Bioactive Essential Oils and Cancer 3319191438, 9783319191430

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Bioactive Essential Oils and Cancer

Damião Pergentino de Sousa Editor

Bioactive Essential Oils and Cancer

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Editor Damião Pergentino de Sousa Federal University of Paraiba João Pessoa, Paraíba Brazil

ISBN 978-3-319-19143-0 ISBN 978-3-319-19144-7 (eBook) DOI 10.1007/978-3-319-19144-7 Library of Congress Control Number: 2015942822 Springer Cham Heidelberg New York Dordrecht London © Springer International Publishing Switzerland 2015 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, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper Springer International Publishing AG Switzerland is part of Springer Science+Business Media (www.springer.com)

I dedicate this volume to my family, especially my little daughter Julia, my son Pedro, and my wife Sheila for her patience and encouragement.

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Acknowledgements

I would like to thank Springer for inviting me to edit this volume. I would like to thank all contributors of the book for accepting the challenge of preparing the contents of the chapters. Their effort made possible the publication of the volume.

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Preface

This volume, intended for professionals and researchers in the fields of life sciences, biotechnology, and drug discovery, contains discussions of several aspects of bioactive essential oils and their potential clinical use in treating or preventing cancer. It is a valuable source of information and studies for all people who work with natural products, especially with applications in cancer. We present chemical structures designed to help the reader understand the key concepts of the chemical and pharmacological properties of essential oils. The content of the book is divided into 15 chapters. Chapter 1 provides an overview of cancer pathogenesis and therapies. Several genetic aspects are covered, including critical genes and epigenetics in cancer, metastasis, epidemiology, and environmental risk factors. This chapter also discusses the main therapeutic approaches in cancer, such as surgery, radiotherapy, and chemotherapy. In Chap. 2, the reader is introduced to the chemistry of essential oils. Key concepts, classification of the constituents of essential oils, and methods of analysis are some of the topics discussed. In short, the authors describe the relevant chemical aspects of aromatic plants and the chemical structures of the major compounds found in their essential oils. Chapter 3 describes the features of botanical classification of the main families containing plants that produce essential oils. Pharmacobotanical aspects of selected species are described. Chapter 4 presents recent developments in the synthesis of anticancer drugs from essential oil constituents, demonstrating the potential of this class of natural products as starting materials for the development of anticancer drugs. Chapter 5 introduces some successful examples of the use of medicinal chemistry tools applied to essential oils constituents focusing on antitumor activity. Chapter 6 reports some clinical studies with essential oil and chemical constituents. Chapter 7 describes the selected studies on antitumor activities of essential oils obtained from aromatic plants. A description of the general aspects of these plants and chemical composition of their essential oils is also presented. The antitumor monoterpenes found in essential oils and their possible mechanisms of action are reported in Chap. 8. The antitumor sesquiterpenes found in essential oils are described in Chap. 9; selected studies containing the mechanisms of action of essential oils are presented in the chapter. Chapter 10 presents a literature review on phenylpropanoids from essential oils which have antitumor activity. Chapter 11 ix

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presents a comprehensive description of synergy and interaction of essential oil and the chemical constituents with drugs used in cancer therapy or other antitumor constituents. The use of essential oil components as cancer preventive agents is outlined in Chap. 12. Geraniol and farnesol are two selected constituents discussed in this topic as dietary bioactive food components with promising applications in cancer chemoprevention. Chapter 13 discusses the use of aromatherapy in promoting well-being of patients with cancer. Depression and anxiety, commonly found in people suffering from cancer, can affect the immune system and worsen the health of the patient. The use of psychotherapeutic essential oils for inhalation can help in the treatment of these patients by improving the quality of life. The perillyl alcohol is a monoterpene found in essential oils with significant antitumor activities. The scientific reports are promising and contain several clinical studies. Chapter 14 describes the studies of a research group using perillyl alcohol monoterpene. The experience of many years of research with perillyl alcohol is demonstrated in the results of clinical analyses and magnetic resonance imaging of patients undergoing treatment with perillyl alcohol, notably via inhalation. Chapter 15 comments on the potential of essential oils for use in cancer therapy and future prospects. I hope that this book will stimulate readers to appreciate the importance of natural products as health-promoting resources and generate a discussion and reflection on new approaches in cancer therapy. Damião Pergentino de Sousa João Pessoa, Brazil

Contents

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Principles of Cancer Pathogenesis and Therapies: A Brief Overview ........................................................................................ Rosane Borges Dias, Ludmila de Faro Valverde, Clarissa Araújo Gurgel Rocha and Daniel Pereira Bezerra

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Essential Oils Chemistry ........................................................................... Mónica Zuzarte and Lígia Salgueiro

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Pharmacobotanical Aspects of Aromatic Plants ..................................... Basílio I.J.L.D., Nathalia Diniz Araujo and Rafael Costa Silva

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Essential Oils as Raw Materials in the Synthesis of Anticancer Drugs ................................................................................... Timothy J. Brocksom, Kleber T. de Oliveira, Marco A. B. Ferreira and Bruno M. Servilha

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5 Antitumor Essential Oils: Progress in Medicinal Chemistry ................. 111 Sócrates Cabral de Holanda Cavalcanti, Rafael dos Reis Barreto de Oliveira and Damião Pergentino de Sousa 6

Clinical Advances in Anticancer Essential Oils ....................................... 125 Ammad Ahmad Farooqi, Rubina Sohail, Sundas Fayyaz and Iryna Shatynska-Mytsyk

7 Antitumor Essential Oils ........................................................................... 135 Hemerson Iury Ferreira Magalhães and Élida Batista Vieira de Sousa 8 Antitumor Monoterpenes .......................................................................... 175 Janaina Fernandes

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Sesquiterpenes from Essential Oils with Promising Antitumor Properties ................................................................................. 201 Fayaz Malik and Suresh Kumar

10 Antitumor Phenylpropanoids.................................................................... 215 Miriam Teresa Paz Lopes, Dalton Dittz Júnior and Fernanda de Oliveira Lemos 11 Antitumor Essential Oils: Synergy and Chemotherapeutic Interactions ................................................................................................. 231 Rogerio Correa Peres, Carolina Foot Gomes de Moura, Flavia Andressa Pidone Ribeiro and Daniel Araki Ribeiro 12 Dietary Essential Oils and Cancer Chemopreventive Potential ............ 237 Thomas Prates Ong 13 Cancer and Aromatherapy: A View of How the Use of Essential Oils Applies to Palliative Care .................................................. 251 Rita de Cássia da Silveira e Sá 14 Perillyl Alcohol: A Pharmacotherapeutic Report ................................... 267 Clovis O Da Fonseca and Thereza Quirico-Santos 15 Conclusion and Future Perspectives ........................................................ 289 Damião Pergentino de Sousa Index .................................................................................................................. 291

Contributors

Clarissa Araújo Gurgel Rocha Gonçalo Moniz Research Center, Oswaldo Cruz Foundation (CPqGM-FIOCRUZ/BA), Salvador, Brazil Department of Propedeutics, Federal University of Bahia, Salvador, Bahia, Brazil Rosane Borges Dias Gonçalo Moniz Research Center, Oswaldo Cruz Foundation (CPqGM-FIOCRUZ/BA), Salvador, Brazil Timothy J. Brocksom Department of Chemistry, Federal University of São Carlos, São Carlos, Brazil Ludmila de Faro Valverde Gonçalo Moniz Research Center, Oswaldo Cruz Foundation (CPqGM-FIOCRUZ/BA), Salvador, Brazil Élida Batista Vieira de Sousa Department of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa, Brazil Nathalia Diniz Araujo Postgraduate Program in Bioactive Natural and Synthetic Products, Federal University of Paraíba, João Pessoa, Brazil Dalton Dittz Júnior Department of Pharmacology, Federal University of Minas Gerais, Belo Horizonte, Brazil Ammad Ahmad Farooqi Laboratory for Translational Oncology and Personalized Medicine, Rashid Latif Medical College, Lahore, Pakistan Sundas Fayyaz Laboratory for Translational Oncology and Personalized Medicine, Rashid Latif Medical College, Lahore, Pakistan Janaina Fernandes NUMPEX-BIO—Pólo Xerém, Universidade Federal do Rio de Janeiro and National Institute for Translational Research on Health and Environment in the Amazon Region—INPETAM Centro de Ciências da Saúde, Rio de Janeiro, RJ, Brazil Marco A. B. Ferreira Department of Chemistry, Federal University of São Carlos, São Carlos, Brazil

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Clovis O Da Fonseca Faculty of Medicine, Fluminense Federal University, Niterói, RJ, Brazil Carolina Foot Gomes de Moura of São Paulo, São Paulo, Brazil

Departament of Pathology, Federal University

Sócrates Cabral de Holanda Cavalcanti Department of Pharmacy, Federal University of Sergipe, São Cristóvão, Brazil Basílio I.J.L.D. Department of Pharmaceutical Sciences, Federal University of Paraiba, Paraíba, Brazil Suresh Kumar Department of Cancer Pharmacology, CSIR-Indian Institute of Integrative Medicine, Jammu, India Fernanda de Oliveira Lemos Department of Pharmacology, Federal University of Minas Gerais, Belo Horizonte, Brazil Hemerson Iury Ferreira Magalhães Department of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa, Brazil Fayaz Malik Department of Cancer Pharmacology, CSIR-Indian Institute of Integrative Medicine, Jammu, India Kleber T. de Oliveira Department of Chemistry, Federal University of São Carlos, São Carlos, Brazil Rafael dos Reis Barreto de Oliveira Department of Pharmacy, Federal University of Sergipe, São Cristóvão, Brazil Thomas Prates Ong Laboratory of Nutrigenomics and Programming, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo and Food Research Center (CEPID/FAPESP), São Paulo, Brazil Miriam Teresa Paz Lopes Department of Pharmacology, Federal University of Minas Gerais, Belo Horizonte, Brazil Daniel Pereira Bezerra Gonçalo Moniz Research Center, Oswaldo Cruz Foundation (CPqGM-FIOCRUZ/BA), Salvador, Brazil Rogerio Correa Peres Department of Biosciences, Federal University of São Paulo, São Paulo, Brazil Flavia Andressa Pidone Ribeiro Department of Biosciences, Federal University of São Paulo, São Paulo, Brazil Thereza Quirico-Santos Department of Cell and Molecular Biology Institute of Biology, Fluminense Federal University, Niterói, RJ, Brazil Daniel Araki Ribeiro Department of Biosciences, Federal University of São Paulo, São Paulo, Brazil Departament of Pathology, Federal University of São Paulo, São Paulo, Brazil

Contributors

Lígia Salgueiro

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Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal

Bruno M. Servilha Department of Chemistry, Federal University of São Carlos, São Carlos, Brazil Iryna Shatynska-Mytsyk Diagnostic Imaging and Radiation Department, Lviv National Medical University, Lviv, Ukraine

Therapy

Rafael Costa Silva Graduate Program in Plant Biology, Federal University of Pernambuco, Recife, Brazil Rita de Cássia da Silveira e Sá Department of Physiology and Pathology, Health Science Center, Federal University of Paraíba, João Pessoa, PB, Brazil Rubina Sohail

Services Institute of Medical Sciences, Lahore, Pakistan

Damião Pergentino de Sousa Department of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa, PB, Brazil Mónica Zuzarte Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal

Chapter 1

Principles of Cancer Pathogenesis and Therapies: A Brief Overview Rosane Borges Dias, Ludmila de Faro Valverde, Clarissa Araújo Gurgel Rocha and Daniel Pereira Bezerra

Abbreviations CDKs EBV EMT HBV HCV HIV HPV HTLV-1 KSHV miRNA NCI TP53 TSG UV

Cyclin-dependent kinases Epstein–Barr virus Epithelial–mesenchymal transition Hepatitis B virus Hepatitis C virus Human immunodeficiency virus Human papillomavirus Human T-lymphotropic virus Kaposi's sarcoma-associated herpesvirus microRNA National cancer institute P53 protein Tumor suppressor genes Ultraviolet radiation

Introduction Cancer was first described thousands of years ago in the Egyptians Papyrus in 3000 BC. However, knowledge of this disease came long back only from observations of aggregated familial cases and social groups. Over the past four decades, a great D. Pereira Bezerra () · R. Borges Dias · L. de Faro Valverde · C. Araújo Gurgel Rocha Gonçalo Moniz Research Center, Oswaldo Cruz Foundation (CPqGM-FIOCRUZ/BA), Salvador, Brazil e-mail: [email protected] C. Araújo Gurgel Rocha Department of Propedeutics, Federal University of Bahia, Salvador, Bahia, Brazil © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_1

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deal of cancer research has focused on uncovering the critical genes necessary for cancer development and understanding the concepts related to tumor initiation and progression, gene signatures, epigenetics, invasion, and metastasis (Cao et al. 2011). Hanahan and Weinberg (2011) have compiled cancer hallmarks related to the general biological properties of tumor cells and their microenvironment, which have contributed greatly to the understanding of cancer biology and current research challenges. In this chapter, we provide an overview of cancer pathogenesis and therapies

Cancer Biology Cancer is a group of complex genetic diseases characterized by uncontrolled growth and spread of abnormal and proliferating cells that have undergone a plethora of changes in multiple genes (Cao et al. 2011). Most tumors are monoclonal and are named carcinomas or sarcomas, depending on whether an epithelial or mesenchymal cell, respectively, gave rise to the tumor. Cancer cells exhibit many morphological patterns such as aberrant nuclei, abundant chromatin, and atypical mitosis. Human carcinogenesis is a complex and multistep process that may take several decades before a primary tumor is established. Tumor progression involves a sequence of modifications in critical genes and epigenetic alterations that promote sustained proliferation, immortalization, resistance to cell death, insensitivity to inhibitory factors, invasion, metastasis, and angiogenesis in the context of genomic instability (Hanahan and Weinberg 2011). The acquisition of these biological properties results from the interaction between the cell and environmental carcinogens (tumor viruses, radiation, chemical carcinogens, etc.), or they can be inherited in germ cells, especially in tumor suppressor genes (TSG).

