoriasis, 238, 437 Psoriasis Area and Severity Index (PASI) score, 238 Psychrotrophic Bacteria Counts (PBC), 446 Pulses, 32 Purity gum ultra (PGU), 112 Pyrazine, 101 Pyrolysis, 396 Pyrularia pubera, 60 Q Qualitative and quantitative composition, 154 Quality control (QC), 133–134, 143–145 Quantitative and qualitative analysis, 139 Quercetin, 413 Quinone content, 100 Quinones, 101 R Radical scavenging activity (RSA), 157, 329, 351 Radical scavenging assays, 329 Radical scavenging tests, 329 Raffinose, 69 Ranunculaceae, 2, 12, 25, 46, 99, 132, 191, 234, 336 Rapeseed oil (RO), 353 Reactive oxygen species (ROS), 87, 115–117, 283, 301, 461 Red blood cells (RBCs), 307 Reduced graphene oxide (RGO), 182, 183 Refractive index (RI), 320, 343 Remedies, 23 Remedy, 12

554 Renal disorders, 525 Renonculaceae, 68 Respiratory, 525 Respiratory burst activity, 309 Respiratory diseases, 525 Response surface methodology (RSM), 446 Reversed phase high performance liquid chromatography (RP-­ HPLC), 62, 111 Rhizosphere soil, 15 Ribulose-1,5-bisphosphate carboxylase, 14 Roasting, 339 Ruminant diets ammonia, 258 animal-derived products, 246 antimethanogenic activity, 258 beneficial effects, 246 bioactive compounds, 247 carbohydrates, 258 carcass traits, 251–253 effect of dietary, 247 energy and protein, 247 feeding trials, 246 fermentation and methane production, 253–255 growth performance, 247–249 growth promoters, 246 health and blood metabolites, 255–258 herbs, 246 meat quality, 251–253 medicinal plants, 246 milk production and composition, 249–251 natural feed additives, 246 nutrients digestibility, 247–249 nutritional interventions, 246 phytochemicals, 246 phytogenic products, 258 protein, 247 supplements, 246 S Sabinene, 414 Safety Regulations, 232 Saline-treated group, 167 Salinity, 13, 14, 19 Saponification values (SV), 321, 343 Saponins, 3, 53, 132 Saturated fatty acids (SFA), 17, 155, 238, 323, 341, 357 Scale profiling, 134 Scanning electron microscopy (SEM), 495 SCE-assisted hydrodistillation, 412

Index SCE-extracted oil, 412 Scientific classification, 24 Scopolamine-induced memory test model, 528 Scopus database, 4, 5 Screw and cold pressed effects antiradical activity, 427 DSC, 427 extinction coefficient, 426 ferric reducing activity, 427 FFA and PV, 426 FRAP assay, 427 melting curves, 427 phenolic compounds, 427 saponification values, 426 Soxhlet oil, 426 thermogravimetric analysis, 428 time-consuming, 427 TPC, 427 TQ, 427 viscosity, 426 Screw pressed methods, 426 Secondary metabolites, 59, 132, 134, 137, 138, 140, 141, 143, 144, 146, 195, 485 Sedentary lifestyle, 494 Seed-ethanol mixture, 182 Seed extracts alcohol, 484 amino acids, 486 antibacterial activity, 507 anti-cancer effects, 511, 512 anti-diabetic effects, 508, 509 antifungal effects, 508 anti-inflammatory, 510 antioxidant activity, 485, 486, 507 antiviral activity, 508 aroma-active compounds, 487, 488 aroma compounds, 487 bioactive agent, 504 bioactive compounds, 502, 505, 506 biological activities, 503 by-products, 482 chemical composition, 485 chromatography analysis, 487 components, 522 diverse solvents, 485 essential oil composition, 506 ethanolic, 483 extraction methods, 484 flavonoid content, 486 functional properties (see Functional properties, seed extract) fungal biomass reduction, 491

Index glutamine amino acid, 485 health promotive agent, 522 high-value compounds, 504 hydroalcoholic extraction method, 483 immuno-modulatory activities, 510, 511 methanolic, 485 methodology, 483 nutritional values, 503 odors, 489 organic phase, 482 percolation-assisted extraction method, 485 pharmacological effects, 522 phenolic compounds, 485, 486 phenolic profile, 486 Prophetic Medicine, 502 secondary metabolites, 485 sensory, 488 skin pigmentation, 512 therapeutic aspects, 502 TPC, 485, 487 treatment, 503 treatment, ailments, 502 wound healing, 512 Seed oils antibacterial activity, 507 anti-cancer activity, 511 anti-diabetic effects, 508, 509 antifungal activities, 508 anti-inflammatory, 510 antioxidants, 507 antiviral activity, 508 bioactive agent, 504 bioactive compounds, 505, 506 biological activities, 503 conventional Indian medical system, 503 high-value compounds, 504 immuno-modulatory activities, 510, 511 skin pigmentation, 512 treatment, ailments, 502 wound healing, 512, 513 Semen parameters, 168 Semi-arid and arid regions, 12 Semiquinone, 87 Sensory alteration, 307 Sensory profiles, 488 Serum globulin concentration, 257 Serum/glucose deprivation (SGD)-induced cytotoxicity, 367 Serum immunoglobulin, 308 Serum lysozyme, 309 Sesquiterpenes, 414 Sexuality, 494 SFE-extracted oil, 427, 428