Critical Genes for Cancer—Oncogenes Oncogenes and their normal counterparts (proto-oncogenes) are a class of genes related to cell growth, proliferation and apoptosis (Croce 2008). The first human oncogene to be described was src (in reference to the src portion of the Rous Sarcoma Virus) by Varmus and Bishop (Stehelin et al. 1976). In cancer development, oncogenes are dominant genes, and the genetic alterations that lead to activation of these genes include point mutations, translocations, gene amplifications (Croce 2008), and epigenetic modifications (Sandoval and Esteller 2012). All of these alterations result in overexpression of oncoproteins and a proliferation advantage to the cell. A single-base substitution in the ras gene (guanine replaced by a thymidine residue) is sufficient to convert the normal gene into an important oncogene for human cancers (Bos 1989). This point mutation results in the substitution of valine with glycine, which results in the loss of intrinsic GTPase activity and unregulated

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signal transduction for several mitogenic pathways, such as the MAP kinase and PI3K pathways (Relógio et al. 2014; Stout et al. 2014). The best example of a gene translocation occurs in Burkitt’s lymphoma (t8;14) in which the myc gene on chromosome 8 is under the control of the immunoglobulin heavy-chain sequences on chromosome 14, which results in higher levels of normal Myc protein expression (Croce et al. 1983; Leder et al. 1983). In chronic myelogenous leukemia (t9;22), chromosome translocation results in a hybrid tyrosine kinase protein called BCRABL1 that promotes strong signals for growth and proliferation (Pasternak et al. 1998). The activation and overexpression of oncogenes can also occur by gene amplification, which results from repetitions of hundreds of kilobases of DNA ( double minutes; e.g., Myc, Ccnd1, Egf-r) or epigenetic mechanisms (e.g., histone acetylation; Suvà et al. 2013). According to Croce (2008), oncogene products can be classified into six distinct groups: growth factors (e.g., Pdgf, Egf, Vegf), growth factor receptors (e.g., Egfr, Vegf-r), signal transducers (e.g., Ras, Abl, Raf), apoptosis regulators (e.g., Bclx, Bcl2), chromatin remodelers (e.g., All1), and transcription factors (e.g., Fos, Jun, Myc).

Critical Genes for Cancer—Tumor Suppressor Genes The first evidence that cancers could be recessive at the cellular level came from studies that fused tumor cells with normal cells. The resulting hybrid cells were non-tumorigenic, which indicated that, in some way, growth-controlling genes contributed to normal cell proliferation. In addition, rare familial cancers (e.g., retinoblastoma) showed an autosomal dominant inheritance pattern, which was explained by the Two Hits Theory ( see Inherited Susceptibility to Cancers) and corroborated the existence of TSG (Knudson 1971). In general, TSG act as regulators of the cell cycle, maintain DNA integrity, and control cell senescence and death by apoptosis. Thus, when TSG are inactivated or lost, carcinogenesis occurs (Vogelstein and Kinzler 2004). Cytogenetic and karyotyping studies in patients with familial retinoblastoma showed a recurrent deletion on chromosome 13q14, and linkage map analysis revealed that the Rb gene was in this location (Friend et al. 1986). Rb, the first cloned human TSG, encodes a phosphoprotein with the same name (pRB) that participates in regulating the cell cycle from G1 to S phases. In quiescent cells (G0 stage), pRb is found in the hypophosphorylated state, forms a nuclear complex with the E2F transcription factor, and blocks the transcription of early genes such as cyclins and cyclin-dependent kinases (CDKs). Furthermore, pRB and the E2F complex recruits histone deacetylases, which favor chromatin condensation and cell cycle arrest. Under the action of mitogenic stimuli, CDKs phosphorylate pRb allowing it to dissociate from the E2F complex, resulting in complete functional inactivation of pRb and the transcription of genes that encode proteins for the S phase, such as cyclins and DNA polymerase (Giacinti and Giordano 2006). Thus, deletion of this gene or pRb sequestration (E7 protein of oncogenic HPV binds to

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pRb to inhibit its function) results in deregulation of the cell cycle and hyperproliferation. pRb is also a cofactor for the transcription factor HIF-α, which is involved in transcription of genes related to angiogenesis such as Vegf-a (Gabellini et al. 2006; Bakker et al. 2013). The p53 gene is located on chromosome 17, and mutations in this gene are found in 30–50% of commonly occurring human cancers. The P53 protein (TP53) is a negative regulator of the cell cycle and inhibits erroneous cell proliferation (Prives and Hall 1999). This phosphoprotein is known as the “guardian of the genome” and acts as a transcription factor to suppress tumor growth. TP53 is constitutively expressed at low levels and exists in the cell as a homotetramer. As wild-type p53 cannot bind to a mutant-type p53, TP53 tetramer functioning may be inhibited. This event is called a dominant-negative effect (Willis et al. 2004). The wild-type P53 protein has a short half-life, and in normal cells, the MDM2 protein regulates the degradation of TP53 by marking it with ubiquitin for rapid degradation after its synthesis. TP53 plays a central role in recognizing cellular DNA damage (e.g., ionizing radiation, UV, and chemical carcinogens). When DNA damage is present, the sensors of cellular stress, ATM, Chk1, and Chk2 kinases phosphorylate TP53 to inhibit its degradation by preventing the MDM2 from ubiquitinating p53. Consequently, TP53 escapes destruction, accumulates rapidly, and induces a series of downstream cellular programs such as temporary cell cycle arrest and/or senescence through induction of p21 proteins, p27 and p57, which inhibit CDK phosphorylation and consequently the Rb protein, DNA repair proteins with participation of the GADD45 family and apoptosis, especially through the increased expression of BAX (Gottlieb and Oren 1996; Prives and Hall 1999; Lahav 2008).

Epigenetics in Cancer Epigenetics refers to heritable changes in gene expression unrelated to any change in the DNA sequence. In cancer, epigenetic changes regulate gene expression at the level of DNA, histones, and microRNAs (miRNA). DNA hypomethylation, the first epigenetic finding in human cancers, contributes to the reactivation of transposable DNA sequences, the loss of gene imprinting, and genomic instability, which favors disease progression and acquisition of several mutations (Feinberg et al. 1983; Howard et al. 2008). Although oncogenes are generally hypomethylated in cancer, hypermethylation of the CpG islands next to the promoter regions of TSG is the most well-defined epigenetic change in cancer, resulting in the silencing of this class of genes (Baylin and Jones 2011). Histone modifications occur with many different chemical groups (e.g., methyl and acetyl) and on different histone residues such as lysine, arginine, and serine. These chemical modifications affect biological processes such as chromatin organization, DNA replication, transcription, and DNA repair (Kouzarides 2007). Histone acetylation is associated with transcriptional activation, but methylation can have

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different functions depending on the type of amino acid involved and the position of the methyl radical. For example, H3K4 methylation is associated with transcriptional activation, but H3K9 methylation is associated with repression. Methylation of lysines 9 and 27 on H3 represses the activity of TSG (Kouzarides 2007; Suvà et al. 2013). In some tumors, epigenetic changes are the result of mutations in the genes that encode DNA and histone methyltransferases (Lokody 2014). On the cytoplasmic level, gene expression can be regulated by miRNA. miRNAs are 20–22 nucleotides of non-coding single-stranded RNAs that are involved in regulating physiological and pathological processes such as differentiation, proliferation, metastasis, and angiogenesis, which makes miRNAs one of the largest regulators of gene expression. miRNAs bind to complementary sequences in the messenger RNA to inhibit their translation. In cancers, miRNAs are usually misregulated in comparison to healthy tissue and are generally considered tumor suppressors (Zhang et al. 2007). For example, loss of expression of the miR-34 family has been associated with metastasis (Rokavec et al. 2014), whereas downregulation of miR15 and miR16 seems to promote cell survival (Calin et al. 2002).

Metastasis Metastasis is defined as the growth of primary cancer cell clones in another and distinct anatomical site (Spano et al. 2012). Until recently, metastatic tumors were considered the final stage of tumor progression. Currently, it is known that the metastasis cascade may occur early, and that there are most likely micrometastases at the time of initial diagnosis (Klein 2009). In recent years, we have seen dramatic and exciting progress in our understanding of metastasis as a disease process (Sleeman 2012), but the metastasis cascade remains a major challenge in cancer research. The metastasis cascade involves a complex sequence of steps—local invasiveness, intravasation, dissemination, extravasation, micrometastasis, and colonization—that enable cancer cells from the primary tumor to invade adjacent tissues and adapt in the microenvironment of the metastatic niche (Spano et al. 2012). For carcinomas, these events are better understood and serve as the primary examples of metastasis. Carcinoma cells acquire an invasive phenotype in a process called the epithelial−mesenchymal transition (EMT) in reference to morphogenetic steps that occur during embryogenesis (Tiwari et al. 2012). Therefore, cancer cells do not make up new pathways but reprogram preexisting molecular pathways. The phenotypic changes that occur in carcinoma cells are coordinated by the transcription factors such as Snail, Slug, and Twist and involve the following: the loss of E-cadherin and cytokeratin, change in cell polarity, the expression of N-cadherin, MMP2, MMP9, and fibronectin secretion, increased resistance to apoptosis, acquisition of motility, and stem cells markers (Chiang and Massagué 2008). The loss of E-cadherin results in destabilization of the cell adhesion complex, with consequent accumulation of β-catenin and translocation of this protein to the nucleus, resulting in the activation of TCF–LEF transcription factors and genes

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related to EMT. Stromal cells, especially macrophages, are also important in metastasis. Tumor macrophages secrete TNF-α and EGF, which contributes to EMT and cell motility, respectively. In addition, the accumulation of macrophages in the vicinity of blood vessels appears to indicate the location of intravasation. In general, metastatic colonization is biologically inefficient and depends on previously activated stroma in the metastatic niche. Stromal activation can be induced by tumor cells themselves through the secretion of CCL9 and CSF-1, which stimulates the differentiation of macrophages and myeloid progenitor cells, respectively. Tumor macrophages, in turn, are resistant to hypoxia, and low oxygen tension induces the secretion of large amounts of IL8 and VEGFA, increasing the angiogenic activity that is critical for successful metastatic colonization (Chiang and Massagué 2008; Nguyen et al. 2009). Some theories attempt to explain the pattern of metastasis in tumors, such as the Paget (1889) “seed and soil” hypothesis and “vascular zip code.” However, despite the influence of vascular architecture on metastatic patterns, molecular and cellular complexities involved in this process are still not fully understood.

Inherited Susceptibility to Cancers Most tumors affecting humans are sporadic and occur in families without a consistent history of cancer (Foulkes 2008). However, the observation that some rare tumors, such as retinoblastoma and Wilms’ tumors, occurred more frequently in specific groups suggested a familial predisposition to some types of tumors. In 1971, Knudson published a mathematical model for patients with familial and sporadic retinoblastoma, which became known as the “Two Hits Theory.” According to this model, both familial and sporadic retinoblastoma occur through the same genetic mechanism; however, the sporadic form of the disease requires two mutational events in a specific gene, but the first hit was inherited from a relative in the familial form. This preliminary Knudson analysis served as the basis for the investigation of other tumors with a same pattern of inheritance. Hereditary cancers account for 5–10% of all tumors and have an autosomal dominant pattern of inheritance that predisposes patients to early, multiple, and different primary tumors (Nagy et al. 2004). In hereditary tumors, the genetic alteration affects TSG primarily, and the mutated allele, found in both germ and somatic cells, is related to an unstable genome. The following are examples of hereditary cancer syndromes: breast and ovarian cancer syndrome, polyposis hereditary syndrome, and hereditary nonpolyposis syndrome—Lynch syndrome. In breast and ovarian cancer syndrome, mutations occur mainly in genomic stability genes, BRCA1, and BRCA2. Mutations in BRCA1 and BRCA2 increase the probability of developing bilateral breast cancer by 30 and 10% by the age of 50 years for women and men, respectively. In polyposis hereditary syndrome, APC is mutated, which leads to disturbances in the WNT pathway and consequent stimulation of proliferation, migration, and degradation of the cell adhesion complex. In Lynch syndrome, the inherited

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mutations involve genes implicated in mismatch repair (Nagy et al. 2004; Foulkes 2008). Other mutations are also associated with cancer susceptibility: Gorlin syndrome (inherited mutations in the PTCH1 gene), Li–Fraumeni syndrome (p53 gene mutation), and xeroderma pigmentosum (mutations in DNA repair genes).

Epidemiology Cancer is a major public health problem worldwide. However, this disease differs locally and nationally, particularly when considering specific types of cancers (WHO 2007). Epidemiological data on the incidence and deaths caused by cancer vary in terms of coverage and quality of health care among the countries. In 2012, the World Health Organization estimated 14.1 million new cases of cancer worldwide. This number is predicted to increase to 24 million by 2035. In the United States, it is estimated that there will be 1,665,540 new cases of cancer, 13.7 million people living with cancer, and 585,720 cancer deaths, which is approximately 1,600 deaths per day, in 2014 (Siegel et al. 2014). The estimated new cases of major cancers worldwide and the United States are detailed in Table 1.1. Table 1.2 shows the estimated number of new cases of the most common cancers in men and women worldwide.

Environmental Risk Factors In a given population, the risk of cancer depends on social, environmental, political, and economic conditions as well as on the biological characteristics of the individual. Cancer is caused by both external factors (e.g., tobacco, infectious organisms, chemicals, and radiation) and internal factors (e.g., inherited mutations, hormones, immune conditions, and mutations that occur from metabolism). However, most cancers are caused by environmental factors, which pose a significant number of risks. These risk factors are responsible for the initiation, promotion, and progression of cancer (WHO 2007; American Cancer Society 2014). Tobacco is the leading cause of preventable deaths worldwide and is directly responsible for approximately 30% of all cancer-related deaths (Carbone 1992; Sasco et al. 2004). Smoking is the primary cause of many cancers including oral, laryngeal, lung, and esophageal. Furthermore, tobacco plays an important role in the development of other cancers, such as bladder, myeloid leukemia, pancreas, uterine cervix, and stomach cancers (Carbone 1992; Sasco et al. 2004; WHO 2007). By far, lung cancer represents the most striking risk imposed by cigarette smoking. Not only does lung cancer account for more deaths per year than any other type of neoplastic disease but 80% of all cases of lung cancer in the United States are believed to be the direct result of cigarette smoking (Carbone 1992).

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Table 1.1  Estimated number of new cases of major cancers worldwide and in the USA Cancer types New cases Worlda USAb Lung 1,825,000 224,210 Breast 1,677,000 235,030 Colorectal 1,361,000 136,830 Prostate 1,112,000 233,000 Stomach 952,000 22,220 Liver 782,000 33,190 Uterine cervix 528,000 12,360 Esophagus 456,000 18,170 Bladder 430,000 74,690 Non-Hodgkin’s lymphoma 386,000 70,800 Leukemia 352,000 52,380 Pancreas 338,000 46,420 Kidney 338,000 63,920 Corpus uteri (endometrium) 320,000 52,630 Lip, oral cavity 300,000 42,440 Thyroid 298,000 62,980 Brain, nervous system 256,000 23,380 Ovary 239,000 21,980 Melanoma of skin 232,000 76,100 Gallbladder 178,000 10,650 All types 12,360,000 1,513,380 a GLOBOCAN 2012, Cancer Incidence and Mortality Worldwide: International Agency for Research on Cancer b The American Cancer Society. Cancer Facts & Figs. 2014. Atlanta: American Cancer Society; 2014 Table 1.2  Estimated number of new cases of the most common cancers in men and women worldwide. Cancer type Cases % Cancer type Cases % (1,000 s)a (1,000 s)a Males ♂ Females ♀ Lung 1,242 22.3 Breast 1,677 34.2 Prostate 1,112 20 Colorectal 614 12.5 Colorectal 746 13.4 Lung 583 11.9 Stomach 631 11.3 Uterine cervix 528 10.8 Liver 554 10 Stomach 320 6.5 Bladder 330 5.9 Endometrium 320 6.5 Esophagus 323 5.8 Ovary 239 4.9 N.H. Lymphoma 218 3.9 Thyroid 230 4.7 Kidney 214 3.8 Liver 228 4.6 Leukemia 201 3.6 N.H. Lymphoma 168 3.4 All types 5,571 100 All types 4,907 100 N.H. non-Hodgkin’sa GLOBOCAN 2012, Cancer Incidence and Mortality Worldwide: International Agency for Research on Cancer

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Involuntary smoking, also called passive smoking, has several deleterious effects on people exposed to it. The chemical composition of secondhand tobacco smoke is quantitatively different from the smoke inhaled by the smoker during smoking, but it also contains several known carcinogens. Thus, nonsmokers exposed to secondhand tobacco smoke have a significant increase (20–30%) in lung cancer risk (Sasco et al. 2004). Alcohol consumption is also a major risk factor for cancers of the oral cavity, larynx, pharynx, esophagus, liver, colon, and rectum (Boffetta and Hashibe 2006; Schütze et al. 2011). The mechanisms by which alcohol exerts its carcinogenic effect have not been fully determined, but plausible events include the following: a genotoxic effect of acetaldehyde, the main metabolite of ethanol; a role as a solvent for tobacco carcinogens; production of reactive oxygen species and nitrogen species; and changes in folate metabolism (Boffetta and Hashibe 2006). Even if moderately consumed, alcohol can increase cancer risk, especially in organs that are part of the aerodigestive system. Several studies indicate that there is a synergistic effect of alcohol and tobacco, further increasing the risk of developing cancer (Marshall et al. 1992; Moreno-López et al. 2000). Diet has also been associated with the development of cancer, especially colon and rectal cancers. Diets low in fiber and high in fat and calories increase the risk of carcinogenesis, possibly because without fiber intake, intestinal pace slows down favoring a more prolonged mucosal exposure to carcinogens. Furthermore, the intake of fat may alter hormone metabolism in the blood, contributing to a breakdown of cellular homeostasis (Wu et al. 1987). Being overweight is a well-known risk factor for cardiovascular disease and diabetes, but epidemiological studies also provide growing evidence for a link between body weight and cancer risk. Thus, excess body weight has been directly associated with the risk of cancer at several organ sites, including the colon, breast (in postmenopausal women), endometrium, esophagus, and kidney (Bianchini et al. 2002; Wolin et al. 2010). In part, these associations with cancer risk may be explained by alterations in the metabolism of endogenous hormones, including sex steroids and insulin, which can lead to distortion of the normal balance between cell proliferation, differentiation, and apoptosis (Bianchini et al. 2002). Ultraviolet radiation (UV) is the principal risk factor for skin cancer (Gilchrest et al. 1999). Upon reaching the skin, UV rays penetrate deeply and trigger immediate reactions, such as burns and more delayed reactions, which have a cumulative effect. Thus, UV radiation contributes to genetic alterations that predispose an individual to cancer (Soehnge et al. 1997; Gilchrest et al. 1999). Some viral infections are risk factors for certain types of cancer (Parkin 2006). According to the American Cancer Society (2014), biological agents known to be carcinogenic in humans include the Epstein–Barr virus (EBV), hepatitis B and C viruses (HBV and HCV), human immunodeficiency virus (HIV), Kaposi's sarcomaassociated herpesvirus (KSHV), human T-lymphotropic virus (HTLV-1), certain types of human papillomavirus (HPV), and non-viral agents, such as Helicobacter pylori, which is associated with gastric cancer. Thus, chronic infections by these agents are associated with neoplastic development.

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Worldwide, approximately 52% of hepatocellular carcinomas are caused by HBV infection, and 20% of hepatocellular carcinomas are caused by HCV infection (Perz et al. 2006). Another viral infection related to cancer is HPV, specifically the high-risk genotypes 16 and 18. These genotypes cause almost all cases of cervical cancer (Schiffman et al. 2007), and they are also associated with the development of anal, vaginal, vulvar, and penile cancers (Muñoz et al. 2004; Watson et al. 2008). Recently, HPV infections, specifically HPV-16 infections, have been found to cause oral cavity and oropharynx cancers (Jayaprakash et al. 2011). Individuals who are infected with HIV have a higher risk of cancer. HIV infection, primarily through immunosuppression, leads to increased replication of oncogenic viruses such as EBV and KSHV (Bouvard et al. 2009). Some sexual habits increase the probability of exposure to carcinogenic viruses. Factors such as sexual promiscuity, early onset of sexual activity, and number of partners are all associated with an increased risk of cervical cancer (Parazzini et al. 1992). Potentially carcinogenic viruses that are sexually transmitted are HPV, HIV, HTLV-1, HBV, and HCV. Although a combination of screening and treatment is increasingly effective in reducing mortality from some cancers, limitations in the availability of clinical interventions for other cancers and access to and use of existing technologies clearly constrain the effects of treatment on population trends in cancer mortality, especially in underdeveloped countries. Thus, primary prevention through lifestyle and environmental interventions might offer the best option for reducing the large and increasing burden of cancers worldwide (Danaei et al. 2005). The study of risk factors, alone or in combination, has permitted the establishment of cause−effect relationships between these factors and certain types of tumors. Thus, the recognition of these risk factors as determinants in the process of carcinogenesis enables the development of strategies for prevention and early diagnosis of tumors, contributing to an increased life expectancy and improved quality of life.

Cancer Therapy An accurate cancer diagnosis and determination of clinical staging is essential for determining the appropriate treatment regimen for each patient. The treatment of cancer is based mainly on three methods: surgery/transplantation, radiotherapy, and chemotherapy. All treatment methods may be used for curative, palliative, or prophylactic purposes. Curative treatments are best reserved for localized tumors without metastases, and one or more methods can be combined to control and cure the disease. Palliative treatment is used to reduce the patients’ symptoms, such as severe pain and bleeding, and to improve their quality of life. In turn, prophylactic treatment is used to control a possible subclinical disease outside the primary tumor site, that is, no tumor present but the possible presence of dispersed neoplastic cells. In addition, these treatment methods may be indicated exclusively (primary

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treatment) or in combination with other therapeutic modalities (adjunctive treatment). Thus, radiotherapy or chemotherapy can be administered pre-surgery, for example, to decrease tumor size so that it can be removed completely by surgery or post-surgery to reduce the risk of recurrence (Holm et al. 1996). The efficacy of cancer therapy has been associated with a multidisciplinary approach and involves a combination of therapeutic modalities that vary depending on the patient’s cancer type.

Surgery/Transplantation Surgery was the first effective therapeutic modality for treating malignant neoplasms. Historically, Ephraim McDowell performed the first surgical excision of a tumor in ovary in 1809. Earlier in the seventh century, however, the ancient Egyptians described techniques for the removal of breast tumors (Hayward 1965). The latest advances in surgical techniques, knowledge of cancer biology, and a multidisciplinary approach has transformed surgical oncology. The choice of a surgical approach varies depending on the type of cancer and its anatomical extent, which necessitates that each patient has an individual surgical approach. Similarly, the effectiveness of surgical therapy depends on several factors, such as the diagnosis and staging of the tumor, understanding the biology of cancer, appropriate surgical techniques, and good postoperative care. Transplantation of bone marrow and liver has been an effective alternative for the treatment of some types of cancers. In this context, bone marrow transplantation or hematopoietic stem cell transplantation is commonly employed for the treatment of various hematological cancers, such as leukemia, lymphoma, and multiple myeloma. This transplant is performed after treatment with chemotherapy and/or radiotherapy (Chen et al. 2013). Liver transplantation is the most comprehensive treatment for liver cancer because it eliminates the tumor and the cause of the disease. Hepatocellular carcinoma was one of the first indications for liver transplantation; however, the success of the transplant depends on the tumor size (Clavien et al. 2012). The indications for liver transplantation are based on the Milan criteria that include the presence of only one lesion < 5 cm or up to three lesions < 3 cm (Mazzaferro et al. 1996).

Radiotherapy In 1895, Wilhelm Conrad Roentgen published the first description of X-rays, which showed a qualitative experimental characterization of the new radiation (Langland and Langlais 1995). In 1896, an American physician, Emil Grubbé, used recently discovered X-rays in an attempt to control the recurrence of breast cancer. Despite being first used as an empirical treatment without any scientific basis, radiotherapy has become a medical specialty in the early twentieth century (Brady et al. 2001).

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Radiotherapy is a therapeutic modality that involves clinical, physical, and biological knowledge with the aim of treating malignant tumors by ionizing radiation. Radiation doses and time of application are calculated according to the type and size of the tumor so that the radiation is effective in destroying neoplastic cells with the least possible damage to the surrounding normal cells, which will regenerate in the irradiated area (van Loon et al. 2012). Ionizing radiation is electromagnetic or corpuscular and carries energy. By interacting with the tissue, the radiation gives rise to electrons, which ionize and create various chemical effects such as water hydrolysis and DNA-strand breaks. Thus, cell death can occur by different mechanisms resulting in the inactivation of vital cell systems that prevent the cell from dividing. The tissue response to radiation depends on several factors, such as tumor sensitivity to radiation, its location and oxygenation, as well as the quality and quantity of radiation and the total time taken for administration (American Cancer Society 2013). Ionizing radiation most commonly used in medical practice is electromagnetic in nature, such as X-rays and gamma rays, and corpuscular, such as electrons, protons, and neutrons. Depending on how it is applied, radiotherapy can be internal, known as brachytherapy, where radiation sources are placed within the tumor or around them, or external, known as teletherapy, where the radiation source is far from the patient (Short and Griffiths 1996). The type of radiotherapy chosen depends on factors such as the type of cancer, tumor location, tumor stage, and the patient's general health. However, to biologically impact the greatest number of neoplastic cells without affecting normal tissues, the total radiation dose applied is generally fractionated into equal daily doses when using external therapy (Cooper et al. 2004). The collateral effects of treatment with ionizing radiation can be classified as immediate or delayed. Immediate effects are those observed in tissues that have higher proliferative capacity, such as the gonads, skin, stomach, urinary and intestinal mucosa, and bone marrow. Conversely, mediate effects occur when radiation doses exceed in normal tissue causing atrophy and fibrosis (Holm et al. 1996). However, collateral effects will occur only in tissues that are included in the irradiation field (Lindberg et al. 1981) and can be aggravated by the simultaneous administration of chemotherapy (Cooper et al. 2004).

Chemotherapy In 1955, the National Cancer Institute (NCI) of the Unites States of America began a large-scale screening program for the discovery and development of cancer chemotherapies. In first 25 years, the animal leukemia L1210 model was used. This model of screening against a rapidly growing animal leukemia resulted in the preferential selection of drugs that were active against only rapidly growing tumors. As a consequence, the clinical response of human leukemias and lymphomas but not solid tumors improved substantially. In the following years, the division of cancer treatment at the NCI introduced to its cancer cell line panel solid tumor models that were representative of the most prevalent histological types of cancer. Next, the growth

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Principles of Cancer Pathogenesis and Therapies: A Brief Overview

Table 1.3  Major classes of natural anticancer agents. Classes of anticancer Agents drugs Taxanes Paclitaxel and docetaxel Vinca alkaloids Camptothecins Epipodophyllotoxin Cytotoxic antibiotics

Vinblastine, vincristine, and vinorelbine Irinotecan and topotecan Etoposide and teniposide Doxorubicin, mitomycin, idarubicin, bleomycin, and dactinomycin

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Mechanism of action Stabilizes tubulin polymerization Inhibits tubulin polymerization Inhibits topoisomerase I Inhibits topoisomerase II Oxygen free radicals bind to DNA causing single- and double-stranded DNA breaks; some of them inhibit topoisomerase II and/or intercalate into the DNA

of human tumor xenografts became available (Suggitt and Bibby 2005; DeVita and Chu 2008). This model continues to be used today. In recent years, new classes of anticancer agents have improved cancer treatments in patients. Currently, the major classes of anticancer agents include the following: antimetabolites (5-fluorouracil, gemcitabine, methotrexate, 6-mercaptopurine, etc.); alkylating agents (cyclophosphamide, dacarbazine, carmustine, etc.); epipodophyllotoxins (etoposide and teniposide); taxanes (paclitaxel and docetaxel); vinca alkaloids (vinblastine, vincristine, and vinorelbine); cytotoxic antibiotics (doxorubicin, mitomycin, bleomycin, dactinomycin, etc.); camptothecins (irinotecan and topotecan); platinum analogs (cisplatin, carboplatin, and oxaliplatin); hormones/antagonists (tamoxifen, cyproterone, flutamide, etc.); and the newer anticancer drugs, such as protein kinase inhibitors (erlotinib, imatinib, sorafenib, etc.); and monoclonal antibodies (bevacizumab, panitumumab, trastuzumab, etc.; Chabner and Longo 2006; Chu and DeVita 2009; Gomez-Martín et al. 2014; Akhtar et al. 2014; Grisham et al. 2014; Jaffe 2014; Kuo et al. 2014). Natural products (epipodophyllotoxins, taxanes, vinca alkaloids, camptothecins, and cytotoxic antibiotics) are among the most effective cancer chemotherapeutics currently available (Cragg et al. 2009; Newman and Cragg 2012; Cragg and Newman 2013; Cragg et al. 2014). Table 1.3 summarizes the major classes of natural products that are used as anticancer agents. In cancer chemotherapy, anticancer drugs are often used in combinations. This type of treatment provides maximal killing of tumor cells in a tolerable toxicity range. Moreover, this allows the interaction of drugs with different mechanisms of action to target a heterogeneous population of tumor cells with different genetic abnormalities. Finally, it may prevent or slow down the subsequent development of cellular drug resistance (Chabner and Longo 2006; DeVita et al. 2008). Cancer chemotherapy is also associated with a high incidence of toxicity. In fact, the primary objective of chemotherapy is to destroy the cancer cells while preserving the normal tissue; however, most chemotherapeutic agents act non-specifically and damage both malignant and normal cells. This explains most of the observed

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side effects of chemotherapy, such as nausea, hair loss, and increased susceptibility to infections. Because of the high rates of toxicity, hospital admissions are often necessary for the treatment of complications (especially infections), which pose an imminent risk and interruption of chemotherapy, thus increasing the probability that the chemotherapy will be ineffective (Chabner and Longo 2006; DeVita et al. 2008). As current anticancer drugs have high toxicity and high resistance rates and are ineffective in some types of tumors, the discovery and development of new classes of chemotherapies are needed. Additionally, patients should be treated in a multidisciplinary and humanized way and assisted by experts from different areas with the common goal of improving the patient’s well-being and quality of life.

Conclusion In conclusion, scientific and technological progress in all areas of oncology has provided earlier and more accurate diagnoses, which enables improved cancer treatment in patients. Moreover, new classes of anticancer agents have been effective. In this context, natural products are among the most effective cancer chemotherapeutics that are currently available. Prevention of new cases of cancer, increased survival, and improved quality of life for people with cancer are the current challenges for oncology research.