555 SiHa and CaSki cancer cell lines, 370 Silver nanoparticles (AgNPs), 181 Silver nanorods, 161 Silver NPs, 495 Sitosterol, 195 Skin infections, 118 Skin pigmentation, 119, 512 Small scale extraction, NS oil biodiesel derivatization/methylation, GLC, 402 FAME quantification, 402, 403 Soxtec-extraction, 400 TL extraction, 401, 402 Soaking, 339 Solid-lipid nanoencapsulation, 385 Solid-phase micro extraction (SPME), 106, 108 Solubility test, 33, 38 Solvent-based extractions, 351 Solvent evaporation, 112 Solvent-extracted oil, 343 Solvent extraction (SE), 108, 196 Solvent NSO extraction, 364 Sorghum sheaths, 72 Soxhlet apparatus, 364 Soxhlet extraction method, 196 Soxhlet oil, 426 Soxtec-extraction, 400 Soybean meal (SBM), 225, 227 Specific growth rate (SGR), 301 Spontaneous emulsification, 112 Staphylococcus aureus, 239, 415, 441, 491, 492, 532 STAT3 signaling pathway, 369 Steam distillation (SD), 85, 106, 197, 198, 365, 425 Sterol composition, 341 Sterols, 132, 326 Stigmasterol, 195, 326 Streptozotocin (STZ), 217 Stress-induced oxidative damage, 75 Structural properties BCSPs, 72, 73 STZ-induced albino rats, 161, 168 Sunflower oil (SO), 338, 352 Sun protection, 119 Supercritical carbon dioxide extraction (SCE), 411 Supercritical fluid carbon dioxide extraction (SCFE-CO2), 424 Supercritical fluid extraction (SFE), 70, 84, 106, 109, 110, 198, 199, 337, 341, 343, 364, 423 Super critical transesterification, 399

556 Superoxide dismutase (SOD), 115, 117, 217, 301, 366 Supplementation, 164 Surface-active compounds, 74 Surfactant micelles, 384 Synthesis TQ, 89, 90 Synthetic antioxidants, 507, 524 Systolic blood pressure, 163 T Taenia saginata, 216 Taguchi method, 424 t-anethol, 3 Terpene-terminated 6-alkyl residues, 159 Terpenoids, 212 Testosterone, 168, 257 Therapeutic management, 164 Thermal oxidation condition, 354 Thermal pre-treatment AV and PV, 339 DA and IR, 339 enzymes, 338 MAE, 338 microwave radiation, 339 moisture content, 339 oil stability, 339 roasting, 339 soaking, 339 Thermogram curves, 428 Thermo gravimetric analysis (TGA), 428 Thin-layer chromatography (TLC), 85, 110, 133 Thiobarbituric acid, 357 Thiobarbituric acid reactive substances (TBARS), 357, 367 Thionins antimicrobial effects, 61 with antimicrobial potential, 60 cell vacuoles, 60 mono- and di-cotyledonous plants, 60 N. sativa isolation, 61, 62 mode of action, 62, 63 plant defensins, 60 sources, 60, 61 Thymohydroquinone (THQ), 3, 155, 192, 194, 195, 363–365 Thymol, 90 Thymoquinone (TQ), 3, 14, 100, 101, 132, 133, 137–140, 142, 144, 145, 155–160, 162, 166–168, 192, 194,

Index 195, 211–213, 224, 235, 239, 256, 351, 356, 358 angiogenesis and metastasis, 370 anthelmintic activity, 469 anti-arthritic activity, 467 antibiotic role, 372 anticancer, 463 anti-cancer mechanisms, 369 anticancer properties, 86, 87 anticancirogenic, 463 antidot and antitoxic properties, 417 anti-immunologic benefits, 371 anti-inflammatory, 466 antimicrobial spectrum, 464 antimutagenic, 463 antineoplastic effect, 365 antioxidant agent, 462 antioxidant properties, 366, 461, 463 anti-proliferative potential, 368 apoptosis, 367, 368 bioactive compound, 502 biological activities, 86, 87 black cumin, 459 cancer cells, 366 caspases activation, 367 chemical structure, 363 chemosensitizing properties, 463 chromatography analysis, 85 content, 82–85 cytoprotective enzymes, 366 detection, 85 EMT, 370 essential oil components, 365 GATA, 368 glycogenolysis reduction, 371 hepatic carcinogenesis, 367 hepatoprotective action, 465 immunosuppressive agent, 511 inflammatory cytokines, 510 isolation, 89, 90 liver tissues, 461 lung injury, 467 MCA-induced fibrosarcoma tumors, 365 medicinal field, 365 N. sativa oil, 461 neuroprotective, 372 NF-κB, 368 oil's general stability, 427 origin, 82–85 pancreatic cancer, 512 physical properties, 88, 89 phytochemical compound, 469 plant-derived secondary metabolite, 82