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Chapter 2

Essential Oils Chemistry Mónica Zuzarte and Lígia Salgueiro

What are Essential Oils? Essential oils, also known as essences, volatile oils, etheric oils, or aetheroleum, are natural products formed by several volatile compounds (Sangwan et al. 2001; Baser and Demirci 2007). According to the International Standard Organization on Essential Oils (ISO 9235: 2013) and the European Pharmacopoeia (Council of Europe 2004) an essential oil is defined as the product obtained from plant raw material by hydrodistillation, steam distillation or dry distillation or by a suitable mechanical process (for Citrus fruits). Cold pressing without heat is usually used for Citrus fruit oils because their constituents are thermosensitive and unstable, converting into artifacts under heat and pressure. Moreover, essential oils are frequently associated with gums and resins that are separated by the distillation process (Baser and Demirci 2007). The definition of an essential oil excludes other aromatic/volatile products obtained by different extractive techniques like extraction with solvents (concretes, absolutes), supercritical fluid extraction, and microwave-assisted extraction. Essential oils also differ from fixed oils or fatty oils in both chemical and physical properties. Fatty oils contain glycerides of fatty acids and leave a permanent stain on filter paper, whereas essential oils contain volatile compounds and vanish rapidly without leaving any stain. In nature, essential oils play very important roles in plant defense and signaling processes (Harborne 1993; Bowsher et al. 2008; Taiz and Zeiger 2010). For example, essential oils are involved in plant defense against microorganisms, insects, and herbivores, attraction of pollinating insects and fruit-dispersing animals, water regulation and allelopathic interactions (Fahn 1979; Harborne 1993; M. Zuzarte () · L. Salgueiro Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal e-mail: [email protected] L. Salgueiro e-mail: [email protected] © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_2

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Pichersky and Gershenzon 2002; Bakkali et al. 2008). Also, they are valuable natural products used as raw materials in many fields, such as pharmaceutical, agronomic, food, sanitary, cosmetic, and perfume industries (Buchbauer 2000). Essential oils can be found in various plant organs (flowers, fruits, seeds, leaves, stems, and roots) being produced and stored in secretory structures that differ in morphology, structure, function, and distribution. These specialized structures minimize the risk of autotoxicity and can be found on the surface of the plant organs or within the plant tissues, being classified as external or internal secretory structures, respectively. Internal secretory structures include secretory cells (often idioblasts), secretory cavities, and secretory ducts whereas external ones include glandular trichomes, epidermal cells, and osmophores (Svoboda and Svoboda 2000). Some plant organs and tissues, such as roots, tubers, and wood, are very hard and need to be broken down to expose the oil-containing cells and cavities for extraction. Essential oils are complex mixtures of volatile (around 100 u) to semi-volatile compounds (around 300 u), usually with a strong odor, rarely colored, soluble in organic solvents, and insoluble in water. They comprise volatile compounds of terpenoid and non-terpenoid origin, synthetized through different biosynthetic routes and with distinct primary metabolic precursors. Terpenoids biosynthesis involves both the mevalonate and non-mevalonate (deoxyxylulose phosphate) pathways, whereas phenylpropanoids are formed via the shikimate pathway (Litchenthaler 1999; Dewick 2002a; Baser and Demirci 2007; Sell 2010). Monoterpenes and sesquiterpenes are usually the main group of compounds found in essential oils. In addition, phenylpropanoids are also very frequent. Moreover, some essential oils may also contain fatty acids and their esters and, more rarely, nitrogen and sulfur derivatives (Baser and Demirci 2007; Bakkali et al. 2008). In aromatic plants, the composition of essential oils usually varies considerably because of both intrinsic (sexual, seasonal, ontogenetic, and genetic variations) and extrinsic (ecological and environmental aspects) factors (Figueiredo et al. 2008a; Taiz and Zeiger 2010). Genetic variations may result in the expression of different metabolic pathways and, consequently, quantitative and qualitative variations in essential oil composition may occur. When significant differences are found, an intraspecific category (chemotype) is defined. Essential oil quality strongly depends on all these factors that may interfere and also limit plant yield. Analytical guidelines published by several institutions such as the European Pharmacopoeia, International Standard Organization (ISO), and World Health Organization (WHO) are available and must be followed to assure the good quality of the commercialized essential oils and of the plants from which they are obtained. In general, the industries choose the chemotypes that have most commercial interest, in order to obtain high-quality end products as well as efficient biological activities. Quality assessments of essential oils include sensory evaluations, very common in perfumery houses; physical and chemical tests, required in standards, pharmacopoeias, and codices; and chromatospectral techniques for oil analysis. Hyphenation of gas chromatography (GC) separation step with spectroscopic techniques is often required for accurate compound identification, gas chromatography–mass spectrometry (GC–MS) being one of the most popular hyphenated techniques for

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characterization and identification of complex volatile compounds. A flame ionization detector is usually used for quantitative analysis, while a quadropole mass detector or ion-trap detector is necessary to characterize essential oil constituents (Baser and Demirci 2007). Identification of the compounds is made by comparison of both chromatographic data (e.g., Kováts indices and linear retention indices) and mass spectra data with those of authentic samples and library reference spectra. Notwithstanding the achievements in analytical techniques, the total separation and identification of all compounds of the volatile mixture remains unattainable because of the large number of compounds, structural similarities, isomeric forms, and concentration range of the compounds present in essential oils (Gomes da Silva et al. 2008). In this way, similar retention times may occur and confirmation on two columns of different polarity is advised in order to avoid misleading identifications. Taking into account that essential oils may contain hundreds of constituents, co-elutions are inevitable and therefore new analytical strategies have been developed to maximize compound separation, namely multidimensional GC (MD-GC) and comprehensive two-dimensional GC (GCxGC) (Gomes da Silva et al. 2008). Moreover, compounds with similar mass spectra and identical retention indices make essential oil characterization a very difficult task. In these cases, other methodologies like GC in tandem with Fourier transform infrared (GC-FTIR) and nuclear magnetic resonance spectroscopy (13C-NMR) should be considered (Gomes da Silva et al. 2008; Tomi et al. 1995). Several plant families comprise well-known aromatic species, many of them included in the Generally Recognized as Safe (GRAS) list fully approved by the US Food and Drug Administration (FDA) and Environmental Protection Agency (EPA, USA) for addition to food and beverages. The major essential oil bearing plant families include Apiaceae, a widely distributed group of annual, biennial, and perennial plants, with essential oils in tubular ducts; Asteraceae, comprising over 30,000 species of evergreen shrubs, rhizomatous herbs, tuberous perennials, and tree herbs; Cupressaceae, a group of conifers usually resinous trees and shrubs producing essential oils within woods; Lamiaceae, a very diverse group of aromatic herbs and shrubs with volatile compounds normally accumulated in glandular trichomes; Lauraceae, comprising flowering plants and a number of aromatic trees with volatiles present in cells within the bark and wood; Myrtaceae, a highly aromatic group, including several fruit species; Pinaceae, a group of high growing conifers with resinous aromatic materials with acids, turpentine, and terpenoids; Piperaceae, a small family of flowering plants; Santalaceae with only a few aromatic species of interest; and Zingiberaceae, the ginger family with several aromatic rhizomes (Hunter 2009).

Biosynthetic Pathways In nature, two main groups of metabolites can be found: primary and secondary metabolites. Primary metabolites are universal compounds, present in all living organisms, and include proteins, carbohydrates, lipids, and nucleic acids. Secondary

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M. Zuzarte and L. Salgueiro

metabolites are found only in some species and are classified as terpenoids, shikimates, polyketides, and alkaloids, the first two being the most relevant in essential oils (Sell 2010). Although terpenoids are more frequent and abundant in essential oils, certain species contain high quantities of shikimates, namely phenylpropanoids and when these compounds are present, they provide specific odor and flavor to the plants (Sangwan et al. 2001).

Terpenes Terpenes result from the condensation of a pentacarbonate unit with two unsaturated bonds, isoprene (2-methyl-1,3-butadiene), and therefore are many times called isoprenoides. The designation “terpenes” was first used by Kekulé in 1880 to name C10H16 compounds found in turpentine (Baser and Demirci 2007). In 1887, his assistant Otto Wallace formulated the “isoprene rule” suggesting that terpenes were formed by two or more isoprene units. Later, Robinson suggested that the isoprene units were connected in a head-to-tail way (Fig. 2.1). In 1950, Leopold Ruzicka replaced this rule by the “biogenetic isoprene rule” which states that a compound is an isoprenoid if it is derived biologically with or without rearrangements from an isoprenoid precursor (Little and Croteau 1999). In summary, terpenoids are derived from aliphatic precursors such as geraniol for the formation of monoterpenes, farnesol for sesquiterpenes, and geranylgeraniol for diterpenes (Baser and Demirci 2007). Terpenes are classified into different structural and functional classes. According to the number of isoprene units in their structure, terpenes can be classified into hemiterpenes (1 unit), monoterpenes (2 units), sesquiterpenes (3 units), diterpenes (4 units), and so on. The terpenes most often found in essential oils are monoterpenes (C10H16) and sesquiterpenes (C15H24). These compounds have many isomeric cyclic or linear structures, various degrees of unsaturations, substitutions, and oxygenated derivatives, being generally called terpenoids.

Fig. 2.1 Head-to-tail coupling of two isoprene units to form linalyl acetate

OAc OAc

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The biosynthesis of terpenes involves two universal precursors: isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP). In higher plants, IPP is biosynthesized through two pathways: the mevalonate pathway (MVA) and the non-mevalonate (mevalonate independent) or deoxyxylulose phosphate pathway, schematized in Fig. 2.2.

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24

M. Zuzarte and L. Salgueiro

In the mevalonate pathway, IPP is formed through mevalonic acid that results from the condensation of 3 acetylcoenzyme-A moieties. In the non-mevalonate pathway, 2 C-metil-D-erythritol-4-phosphate (MEP) and 1-deoxy-d-xylulose5-phosphate (DOXP) are involved, resulting from the condensation of glyceraldehyde phosphate and pyruvate (Baser and Demirici 2007). The former takes place in the cytoplasm and leads to the formation of most sesquiterpenes whereas the latter occurs in the chloroplasts, producing primarily monoterpenes and diterpenes (Bouwmeester 2006). IPP and DMAPP lead to geranyl diphosphate (GPP), the immediate precursor of monoterpenes. The condensation of GPP with IPP leads to farnesyl diphosphate (FPP), the immediate precursor of sesquiterpenes, and the condensation of FPP with IPP results in geranyl geranyl diphosphate, the precursor of phytol, other diterpenes, and carotenoids. Monoterpenes and sesquiterpenes are the main compounds found in essential oils (Bakkali et al. 2008). Heavier terpenes, such as diterpenes, may also be present but usually do not contribute to the odor of essential oils (Hunter 2009).

Phenylpropanoids Phenylpropanoids contain one or more C6–C3 units, the C6 being a benzene ring. Many of the phenylpropanoids found in essential oils are phenols or phenol ethers and in some cases, the side chain is shortened (C1) as, for example, in methyl salicylate and vanillin (Tyler et al. 1988). Phenylpropanoids are synthetized via the shikimic acid pathway (Fig. 2.2), their main precursors being cinnamic acid and p-hydroxycinnamic acid, originated from the aromatic amino acids phenylalanine and tyrosine, respectively (Dewick 2002a; Sangwan et al. 2001). Shikimic acid is synthesized from erythrose 4-phosphate and phosphoenolpyruvate. Elimination of one of the ring alcohols of shikimic acid and reaction with phosphoenol pyruvate gives chorismic acid. This compound forms the phenylpropionic acid skeleton. Amination and reduction of the ketone function produces the amino acid phenylalanine while reduction and elimination leads to cinnamic acid that produces oand p-coumaric acids. Moreover, aromatization of shikimic acid gives benzoic acid derivatives, present in several essential oils (Sell 2010).

Chemical Composition The constituents of plant essential oils fall mainly into two distinct chemical classes: terpenoids and phenylpropanoids. Terpenoids are extremely variable, showing different carbon skeletons and a wide variety of oxygenated derivatives, including alcohols, esters, aldehydes, ketones, ethers, peroxydes, and phenols. There is a very little difference between the molecular weights of terpenes and their oxygenated products. The similarity of many of these structures reflects the difficulty of their chemical characterization. There is also the problem of stereoisomerism, whereby

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one or more groups are arranged in a mirror image style in space compared with their isomer. Both enantiomers (optically active isomers) of many terpenoids occur in nature. However, some species produce only one enantiomer whereas in other cases both are produced (racemic mixture). Examples of the most relevant essential oil constituents, namely hydrocarbon and oxygenated derivatives of monoterpenes, sesquiterpenes, and phenylpropanoids, are presented in detail. Moreover, other compounds found in essential oils such as diterpenes, sulfur- and nitrogen-containing constituents and lactones are also referred.

Monoterpenes (C10 H16 ) Monoterpenes can be found in nearly all essential oils and are the most representative constituents, attaining around 90% of many oils (Bakkali et al. 2008). They are formed by the attachment of two isoprene units (10 carbon atoms and at least one double bond). These compounds oxidize easily because of their rapid reaction to air and heat sources. Monocyclic monoterpenic hydrocarbons are the most common in essential oils but linear (acyclic) and bicyclic compounds also occur. The main linear monoterpenic hydrocarbons found in essential oils have a typical 2,6-dimethyloctane structure with three double bonds while bicyclic compounds have a second ring with three, four, or five carbons besides the hexane ring. Biochemical modifications including oxidations or rearrangements produce several other compounds, generally called monoterpenoids. These compounds include highly functionalized chemical entities (Hunter 2009).

Hydrocarbons Limonene Terpene hydrocarbon widely spread among essential oils, being very abundant in citrus oils. (+)-Limonene, the enantiomer of orange and lemon peels, has a lemon-like odor making it attractive as an additive in cosmetics and foods (Kim et al. 2013). This enantiomer is the most abundant in plants. It is widely used in household and industrial cleaning solvents, as a paint stripper and botanical insecticide as well as in food flavorings (Hunter 2009). Racemic limonenes are commercially available under the name dipentene (Bauer and Garbe 2001).

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M. Zuzarte and L. Salgueiro

β-Myrcene Has a fresh citrus odor occurring in several essential oils like hop – Humulus lupulus (Vázquez-Araújo et al. 2013), thyme – Thymus serpyllum (Raal et al. 2004), and Margotia gummifera (Valente et al. 2013). This compound is very relevant in perfumery as an important intermediate in the synthesis of menthol, citral, citronellol, citronellal, geraniol, nerol, and linalool as well as vitamins A and E (Behr and Johnen 2009). The isomer α does not occur in nature.

Phellandrene  (−)-α-Phellandrene is primarily found in dill – Anethum graveolens and in Eucalyptus dives oils. It has a citrus odor with a slight peppery note. (−)-β-Phellandrene is characteristic of lodgepole pine – Pinus contorta (Sell 2010) and sea fennel – Crithmum maritimum oils (Ozcan et al. 2006).

α

β

Pinene There are two structural isoforms found in nature: α- and β-pinenes. Pinenes are the most important naturally occurring hydrocarbons (Bauer and Garbe 2001). Both l- and/or d-forms as well as racemic forms may occur. As the name suggests, both isoforms are important constituents of pine resin as well as resin of many other conifers. High concentrations of these compounds are also present in a wide variety of essential oils such as ironwort – Sideritis erythrantha (Kose et al. 2010), sage – Salvia rosifolia (Ozek et al. 2010), lemon – Citrus limon (Vekiari et al. 2002), Eucalyptus sp. (Juan et al. 2011), juniper – Juniperus communis berries and needles (Gonny et al. 2006), and rosemary – Rosmarinus officinalis (Wang et al. 2012) oils. The fresh pine odor makes pinenes interesting compounds for household perfumery. α-Pinene is also used in the synthesis of other compounds like terpineol, borneol, and camphor (Bauer and Garbe 2001).

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α

β

Ocimene β-Ocimene ( cis and trans) is the isomer most frequently found in essential oils like Lavandula multifida. It has a pleasant odor highly appreciated in perfumery and has showed a potent effect on the inhibition of Candida albicans filamentation (Zuzarte et al. 2012b).

Sabinene An important compound in carrot – Daucus carota seed (Marzouki et al. 2010) and in Juniperus communis oils (Ottavioli et al. 2009) that also contributes to the spiciness of black pepper. This compound is used as a perfume additive (Zhang et al. 2014) and has been explored as a component for the next generation of aircraft fuels (Rude and Schirmer 2009).

Terpinene α-Terpinene is an important compound in cardamom – Elettaria cardamomum oil (Abbasipour et al. 2011) and is one of the compounds responsible for the antioxidant activity of tea tree – Melaleuca alternifolia oil (Rudbäck et al. 2012). γ-Terpinene has a herbal citrus odor and occurs frequently in several Thymus

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species (Jamali et al. 2012). δ-Terpinene or terpinolene is relevant in tea tree oil (Homer et al. 2000). It has a sweet piny odor with citrus characteristics and is used in household perfumes (Hunter 2009).

α

γ

δ

Oxygenated derivatives Ascaridole A terpene peroxide with a pungent smell and taste. Present, as a major constituent, in wormseed – Chenopodium ambrosioides oil. Ascaridole has anthelmintic properties but is very toxic to mammals, and oils with this compound should be treated carefully since they can explode when heated or treated with acids (Harborne and Baxter 2001).

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Borneol  Occurs  as  an  enantiomer  or  a  racemic  mixture.  (−)-Borneol  is  found  in Pinus sp., Abies nordmanniana, and Artemisia sp. oils. (+)-Borneol occurs in camphor – Cinnamomum camphora, rosemary – Rosmarinus officinalis, lavender – Lavandula sp., and olibanum – Boswellia sp. oils. The (+) isomer has a more evident camphor-like odor, with a slightly sharp, earthy-peppery note (Bauer and Garbe 2001).

Bornyl Acetate A characteristic component in several pine – Pinus sp. oils (Wagner and Bladt 2009). Has a pleasant pine needle odor and is used for perfuming soaps, bath products, air refreshers, and pharmaceutical products (Bauer and Garbe 2001).

Camphor A bicyclic monoterpene extracted from Cinnamomum camphora (Hunter 2009). Solid and with a characteristic penetrating camphoraceous odor. Used as a plasticizer (Bauer and Garbe 2001) and in medicinal preparations, low-cost perfumes, shoe polish, and as a solvent for paints (Hunter 2009).

Carvacrol An isomer of thymol found in high amounts in savory – Satureja hortensis and S. montana (Bauer and Garbe 2001), oregano – Origanum vulgare (Teixeira et al. 2013), O. virens (Salgueiro et al. 2003), lavender – Lavandula multifida (Zuzarte et al. 2012b), and thyme – Thymus sp. (Figueiredo et al. 2008b) oils. Has a dry medicinal, herbaceous, phenolic odor and is widely used in dental preparations as an antiseptic (Hunter 2009). Carvacrol is irritating and can produce sensitization. It lends a moderate toxicity to those oils containing it in quantity.

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Carvone  (−)-Carvone has a herbal scent and is found in caraway seed and dill oils;  (+)-carvone with a spicy-minty odor can be found in spearmint – Mentha spicata, Eucalyptus sp., and mandarin – Citrus reticulata oils (Hunter 2009; Bauer and Garbe 2001). Used in food flavor and fragrances (Hunter 2009).

1,8-Cineole (Eucalyptol) Obtained primarily by isolation from Eucalyptus sp. oils. Also present in high amounts in tea tree – Melaleuca alternifolia and sage – Salvia officinalis oils (Hunter 2009). Has a characteristic camphoraceous odor and a pungent, cooling, spicy taste. Used in a wide variety of products such as nasal inhalers and sprays, external analgesics, and mouthwashes (Tyler et al. 1988).

Citral Formed by two isomers, geranial (citral a) and neral (citral b). In the isomeric mixtures geranial is usually the predominant isomer. An important compound of several essential oils including lemon myrtle – Backhousia citriodora, lemongrass – Cymbopogon citratus, exotic verbena – Litsea cubeba and Citrus sp. fruits (Hunter 2009). Its strong lemon scent (Breitmaier, 2006) makes it an interesting

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compound in perfumery and food industries, as a flavor. It is also used as a starting material in the synthesis of β-ionone and vitamin A (Bauer and Garbe 2001).

geranial

neral

Citronellal (+)-Citronellal is present in citronella – Cymbopogon sp. oils. (−)-Citronellal in lemon myrtle – Backhousia citriodora oil and the racemic mixture in lemon-scented eucalyptus – Eucalyptus citriodora oil (Bauer and Garbe 2001). Compound with a lemon citronella, slightly rosy aroma (Hunter 2009) widely used as a repellant (Kim et al. 2005) and starting material for the production of isopulegol, citronellol, and hydroxydihydrocitronellal (Bauer and Garbe 2001). It is a main constituent in many pharmaceutical preparations as a mild sedative or stomachicum. Also used in household and industrial perfumes (Hunter 2009).

Citronellol Occurs in a number of species including citronella – Cymbopogon sp., geranium – Pelargonium graveolens, oakmoss – Evernia prunastri, palmarosa – Cymbopogon martinii, rose – Rosa sp., and several Eucalyptus species. Has a sweet rose-type odor and is widely used as a fragrance compound (Bauer and Garbe 2001).

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Fenchone Occurs in high amounts in several lavender species such as Lavandula pedunculata (Zuzarte et al. 2009), Lavandula stoechas subsp. stoechas (Tzakou et al. 2006), and bitter and sweet fennel – Foeniculum vulgare (Coşge et al. 2008) oils. It has a camphoraceous odor and is used to prepare artificial fennel oils and in technical perfumery (Bauer and Garbe 2001).

Geraniol A major compound of citronella – Cymbopogon sp., citronella java – C. winterianus, rose – Rosa sp., palmarosa – Cymbopogon martinii, and geranium – Pelargonium graveolens oils. In nature, geraniol is often found with its isomer nerol  ( cis), name derived because of its occurrence in neroli – Citrus aurantium subsp. amara oil (Bauer and Garbe 2001). Geraniol has a sweet rose-type odor being widely used in floral and oriental fragrances (Hunter 2009), as a flavor agent in flavors like peach, raspberry, grapefruit, red apple, plum, lime, orange, lemon, watermelon, pineapple, and blueberry and as a mosquito repellant (Barnard and Xue 2004).

geraniol

nerol

Linalyl Acetate Important compound in lavender – Lavandula angustifolia (Prashar et al. 2004) and bergamot – Citrus bergamia (Nabiha et al. 2010). Sweet fruity aroma that resembles terpenless bergamot oil (Hunter 2009). Widely used in perfumery for bergamot, lilac, lavender, linden, neroli, and ylang-ylang notes (Bauer and Garbe 2001).

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Linalool An important compound in several essential oils including English lavender – Lavandula angustifolia (Prashar et al. 2004), bay laurel – Laurus nobilis (Saab et al. 2012), sweet basil – Ocimum basilicum (Hussaina et al. 2008), coriander – Coriandrum sativum seed (Bauer and Garbe 2001), Cymbopogon sp. (Ganjewala 2009), and sweet orange – Citrus sp. flower (Miguel et al. 2008) oils. This compound has a fresh and light floral aroma with a slight citrus impression reminiscent of lily of the valley and lavender (Hunter 2009). Widely used in perfumery, soaps and detergents, as a fixative, and in the synthesis of vitamin E (Bauer and Garbe 2001; Hunter 2009).

Menthofuran Found in several mint species such as Mentha aquatica (Andro et al. 2013) and in very low amounts in pennyroyal – M. pulegium (Derwich et al. 2010) and peppermint – M. × piperita (Saharkhiz et al. 2012). It contributes to the odor of peppermint (Sell 2010). Menthofuran is a toxic metabolite of pulegone. It is hepatotoxic and destroys the enzyme cytochrome P 450. Therefore, the amounts of menthofuran present in peppermint oil should be less than 9% (Council of Europe 2004).

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Menthol A solid compound at room temperature, present in several Mentha species. Currently, menthol is isolated from Mentha canadensis ( Mentha arvensis) and primarily used as a flavoring additive in a variety of products (Kamatou et al. 2013). It is known for causing a cooling sensation when inhaled, eaten, or applied to the skin (Eccles 1994) and has been suggested to possess several biological properties, including antibacterial, antifungal, antipruritic, anticancer, and analgesic effects (Kamatou et al. 2013). Used in after shaves and eau de colognes, balms, cough medicines, topical analgesics, mouthwashes, and as a flavor agent in gums and toothpastes (Hunter 2009).

Menthone Exists as two isomers, menthone and isomenthone. These compounds are found in Mentha × piperita (peppermint) and M. spicata (spearmint) oil. (Bauer and Garbe 2001). Compound with a refreshing menthol odor that resembles peppermint, with slight wood traces (Hunter 2009). Used for synthetic peppermint oils and bases.

Pulegone A major component of pennyroyal – M. pulegium (Sardashti and Adhami 2013) essential oil. In higher doses, pennyroyal oil resulted in central nervous system toxicity, hepatic and renal failure, pulmonary toxicity and death. Pulegone is hepatotoxic because it is metabolized to epoxides. It also has a folklore history as an abortifacient (Dewick 2002b). It is used in perfume compositions for soap and mouth care products (Bauer and Garbe 2001).

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Terpineol Terpenoid alcohol that exists in three isomeric forms (α, β, and γ), α-terpineol being found in higher concentrations. A major component of thyme – Thymus caespititius (Salgueiro et al. 1997; Pereira et al. 2000) and black cardamom – Amomum subulatum (Joshi et al. 2012) oils. Based on its pleasant odor similar to lilac, α-terpineol is widely used in the manufacture of perfumes, soaps, cosmetics, and antiseptic products (Hunter 2009).

Terpinene-4-ol An important constituent of tea tree – Melaleuca alternifolia (Carson et al. 2006), juniper – Juniperus wallichiana (Lohani et al. 2013), and Pandamus odoratissimus (Raina et al. 2004) oils. It is the primary antibacterial component of tea tree oil ( M. alternifolia) (Dewick 2002b) being used in cosmetic and pharmaceutical preparations (Hunter 2009). Also used in artificial geranium and pepper oils and sometimes in perfumery creating herbal and lavender notes (Bauer and Garbe 2001).

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Thujone Terpene ketone existing in two isomeric forms, α-thujone and β-thujone. The isomer ratio depends on the plant source, with high content of α-thujone in cedar – Cedrus sp. leaf oil and β-thujone in wormwood – Artemisia absinthium oil. The α-isomer is more toxic than the β one and is the active ingredient of the alcoholic beverage absinthe, a drink banned in most countries (Dewick 2002b; Pinto et al. 2007). These compounds have a herbaceous aroma similar to Artemisia and are also used in fine fragrances (Hunter 2009).

α

β

Thymol Monoterpene phenol characteristic of thyme – Thymus sp. (Figueiredo et al. 2008b; Mota et al. 2012), oregano ( Origanum sp.) and ajowan seed and foliage (Davazdahemami et al. 2011) oils. It has a spicy-herbal, slightly medicinal aroma, reminiscent of thyme. Used in men’s fragrances, in soaps and household products, as a disinfectant in mouth care products, and as a fungicide in some medicinal products and some cosmetics (Bauer and Garbe 2001; Hunter 2009). Thymol is also used as a flavor additive in a number of foods and beverages. Several studies have shown its potent antimicrobial effect against several human and foodborne pathogens.

Sesquiterpenes (C15 H24 ) Farnesol is the precursor of all sesquiterpenoids. These compounds are formed by three isoprene units. This results in lower volatilities and higher boiling points than monoterpenes and allows several cyclizations responsible for the high diversity of structures (Sell 2010). Besides linear compounds, monocyclic, bicyclic, and tricy-

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clic structures can occur. Sesquiterpenes are unsaturated compounds (Baser and Demirci 2007). Examples of important sesquiterpenes found in essential oils are presented.

Hydrocarbons Cadinene Name derived from Cade juniper – Juniperus oxycedrus since the wood of this species yields an oil from which cadinene isomers (α, γ, and δ) were first isolated. A mixtute of cadinene isomers is used as a flavoring agent/flavor modifier (Yannai 2004).

α

γ

δ Caryophyllene β-Caryophyllene is the most widely distributed isomer, usually together with isocaryophyllene (Hunter 2009). It has a clove-type turpentine odor and occurs as an important compound in the essential oils of different spice and food plants, such as oregano – Origanum vulgare (Mockute et al. 2001), cinnamon – Cinnamomum zeylanicum (Jayaprakasha et al. 2003), and black pepper – Piper nigrum (Orav et al. 2004) as well as in Cannabis sativa (Hendriks et al. 1975) and Humulus lupulus (Katsiotis et al. 1990) oils. Used as a food additive and in cosmetics (Skold et al. 2006).

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Chamazulene The blue coloring principle of chamomile oil, with a very unusual molecular structure (seven-membered ring fused to a five-membered ring). This compound is formed from matricine during distillation (Baser and Demirci 2007). Chamazulene contributes to the anti-inflammatory activity of chamomile extracts by inhibiting leukotriene synthesis (Safayhi et al. 1994).

Elemene Different isomers (α-, β-, γ-, and δ-elemenes) occur in a variety of plant oils, contributing to their aroma and are used as pheromones by some insects. β-Elemene found in high amounts in Curcuma aromatica oil is one of the most relevant. A recent review pointed out its use as an adjunctive treatment in lung cancer since the effectiveness of chemotherapy seems to improve when combined with an injection of this compound (Wang et al. 2012b).

Farnesene α-Farnesene is found in the coating of fruits and is responsible for the characteristic green apple aroma. β-Farnesene is a natural insect repellant (Avé et al. 1987). It has citrusy notes with hints of lavender being frequently used in perfumery, masks, and powders. Also used to aromatize beer and as a flavoring agent in teas and juices.