Index potent anti-oxidant, 365 proliferation, 367 protective effect, 366 quantification, 85 quinone reductase enzyme, 363 regulation, 371 role, 369, 370 ROS, 461 STAT3 signaling, 369 structure, 36, 39, 82 synchronized LNCaP cells, 368 synthesis, 89, 90 Tamoxifen, 370 toxicity, 88, 89 toxicology, 87, 88 transcription factor, 510 upstream kinases phosphorylation, 369 volatile oil composition, 82 Wnt signaling, 369 wounding process, 512 Thymus vulgaris, 493 Thyroid hormones, 257, 527 Tibb-e- Nabwi, 24 Time of harvest, 155 TLC screening technique, 117 Tocochromanols, 325, 326, 353 Tocols, 338, 341, 344 Tocopherols, 325, 336, 353 Tocotrienols, 325 Tooth caring, 119 Topical preparations, 232, 237–239 Total aerobic mesophilic bacteria (TAMB), 357 Total Aerobic Mesophilic (TAMC), 446 Total cholesterol (TC), 163, 164 Total digestible nutrients (TDN), 248 Total glycolipids (TGL), 322 Total lipids (TL) extraction, 401, 402 Total phenolic compounds (TPC), 201, 327 Total phenolic contents (TPC), 427 Total unsaturated fatty acids (TFAs), 101 Total volatile basic nitrogen (TVB-N), 357, 358 Toxic oxygen metabolites, 523 Toxicity, 168 TQ, 88, 89 Toxicology TQ, 87, 88 TQ-analogs, 89, 90 TQ content agriculture treatments, 82 genetic factor, 83, 84 geographical location, 82, 83

557 oil extraction method, 84, 85 Traditional medicine and food applications, 336 Traditional volatile oil extraction methods, 107, 108 trans-anethole, 414, 425, 435 Transesterification catalytic, 397–399 definition, 397 reaction, 399–402 reactor, 400, 401 super critical, 399 Triacylglycerols (TAG), 50, 321, 426 Trichomes, 33 Trifolium alexandrinum, 248 Triglycerides (TG), 256, 493, 494 Triple-negative breast cancer (TNBC), 159 Tumor, 12 Tumor necrosis factors (TNF), 309 Turkish cold pressed oil, 330 Turmeric (Curcuma Longa), 217 Two-dimensional honeycomb structure, 182 Type 2 diabetes, 163 U Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), 137–139 Ultra-performance liquid chromatography MS (UPLC/MS), 135 Ultrasonication, 112 Ultrasound, 340 Ultrasound treatment acoustic and hydrostatic pressure, 340 application, 340 cell membranes and enhancement, 340 lipolytic enzyme activity, 340 Unani, 32 Unconventional oils, 350 Unconventional seed, 350 Uncultivated land pressure, 404 Undecylenic acid, 237 Un-encapsulated TQ, 87 The United Nations Sustainable Development Goals (UNSDG), 2 Unsaturated fatty acids (USFA), 155, 300 UPLC-FTMS profiling, 138 UPLC/PDA/(-)ESI-qTOF-MS, 138 Urea and cattle manure, 16 Urinary calculi, 526 UV spectrophotometry, 135

Index

558 V Vaginal moniliasis, 240 Variable importance plot (VIP), 144 Vegetable oils compounds, 350 nutritional properties, 350 physicochemical properties, 337 unconventional, 350 Vegetables, 32 VEGF-induced ERK activation, 370 Very-low-density lipoprotein cholesterol (VLDL-c), 280 Vibrio harveyi, 415 Visual scale (VAS), 237 Vitamins, 48, 49, 132, 156, 194, 195, 482 Vitiligo, 239 Volatile component distribution, 413 Volatile compounds, 328, 355 Volatile fatty acids (VFA) production, 250 Volatile oil, 83, 459Essential oil (EO), see Volatile oil component, 423 Volatile oil fraction, 328, 383 Volatile oxidation compounds, 354

W Water balance methods, 19 Water-deficit treatments, 18 Water immersion restraint (WRS), 166 Water retention capacity (WHC), 74 Water salinity, 412 Water-use efficiency (WUE), 15, 17 Wistar albino rats, 166 Wistar rats, 167 Wnt signaling, 369–370 World Health Organization (WHO), 2, 23 Wound healing, 119, 493, 512, 513 X X-ray diffraction analysis (XRD), 72, 73, 495 Y YAC-1 tumor cells, 159 Z Zinc (Zn), 17 Zinc oxide nanoparticles (ZnONPs), 184 Zinc oxide NPs, 495 Zinc pyrithione, 237