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β

α

Longifolene Longifolene is present in Indian turpentine, obtained from Pinus longifolia. This hydrocarbon has a strained skeleton and treatment with acid causes rearrangements, forming isolongifolene. Longifolene is also one of the most abundant aroma constituents of smoked tea (lapsang sounchan tea) made with leaves smoke-dried over pinewood fires (Yao et al. 2005). Its pleasant odor is responsible for its uses in the food industry.

Zingiberene Both isomers (α and β) are responsible for the characteristic flavor of ginger – Zingiber officinale oil (Baser and Demirci 2007). Recent studies have pointed out the antioxidant, anti-inflammatory, and antinociceptive properties of this oil (Jeena et al. 2013).

α

β

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M. Zuzarte and L. Salgueiro

Oxygentaed Derivatives Bisabolol Found in high quantities in Myoporum crassifolium oil (Sell 2010). Also present in chamomile – Matricaria recutita (Dewick 2002b), lavender, and rosemary oils (Sell 2010). This compound has a faint floral odor and has shown antiinflammatory properties (Kim et al. 2011).

Carotol Is one of the main constituents of carrot – Daucus carota subsp carota seed oil and is used in the alcoholic beverage industry, in food flavoring and perfumery (Bauer and Garbe 2001).

Caryophyllene Oxide A major compound of Didymocarpus tomentosa oil (Gowda et al. 2012). Used as a preservative in food, drugs, and cosmetics (Yang et al. 1999). Component responsible for cannabis identification by drug-sniffing dogs (Russo 2011).

Cedrol A white crystalline solid found in several trees of Juniperus, Cupressus, and Thuja species (Sell 2010). It has a cedarwood, sweet soft aroma and is used as a fixative for soap perfumes, particularly household and industrial products (Hunter 2009).

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41

Farnesol Present in many essential oils such as citronella – Cymbopogon sp., neroli – Citrus aurantium, lemon grass – Cymbopogon citratus, and rose – Rosa sp. Interestingly, it is also produced in humans where it acts on numerous nuclear receptors and has received considerable attention because of its potential anticancer properties. It is used in perfumery to emphasize the odors of sweet floral perfumes (Jager 2010). OH

Nerolidol An important compound of neroli – Citrus aurantium (Baser and Demirci 2007) and Zanthoxylum gardneri (Craveiro et al. 1991) oils. Used as a base note in several delicate flowery odors and as an intermediate in vitamins E and K1 production (Bauer and Garbe 2001).

Patchoulol Main active ingredient and the most odor-intensive component of patchouli oil, the volatile oil of Pogostemon cablin. Patchouli oil is widely used in the cosmetic and oral hygiene industries to scent perfume and flavor toothpaste. Both the oil and its constituents have several pharmacological activities including antiemetic and antimicrobial activities (Jager 2010).

Turmerones ar-Turmerone, α-turmerone, and β-turmerone are characteristic compounds of turmeric rhizome-Curcuma longa oil (Asghari 2009) and are also present in other species including C. sichuanensis and C. aromatica (Tsai et al. 2011). arTurmerone has a characteristic odor of turmeric and a slightly pungent bitter taste.

42

M. Zuzarte and L. Salgueiro

ar

α

β

Diterpenes (C20 H32 ) Head-to-tail rearrangements of four isoprene units form diterpenes (Fig. 2.3). These compounds are generally found in resins but some can occur is essential oils. Since they are heavier than monoterpenes and sesquiterpenes, longer distillations are re-

2+

2+

+

2+

2+

+ +

*HUDQ\OFLWURQHOORO

3K\WRO

+& +

&DPSKRUHQH

6FDUHRO

0DQRRO

&+

&+

&+

&+ &+

+&

&+

+

&+

+&

+

&+

+&

&+

+ &+

&+

/DEGDQH

.DXUHQH

3K\OORFODGHQH

3ULPDUDGLHQH

Fig. 2.3 Examples of some diterpenes present in essential oils

6DQGDUDFRSLPDUDGLHQH

2

Essential Oils Chemistry

43 O

O

Eucarvone

O

Nezukone

HO

HO

Santolinatriene

Artemisia ketone

Chrysanthemol

Necrodol

Fig. 2.4 Irregular monoterpenes found in essential oils

quired. Examples of diterpenes include phytol and geranyl citronellol (acyclic); camphorene (monocyclic); scareol and its derivatives, manool and labdane (bicyclic like) and kaur-15-ene, phyllocladene, pimaradiene, and sandaracopimaradiene (tricyclic) (Baser and Demirci 2007).

Irregular Compounds Two major types of irregular monoterpenes (Fig. 2.4) have been identified. The first are substituted cycloheptane monoterpenes (tropones), such as eucarvone and nezukone. The second group includes those compounds that do not fit the regular head-to-tail coupling. Although IPP and DMAPP are involved in their biosynthesis, geranyl pyrophosphate and neryl pyrophosphate do not appear to be involved (Dewick 2002b). Examples of these compounds are artemisia ketone, santolinatriene, and chrysanthemol (Baser and Demirci 2007). Other irregular monoterpenes namely necrodane derivatives also occur primarily in Lavandula luisieri oils (Zuzarte et al. 2012a).

Phenylpropanoids These compounds contain one or more C6–C3 fragments, the C6 unit being a benzene ring. There is no widely accepted classification for these compounds (Baser and Demirci 2007). The main plant sources of phenylpropanoids include species primarily from the Apiaceae, Lamiaceae, Myrtaceae, and Rutaceae families (Bakkali et al. 2008). Examples of phenylpropanoids found in essential oils are presented. Anethole Occurs in high amounts in anise – Pimpinella anisum, fennel – Foeniculum vulgare (Sharif et al. 2008), and star anise – Illicium verum (Huang et al. 2010).

44

M. Zuzarte and L. Salgueiro

This compound has a sweet anise odor and is used in liquors, soap perfumes (Hunter 2009), and mouth care products (Bauer and Garbe 2001).

Apiol An important compound of celery – Apium graveolens and parsley – Petroselinum crispum essential oils (Bauer and Garbe 2001). Apiol is an antipyretic and, like myristicin, a uterine stimulant and has been used as an abortifacient (Tisserand and Balacs 1995).

Cinnamaldehyde Occurs in cinnamon – Cinnamomum sp. (Ooi et al. 2006) and cassia – Cinnamomum cassia (Chang et al. 2013) oils. Compound with a warm spicy balsamic odor that resembles Cassia. Used in perfumery, as a fragrance “sweetener” and in flavors (Hunter 2009).

Eugenol Main component of clove – Syzygium aromaticum (Jirovetz et al. 2006) and cinnamon – Cinnamomum zeylanicum oils (Chericoni et al. 2005). Used as a flavor, in dentistry as a local anesthetic, in the manufacture of vanillin and clandestine production of phenyllamines (Hunter 2009), and in perfumery for oriental and spicy notes (Bauer and Garbe 2001).

Methyleugenol Found in rose – Rosa sp., basil – Ocimum basilicum, eucalyptus – Eucalyptus sp., ylang-ylang – Cananga odorata, huon pine – Lagarostrobos

2

Essential Oils Chemistry

45

franklinii oils. This compound has a sweet spicy clove–eucalyptus aroma and is used as a substitute of iso-eugenol in floral fragrances and in insect traps to lure cockroaches (Hunter 2009).

Myristicine An important compound of nutmeg – Myristica fragrans and parseley – Petroselinum crispum leaf and seed oils (Tyler et al. 1988). Aromatic ether that apparently causes psychotropic effects. However, this effect only occurs in synergy with other compounds also present in nutmeg (Tisserand and Balacs, 1995).

Safrole Found in high amounts in sassafras – Sassafras sp. and nutmeg – Myristica fragrans oils (Tyler et al. 1988). Has a warm, spicy, woody floral note. Banned as a food ingredient by the US FDA because of its mild carcinogenic properties but used in fragrances and as an additive in root beers (Hunter 2009).

Other Compounds Sulfur-Containing Compounds Some plants like garlic, onion, leek, and shallots ( Allium spp.) contain volatile sulfur compounds (Fig. 2.5), namely allyl sulfide, dimethyl sulfide, diallyl disulfide, and dimethylthiophene. Other sulfur-containing compounds like 4-mercapto-4-methyl-pentanone occurs in blackcurrant—Rubus nigrum whereas 1-p-menthene-8-thiol is found in fruit oils (Fig. 2.5; Baser and Demirci 2007; Hunter 2009). These compounds appear to be important in plant defense and in nitrogen detoxication of plants. Although most sulfur compounds have very unpleasant pungent odors, organosulfur compounds present in essential oils can be aromatically very pleasant. It is also known that sulfur compounds are relevant in the flavoring of vegetables, fruits as well as processed foods and beverages. Nitrogen-Containing Compounds Compounds found in only a few essential oils. Examples include methyl anthranilate, skatole, indole, pyridine, and pyrazine (Fig. 2.6). Methyl anthranilate is present in several Citrus oils (orange, lemon, and bergamot) and in ylang-ylang – Cananga odorata oil. Skatole is a compound in the

46

M. Zuzarte and L. Salgueiro

H2C

CH2

S

S

S

S

Allyl sulphide

Dimethyl sulphide

Diallyl disulphide

O

SH

SH

S

Dimethylthiophene

4-Mercapto-4-methyl-pentanone

1-p-Menthene-8-thiol

Fig. 2.5 Examples of sulfur-containing compounds found in essential oils

N NH2

O

O

Methyl anthranilate

N H

N H

Skatole

N

Indole

Pyridine

N

Pyrazine

Fig. 2.6 Nitrogen-containing compounds found in essential oils

form of large crystals or powder. It occurs in orange – Citrus aurantium blossoms and jasmine – Jasminum sp. (Baser and Demirci 2007). This compound has a very interesting aroma with a fecal smell at high concentrations but a floral scent in dilution. Used as a fixative in floral fragrances and a flavor agent in ice-cream and cigarettes. Indole is a white crystalline powder that turns red on exposure to air. It occurs in neroli and some citrus fruit oils. Has a similar odor to skatole and is used in a wide range of fragrances (Hunter 2009). Pyridines and pyrazines occur in black pepper – Piper nigrum, sweet orange – Citrus × sinensis, and vetiver – Chrysopogon zizanioides oils (Baser and Demirci 2007). Lactones Lactones (Fig. 2.7) are cyclic esters that derive from lactic acid. Some occur in essential oils as γ-lactones (five-membered cyclic rings), like γ-decalactone that has a peach-like flavor, δ-lactones (six-membered cyclic rings) such as δ-decalactone with a creamy-coconut odor. Lactones in the form of benzofuran derivatives such as butylphtalide and sedanolide are also found in some plants like celery – Apium graveolens (Marongiu et al. 2013) and Angelica sp. (Hunter 2009) and macrocyclic lactones like ambrettolide occur in ambrette – Abelmoschus moschatus seed oil. Other lactones like coumarin, with spicy green notes, scopoletin, and bergaptene are present in several oils while nepetalactones are characteristic of Nepeta oils (Baser and Demirci 2007).

2

Essential Oils Chemistry

47 O

O

O

O

O

Butylphtalide

γ-Decalactone

O

Coumarin

O O

O

O

OH

O

Scopoletin

O

O

Bergaptene

O

O

Nepetalactone

Fig. 2.7 Lactones of essential oils

Important Trade Essential Oils The quality of essential oils is not easy to ensure and the high chemical variability is one of the main problems that limits quality, safety, and efficacy of oils. Composition of essential oils can vary greatly because of physiological (plant organ, ontogenesis), environmental (soil composition, weather conditions), and genetic factors (Salgueiro et al. 2010). A typical example of the influence of the plant organ occurs in Citrus aurantium subsp. aurantium. The oils obtained from its fruits (peels), flowers, and leaves have different chemical compositions. The first are rich in limonene whereas the flower and leaf oils are characterized by different quantities of linalool and linalyl acetate (Sarrou et al. 2013). Also, a close relationship between ontogenesis and oil composition can be seen in several species. For example, Mentha × piperita accumulates a maximum amount of oil before blooming, being the optimal harvest time when the calyces are only barely visible at the stem extremity (Perrina and Colsona 1991). Plants growing in different geographical regions also exhibit distinct oil compositions, as pointed out by a study comparing Salvia officinalis oils from nine European countries (Raal et al. 2007). Moreover, even in the same geographical region, differences may occur because of other variables such as soil type, sunlight levels, and water availability, or different genetic backgrounds. The latter is responsible for the occurrence of chemically distinct populations, named chemotypes, within species with similar phenotypes. Several species such as Thymus spp. (Figueiredo et al. 2008b), Lavandula pedunculata (Zuzarte et al. 2009), Ferula communis (Rubiolo et al. 2006), and Foeniculum vulgare (Krüger and Hammer 1999) are known to have different chemotypes, named according to the main components of the essential oil. Some chemotypes are commercially more relevant being referred in the ISO standards. Examples include the oil of bitter fennel-Foeniculum vulgare subsp. vulgare with two chemotypes: trans-Anethole type and Phellandrene type (ISO17412) and the oil of Matricaria recutita (syn. Chamomilla recutita, Matricaria chamomilla) also with two chemotypes: Egyptian

48

M. Zuzarte and L. Salgueiro

Table 2.1 Chromatographic profile of Matricaria recutita [Table reprinted with permission from the ISO, copyright ISO 19332:2007 (Table 2.1, page 2)]. Component Egyptian type Hungarian type Minimum (%) Maximum (%) Minimum (%) Maximum (%) 15 35 20 51 β-( E )-Farnesene 8 2 21 α-Bisabolol oxide B 2 Bisabolone oxide A 2 6.5 1 4 1 10 15 40 α-Bisabolol Chamazulene 2 5 5 22 50 2 27 α-Bisabolol oxide A 35

type and Hungarian type. The chromatographic profile for this species is presented in table 2.1 (ISO 19332:2007). Analytical standards and monographs like those of the ISO and Pharmacopoeias have been published and should be considered in order to produce and commercialize consistent and safe products. The ISO/TC 54 on essential oils regulates analytical methods and specifications for essential oils with industrial potential. Normalization of essential oils cover several aspects including those related to transportation, labeling, and nomenclature (use of scientific names). The establishment of physical, chemical, and organoleptic characteristics as well as the chromatographic profile of the essential oils enables the detection of adulterations and controls toxic components, limited by the health sector legislation. Moreover, these standards can be used as a source of information for the industry and as a specification that contributes to the stability of quality and authenticity of the commercialized products. The main producers of essential oils are Brazil (29%), India (26%), United States (17%), and China (19%) (Schmidt 2010), the main trading markets being the United States, Germany, United Kingdom, Japan, and France (Bovill 2010). Overall, 118 species are used for essential oil production worldwide. Fifteen of these species, namely citronella Ceylon ( Cymbopogon nardus), citronella Java ( C. winterianus), clove buds ( Syzygium aromaticum), cornmint ( Mentha arvensis), eucalyptus (  Eucalyptus globulus), lemon-scented eucalyptus ( E. citriodora), lavandin ( Lavandula x intermedia), lemon ( Citrus limon), lime distilled ( C. aurantifolia), sweet orange ( C. sinensis), patchouli ( Pogostemon cablin), peppermint ( M. x piperita), sassafras Brazilian  ( Ocotea odorifera),  sassafras  Chinese  ( Sassafras albidum), and spearmint Scotch ( M. gracilis) are the most relevant with over 1000 t/year of essential oils being produced. The majority of these species are cultured for essential oil extraction but some are collected in their natural habitats (Franz and Novak 2010). Table 2.2 summarizes the most important trade essential oils worldwide. Their trade name, species, family, and parts of the plants used for essential oil extraction are presented as well as the quantity of oil annually produced. The essential oils are grouped according to the chemical group of their main compounds or relevant minor compounds (terpene hydrocarbons, alcohols, esters, aldehydes, ketones, ethers, phenylpropanoids, peroxydes, nitogen (N−) and/or sulfur (S−) compounds, and lactones). Moreover, essential oils with ISO monographs are pointed out, being the standard chemical composition referred.

Apiaceae Zingiberaceae

Ferula galbaniflua Boiss.

Zingiber officinale Roscoe

Dipterocarpus spp.

Juniperus communis L.

Galbanum

Ginger

Gurjum

Juniper berry

< 100

< 100

Resin

Berry

< 100

< 100

< 100

< 100

100–1000

100–1000

Rhizome

Gum

Resin

Leaf/twig

Wood

Wood

< 100

< 100

100–1000

Monoterpene hydrocarbon (limonene); linalyl acetate Sesquiterpene hydrocarbon (cadinene) Monoterpene hydrocarbons (pinenes, camphene, limonene) Sesquiterpene hydrocarbons (α-cedrene, thujopsene, cuperene); cedrol Sesquiterpene hydrocarbons (α-cedrene, β-funebrene, thujopsene); cedrol Monoterpene hydrocarbons (limonene, α-phellandrene, δ-3-carene) Monoterpene hydrocarbons (limonene, α-phellandrene); elemol Monoterpene hydrocarbons (β-pinene, α-pinene, δ-3-carene) Sesquiterpene hydrocarbons (zingiberene, β-sesquiphellandrene) Sesquiterpene hydrocarbons (α-gurjunene, calarene,  α-copaene) Monoterpene hydrocarbons (pinenes, sabinene, myrcene)

Important constituents

ISO 8897:2010

ISO 16928:2013

ISO 14716:1998

ISO 10624:1998

ISO 4724:2004

ISO 4725:2004

ISO 3520:1998

ISO/TC 54

Essential Oils Chemistry

Cupressaceae

Dipterocarpaceae

Burseraceae

Canarium luzonicum Miq.

Elemi

Cupressaceae

Cupressaceae

Cupressus sempervirens L.

Junipenus virginiana L.

Cypress

Cedarwood, Virginia

Cedarwood, Texas Juniperus mexicana Schiede

Cupressaceae

Euphorbiaceae

Cascarilla

Bark

Wood

Cupressaceae

Croton eluteria (L.) W. Wright

Fruit peel

Plant parts Trade quantities (t/year)

Rutaceae

Family

Terpene hydrocarbons Bergamot Citrus aurantium L. subsp. bergamia (Risso et Poit.) Engl. Cade Juniperus oxycedrus L.

Table 2.2 Important trade essential oils Trade name Species

2 49

Myristicaceae Rutaceae Piperaceae

Pinaceae

Pinaceae

Myristica fragrans Houtt.

Citrus aurantium L.

Piper nigrum L.

Pinus mugo Turra

Pinus sylvestris L.

Pinus palustris Mill.

Citrus sinensis (L.) Osbeck

Pinus spp.

Nutmeg

Orange bitter

Pepper

Pine needle

Pine silvestris

Pine white

Sweet orange

Turpentine

Pinaceae

Rutaceae

Pinaceae

Rutaceae

Mandarin

Lime distilled

Rutaceae

Rutaceae

Terpene hydrocarbons Lemon Citrus limon (L.) Burman fil.

Citrus aurantifolia (Christm. et Panz.) Swingle Citrus reticulata Blanco

Family

Table 2.2 (continued) Trade name Species

Resin

Fuit peel

Leaf/twig

Leaf/twig

Leaf/twig

Fruit

Fruit peel

Seed

Fruit peel

Fruit

Fruit

< 100

>1000

< 100

< 100

< 100

< 100

< 100

< 100

100–1000

> 1000

> 1000

Plant parts Trade quantities (t/year) Monoterpene hydrocarbons (limonene, β-pinene, γ-terpinene) Monoterpene hydrocarbons (limonene, γ-terpinene) Monoterpene hydrocarbons (limonene, γ-terpinene) Monoterpene hydrocarbons (sabinene, pinenes); myristicine Monoterpene hydrocarbons (limonene) Monoterpene hydrocarbons (pinenes, sabinene, limonene, δ-3-carene); β-caryophyllene Monoterpne hydrocarbons (pinenes, δ-3-carene, limonene, myrcene, β-phellandrene) Monoterpne hydrocarbons (pinenes, δ-3-carene, limonene, myrcene) Monoterpne hydrocarbons (pinenes) Monoterpene hydrocarbons (limonene) Monoterpene hydrocarbons (pinenes)

Important constituents

ISO 11020:1998

ISO 3140:2005

ISO 21093:2003

ISO 3061:2008

ISO 9844:2006

ISO 3215:1998

ISO 3528:1997

ISO 3519:2005

ISO 855:2003

ISO/TC 54

50 M. Zuzarte and L. Salgueiro

Herb Seed Leaf Fruit Leaf Leaf Flower Flower Herb

Leaf Leaf

Lamiaceae Apiaceae Poaceae Apiaceae Lamiaceae Geraniaceae Lamiaceae Rutaceae Lamiaceae

Poaceae Lamiaceae

Basil (European Ocimum basilicum L. type) Carrot seed Daucus carota L. Citronella, Ceylon Cymbopogon nardus (L.) W. Wats. Coriander Coriandrum sativum L. Cornmint Mentha canadensis L. ( Mentha arvensis L.) Geranium Pelargonium spp. Lavender spike Lavandula latifolia Medik.

Neroli

Patchouli

Palmarosa

Oregano

Wood

Rutaceae

Citrus aurantium L. subsp. aurantium Origanum onites L., O. vulgare L. subsp. hirtum (Link) Iestw. or other Origanum spp., Thymbra spicata L., T. capitata Rechb. Fil., Satureja spp., Lippia graveolens Kunth Cymbopogon martinii (Roxb.) W. Wats Pogostemon cablin (Blanco) Benth.

Amyris balsamifera L.

Alcohols Amyris

> 1000

< 100

< 100

< 100

100–1000 < 100

< 100 > 1000

< 100 > 1000

< 100

< 100

Plant parts Trade quantities (t/year)

Family

Table 2.2 (continued) Trade name Species

ISO 3525:2008

ISO/TC 54

Patchoulol; bulnesene, α-guaiene

Geraniol

Citronellol, geraniol Linalool; linalyl acetate, 1,8-cineole, camphor Linalool; linalyl acetate, β-pinene, limonene Monoterpene phenol (carvacrol); ρ-cymene, γ-terpinene

ISO 3757:2002

ISO 4727:1988

ISO 14717:1999

ISO 3517:2002

ISO 4731:2006 ISO 4719:1999

Carotol Geraniol; methylisoeugenol, ISO 3849:2003 limonene Linalool ISO 3516:1997 Menthol, isomenthol; menthone ISO 9776:1999

Valerianol, elemol, 7-epi-αeudesmol,10-epi-γ-eudesmol Linalool; estragol

Important constituents

2 Essential Oils Chemistry 51

Fir needle, Siberian Lavandin

Vetiver Esters Cardamom Cedarwood, Chinese Clary sage

Thyme

Rosewood Sandalwood, East Indian Marjoram Tea tree

Flowering 100–1000 herb Leaf/twig < 100

Lamiaceae

Salvia sclarea L. Pinaceae

Lavandula angustifolia Mill. × Lamiaceae L. latifolia Medik. ( Lavandula × intermedia)

Abies sibirica Ledeb.

Seed Wood

Zingiberaceae Cupressaceae

Leaf

> 1000

< 100 100–1000

100–1000

< 100

Elettaria cardamomum Maton Cupressus funebris Endl.

Herb

Lamiaceae Root

Herb Leaf

Lamiaceae Myrtaceae < 100 < 100

< 100 100–1000

> 1000 < 100

Poaceae

Wood Wood

Leaf Flower

Plant parts Trade quantities (t/year)

Origanum majorana L. Melaleuca alternifolia (Maiden et Bech) Cheel, M. linariifolia Smith, M. dissitiflora F. Mueller and other species Thymus vulgaris L., T. zygis Loefl. ex. L. Vetiveria zizanioides (L.) Nash

Aniba rosaeodora Ducke Santalum album L.

Lauraceae Santalaceae

Lamiaceae Rosaceae

Alcohols Peppermint Rose

Mentha × piperita L. Rosa × damascena Miller

Family

Table 2.2 (continued) Trade name Species

Bornyl acetate; camphene, α-pinene, δ-3-carene Linaly acetate; linalool, 1,8-cineole

α-Terpenyl acetate; 1,8-cineole Linalyl acetate; α-cedrene, funebrene, thujopsene Linalyl acetate; linalool

Monoterpene phenol (thymol); ρ-cymene, γ-terpinene khusimol, isovalencenol

Terpinen-4-ol; γ-terpinene Terpinen-4-ol; 1,8-cineole, γ-terpinene, α-terpinene

Menthol; menthone Citronellol, geraniol, nerol; nonadecane Linalool cis-α-Santalol, cis-β-santalol

Important constituents

ISO 3054:2001

ISO 10869:2010

ISO 4733:2004 ISO 9843:2002

ISO 4716:2002

ISO 14715:2010

ISO 4730:2004

ISO 3761:2005 ISO 3518:2002

ISO 856:2006 ISO 9842:2003

ISO/TC 54

52 M. Zuzarte and L. Salgueiro

Leaf Flower

Ericaceae Annonaceae

Lemongrass, Indian Lemongrass, West Indian Lemon-scented eucalyptus Litsea cubeba

Cumin

Aldehydes Cinnamon bark, Ceylon Cinnamon bark, Chinese Citronella, Java

Ylang-ylang

Wintergreen

Leaf Leaf

Lamiaceae Rutaceae

Lauraceae

> 1000

Leaf

100–1000

< 100

Leaf

Fruit/leaf

< 100

< 100

> 1000

Leaf

Seed

Leaf

< 100

< 100

100–1000

< 100

100–1000 < 100

ISO 3063:2004

ISO 21390:2005

ISO 3515:2002 ISO 8901:2003

ISO/TC 54

Geranial, neral; limonene

Citronellal

Geranial, neral

Cuminaldehyde, ρ-mentha1,3-dien-7-al; γ-terpinene, ρ-cymene Geranial, neral

ISO 3214:2000

ISO 3044:1997

ISO 3217:1974

ISO 4718:2004

ISO 9301:2003

Cinnamaldehyde, ISO 3216:1997 trans-o-methoxycinnamaldeyde Citronellal; geraniol, citronellol

Cinnamaldehyde

Benzyl acetate, germacrene geranyl acetate, ρ-cresyl methyl ether; linalool, (E, E, α-farnesene)

Methyl salicylate

Linaly acetate; Linalool Linaly acetate; Linalool

Important constituents

Essential Oils Chemistry

Litsea cubeba C.H. Persoon

Cymbopogon flexuosus (Nees ex Poaceae Steud.) J. F. Watson Poaceae Cymbopogon citratus (DC.) Strapf Myrtaceae Eucalyptus citriodora Hook.

Apiaceae

Poaceae

Bark

Lauraceae

Cinnamomum cassia Blume

Cymbopogon winterianus Jowitt. Cuminum cyminum L.

Bark

Lauraceae

Cinnamomum zeylanicum Nees

Lavandula angustiolia Mill. Citrus aurantinum L. subsp. aurantinum Gaultheria procumbens L., G. junnanensis (Franch.) Rehd. Cananga odorata (Lam.) Hook. f. & Thomson

Esters Lavender Petitgrain

Plant parts Trade quantities (t/year)

Family

Table 2.2 (continued) Trade name Species

2 53

Myrtaceae Myrtaceae Lauraceae Lamiaceae

Melaleuca leucadendron L. Eucalyptus globulus Labill. Laurus nobilis L. Salvia fruticosa Mill.

Ethers Cajuput Eucalyptus Laurel leaf Sage, three lobed

Lamiaceae

Salvia officinalis L.

Sage, Dalmatian

Asteraceae

Lamiaceae

Mentha pulegium L.

Pennyroyal

Artemisia absinthium L.

Apiaceae

Anethum graveolens L.

Dill

Wormwood

Asteraceae

Artemisia pallens Wall.

Davana

Lamiaceae Lamiaceae Asteraceae

Apiaceae Asteraceae

Ketones Caraway Carum carvi L. Mugwort common Artemisia vulgaris L.

Spearmint, Native Mentha spicata L. Spearmint, Scotch Mentha × gracilis Sole Tansy Tanacetum vulgare L.

Family

Table 2.2 (continued) Trade name Species

< 100 < 100

Leaf Leaf Leaf Herb

Leaf Leaf Flowering herb Herb

Leaf

Herb

< 100 > 1000 < 100 < 100

< 100

100–1000 > 1000 < 100

< 100

< 100

Flowering < 100 herb Herb/fruit < 100

Seed Herb

Plant parts Trade quantities (t/year) ISO 8896:1987

ISO/TC 54

1,8-Cineole 1,8-Cineole; α-pinene 1,8-Cineole 1,8-Cineole

ISO 770:2002

Carvone; limonene, α-phellandrene, dillapiole Pulegone, piperitenone, piperitone, isomentone, neoisomentone α-Thujone, β-thujone, camphor; ISO 9909:1997 1,8-cineole Carvone; limonene ISO 3033-3:2005 Carvone; limonene ISO 3033-4:2005 α-Thujone, camphor, artemoisia ketone; 1,8-cineole β-Thujone, α-thujone; (Z)epoxy-ocimene, sabinyl acetate, chrysanthenyl acetate, α-phellandrene

Carvone; limonene α-Thujone, camphor; 1,8-cineole cis-Davanone, trans-davanone

Important constituents

54 M. Zuzarte and L. Salgueiro

Cinnamomum zeylanicum Nees Syzygium aromaticum (L.) Merill et L. M. Perry Syzygium aromaticum (L.) Merill et L. M. Perry Foeniculum vulgare Mill. subsp. vulgare var. vulgare Foeniculum vulgare Mill. subsp. vulgare var. dulce Petroselinum crispum (Mill.) Nym. Ex A. W. Hill Ocotea odorifera (Vell.) Rohwer

Sassafras, Brazilian Sassafras, Chinese Sassafras albidum (Nutt.) Nees. Star anise Illicium verum Hook fil. Tarragon Artemisia dracunculus L.

Parsley seed

Fennel sweet

Fennel bitter

Clove leaf

Cinnamon leaf Clove buds

Phenylpropanoids Anise seed Pimpinella anisum L. Basil (Reunion Ocimum basilicum L. type) Calamus Acorus calamus L.

Table 2.2 (continued) Trade name Species

Fruit

Apiaceae < 100

< 100

> 1000 100–1000 < 100

Root bark Fruit Herb

Fruit

Apiaceae

< 100

Lauraceae Illiciaceae Asteraceae

Fruit

Apiaceae

< 100

Root bank > 1000

Leaf

Myrtaceae

< 100 > 1000

< 100

< 100 < 100

Lauraceae

Leaf Bud

Rhizome

Araceae Lauraceae Myrtaceae

Fruit Herb

Plant parts Trade quantities (t/year)

Apiaceae Lamiaceae

Family

ISO 3141:1997

Eugenol; β-caryophyllene

Safrole ( E)-Anethole Estragol; cis-β-ocimene, trans-β-ocimene

Myristicine, apiol; α-pinene, β-pinene Safrole

ISO 11016:1999 ISO 10115:1997

ISO 3527:2000

ISO 17412:2007

ISO 3524:2003 ISO 3142:1997

β-Asarone (triploid and tetraploid variety) Eugenol Eugenol, eugenyl acetate

(E)-Anethole; fenchone, α-phellandrene, limonene ( E)-Anethole

ISO 3475:2002 ISO 11043:1998

ISO/TC 54

( E)-Anethole Estragol

Important constituents

2 Essential Oils Chemistry 55

Apiaceae

Apiaceae

Apiaceae

Angelica archangelica L.

Apium graveolens L.

Levisticum officinale Koch

Lactones Angelica root

Celery seed

Lovage root

Alliaceae Alliaceae

Allium sativum L. Allium cepa L.

Rutaceae Agathosma betulina (Bergius) Pillans, A. crenulata (L.) Pillans

Root

Seed

Root

Bulb Bulb

Leaf

< 100

< 100

< 100

< 100 < 100

< 100

< 100

Asteraceae

Resin

< 100

Plant parts Trade quantities (t/year)

Chenopodiaceae Seed

Family

Garlic Onion

Buchu leaf

Peroxides Chenopodium Chenopodium ambrosioides L. N- and/or S- containing oils Asafoetida Ferula assa-foetida L.

Table 2.2 (continued) Trade name Species

15-Pentadecanolide, 13-tridecanolide as characteristic minor components in addition to terpenoids and sesquiterpenoids 3-Butylphthalide and sedanenolide as characteristic components in addition to terpenoids (limonene and β-selinene) 3-Butylphthalide, ligustilide, ligusticum lactone

R-2-Butyl-1-propenyl disulfide, 1-(1-methylthiopropenyl)1-propenyl disulfide, 2-butyl3-methyl-thioallyl disulfide trans-p-Menthane-8-thiol-3-one and its S-acetate as characteristic minor components Diallyl disulfide Methylpropyl disulfide, dipropyl disulfide, propenylpropyl disulfide, 2-hexyl-5methyl-3(2 H)-furanone

Ascaridole

Important constituents

ISO/TC 54

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Joshi R, Sharma P, Sharma V et al (2012) Analysis of the essential oil of large cardamom ( Amomum subulatum Roxb.) growing in different agro-climatic zones of Himachal Pradesh, India. J Sci Food Agric 93(6):1303–1309 Juan LW, Lucia A, Zerba EN et al (2011) Chemical composition and fumigant toxicity of the essential oils from 16 species of Eucalyptus against Haematobia irritans (Diptera: Muscidae) adults. J Econ Entomol 104(3):1087–1092 Kamatou GP, Vermaak I, Viljoen AM et al (2013) Menthol: a simple monoterpene with remarkable biological properties. Phytochemistry 96:15–25 Katsiotis ST, Langezaal CR, Schefferj JJC (1990) Composition of the essential oils from leaves of various Humulus lupulus L. cultivars. Flavour Frag J 5:97–100 Kim J-K, Kang C-S, Lee J-K et al (2005) Evaluation of repellency effect of two natural aroma mosquito repellent compounds, citronella and citronellal. Ent Res 35(2):117–120 Kim  S,  Jung  E,  Kim  JH,  Park YH,  Lee  J,  Park  D  (2011)  Inhibitory  effects  of  (−)-α-bisabolol on LPS-induced inflammatory response in RAW 264.7 macrophages. Food Chem Toxicol 49(10):2580–2585 Kim YW, Kimm MJ, Chung BY et al (2013) Safety evaluation and risk assessment of d-limonene. J Toxicol Environ Health Sci Part B Crit Rev 16:17–38 Kose EO, Deniz IG, Sarikurkcu C et al (2010) Chemical composition, antimicrobial and antioxidant activities of the essential oils of Sideritis erythrantha Boiss. and Heldr. (var. erythrantha and var. cedretorum P.H. Davis) endemic in Turkey. Food Chem Toxicol 48:2960–2965 Krüger H, Hammer K (1999) Chemotypes of fennel ( Foeniculum vulgare Mill.). J Essent Oil Res 11:79–82 Litchenthaler HK (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 50:47–65 Little DB, Croteau RB (1999) Biochemistry of essential oil terpenes. In: Teranishi R, Wick EL, Hornstein I (eds) Flavor chemistry. Thirty years of progress. Kluwer Academic/Plenum Publishers, New York, pp 239–253 Lohani H, Haider SZ, Chauhan NK et al (2013) Aroma profile of two Juniperus species from Alpine region in Uttarakhand. J Nat Prod 6:38–43 Marongiu B, Piras A, Porcedda S et al (2013) Isolation of the volatile fraction from Apium graveolens L. (Apiaceae) by supercritical carbon dioxide extraction and hydrodistillation: chemical composition and antifungal activity. Nat Prod Res 27(17):1521–1527 Marzouki H, Khaldi A, Falconieri D et al (2010) Essential oils of Daucus carota subsp. carota of Tunisia obtained by supercritical carbon dioxide extraction. Nat Prod Commun 5(12):1955– 1958 Miguel MG, Dandlen S, Figueiredo AC et al (2008) Essential oils of flowers of Citrus sinensis and Citrus clementina cultivated in Algarve, Portugal. Acta Hort 773:89–94 Mockute D, Bernotiene G, Judzentiene A (2001) The essential oil of Origanum vulgare L. ssp. vulgare growing wild in vilnius district (Lithuania). Phytochemistry 57:65–69 Mota KSL, Pereira FO, Oliveira W A et al (2012) Antifungal activity of Thymus vulgaris L. essential oil and its constituent phytochemicals against Rhizopus oryzae: interaction with ergosterol. Molecules 17:14418–14433 Nabiha B, Abdelfatteh EO, Faten K (2010) Chemical composition of bergamot ( Citrus bergamia Risso) essential oil obtained by hydrodistillation. J Chem Chem Eng 4:60–62 Ooi LS, Li Y, Kam SL et al (2006) Antimicrobial activities of cinnamon oil and cinnamaldehyde from the Chinese medicinal herb Cinnamomum cassia Blume. Am J Chin Med 34(3):511–522 Orav A, Stulova I, Kailas T et al (2004) Effect of storage on the essential oil composition of Piper nigrum L. fruits of different ripening states. J Agric Food Chem 52:2582–2586 Ottavioli J, Gonny M, Casanova J et al (2009) Chemical variability of the needle oil of Juniperus communis ssp. alpina from Corsica. Chem Biodivers 6(12):2192–2199 Ozcan MM, Pedro LG, Figueiredo AC et al (2006) Constituents of the essential oil of sea fennel ( Crithmum maritimum L.) growing wild in Turkey. J Med Food 9(1):128–130 Ozek G, Demirci F, Ozek T et al (2010) Gas chromatographic-mass spectrometric analysis of volatiles obtained by four different techniques from Salvia rosifolia Sm., and evaluation for biological activity. J Chromatog 1217:741–748

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Chapter 3

Pharmacobotanical Aspects of Aromatic Plants Basílio I.J.L.D., Nathalia Diniz Araujo and Rafael Costa Silva

Pharmacobotany The pharmacobotany by definition is a branch of pharmacognosy that deals with the botanical knowledge of plant species used in the preparation of extracts and obtaining derivatives such as oils and resins, which can be employed in the production of phytotherapics, phytopharmaceuticals, phytocosmetics, nutraceuticals, and functional foods. Therefore, the topic addressed in this chapter is indispensable in the detection of adulteration by addition or substitution of raw materials in the form of herbal drugs used in these productive sectors. Additionally, this is useful for forensic and toxicological investigations, supporting the elucidation of crimes with traces of plants and poisoning by ingestion of plants. The term pharmacognosy is derived from two words of Greek origin, pharmakon (drug, poison, and remedy) and gnosis (knowledge). Currently, it is divided into two main branches, pharmacobotany and farmacozoology, which address the knowledge of drugs of plant and animal origin, respectively. In this context, vegetal drugs are medicinal plants or their parts, which contain the substances or classes of substances responsible for therapeutic action after collecting or harvesting, stabilizing, and drying, in its entire form, crushed, or powdered. These processes are essential

B. I.J.L.D. () Department of Pharmaceutical Sciences, Federal University of Paraiba, 58051-970 João Pessoa, Paraíba, Brazil e-mail: [email protected] N. Diniz Araujo Postgraduate Program in Bioactive Natural and Synthetic Products, Federal University of Paraíba, João Pessoa, Brazil e-mail: [email protected] R. C. Silva Graduate Program in Plant Biology, Federal University of Pernambuco, Recife, Brazil e-mail: [email protected] © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_3

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for the conservation of raw materials, besides facilitating transport and subsequent large-scale use. To understand in which parts of the plants are the biologically active compounds produced and stored, it is important to understand the morphology and anatomy of the plant and some concepts such as plant systematics and taxonomy.

Plant Systematics Systematics is the science of the diversity of organisms which includes and encompasses the taxonomy. It involves the discovery, description, and interpretation of biological diversity, as well as summary information on diversity in the form of predictive classification systems. According to Judd et al. (2007) systematics is not only a descriptive science, but also seeks to discover evolutionary relationships and real evolutionary entities that are result of the evolutionary process. The historical development of plant classification systems can be divided into two major periods: the descriptive and of systematization. In the beginning, systems emerged based on the habit of plants, grouped into the tree, shrub, and herbaceous subshrubs. Teophrastus, a disciple of Aristotle and considered the Father of Botany, stood out at this time, along with Dioscorides, Pliny, and Albertus Magnus. In the Middle Ages, Brunfels, Bock, Fuchs, Clusius, L’Obel, and Gerard, designated as herbalists, were worried about the medicinal properties of plants, providing descriptions and illustrations of them for easy identification. In the period of systematization, these poorly elaborated systems have given way to artificial ones, so named because they used a few and arbitrary attributes to form groups, showing no affinity relationships between species. The most widespread was the sexual system of Linnaeus, which emphasized floral characters, and distinguished itself by establishing a binomial nomenclature of biological species. Other naturalists who deserve references are: Caesalpinus, Tournefort and Jean, and Gaspar Bauhin brothers. In the second half of the eighteenth century natural systems appeared, having been built taking into account a large number of information, mainly from the accumulated knowledge on plant morphology. Lamarck, Jussieu, Augustin Pyrame and Alphonse de Candolle (father and son), Brown, Lindley, Brogniart, Bentham, and Hooker are important names of the period. Finally, phylogenetic systems succeeded natural systems in the nineteenth century, using all available information to characterize taxa and establish relations of similarity between them on the basis of ancestry and descent. Evolutionary theory postulates that the affinities between living beings are a reflection of phylogenetic evolution, where primitive forms (simpler ones) gave rise to the evolved one (more complex). Naturally, current organisms are descended from others in the past, although there are marked differences between them. The best known phylogenetic systems are Eichler, Engler, Wettstein, Bessey, Hutchinson, and Tippo. Among the most current, which use different knowledge areas—external morphology, anatomy, cytology, embryology, ecology, genetics, chemistry, and statistics—are Takhtajan, Cronquist, Thorne, Banks, and Dahlgren.

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Some of these systems did not survive the effects and demands of cladisitic theory and the emergence of molecular and morphological studies using this theory. The aim of taxonomists is currently focused on proposing phylogenetic hypotheses instead of describing relationships in nature. According to Judd et al. (2007), the classifications are the works of man, not nature. We decided which groups that we want to talk about. And although many important aspects of phylogenies are not clear, for example, the relationship of monocots with other angiosperms, a new arrangement with new general boundaries is evident (Chase 2004; Judd and Olmstead 2004; Bremer et al. 2009).

Plant Taxonomy Taxonomy aims to address the individualization, classification, and nomenclature of species. Characters used in the classification of living things are called taxonomic characters and are attributes of an individual, in isolation or in comparison to other characters of individuals of identical or different species. The ordering of these species in a hierarchical manner, that is, in accordance with adopted criteria, is called classification. The identification is the recognition of a given species as identical as a previously classified one. Taxonomic groupings of any category, for instance, order, family, tribe, genus, species, are designated as taxon (plural: taxa). The basic category of taxonomic hierarchy is the species, which can be defined as the lowest permanently distinct and distinguishable populations, whose genetic exchange is free (possible interbreeding, resulting in fertile offspring). Taxonomy includes four main components: description, identification, nomenclature, and classification. Description is the act of giving attributes to a particular taxon. These attributes are called characters. Two or more forms of a character are called state of character. An example is the character sheet format, whose character states can be oval, elliptical, obovate, or lanceolate. Multiple characters and character states are used in plant systematics and the purpose of these descriptive terms is to use them as communication tools, concisely to categorize and delimit the attributes of a taxon, an organism, or any part of the organism. An accurate and complete list of these resources is one of the main goals and contributions of taxonomy. Identification is the process of association of an unknown taxon with a known taxon, or recognition that the unknown taxon is new to science and justifies the formal description and naming. Plant taxa can be identified in several ways, but the main one is using dichotomous identification keys. A dichotomous identification key is composed of two contrasting assertion series. Each statement is a character or state of character. The choice of these alternative statements determines the next step and so on until identification is achieved. Nomenclature is the formal nomination of the taxa according to a standardized system (International Code of Botanical Nomenclature). The International Code of Botanical Nomenclature establishes criteria for the elaboration of names for different taxa, according to the principles, rules, and recommendations, updated every 4

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years, during the International Congress of Botany. The principles form the basis and establish the philosophy of nomenclature system: the botanical nomenclature is independent of the zoological; the application of names is determined by nomenclature types; the nomenclature of a taxonomic group is based on the priority of publication; each taxon has only one valid name; the names of the taxa are treated as Latin names; and naming rules are retroactive, except when clearly limited (McNeill et al. 2012). The rules, organized into articles, aim to sort the existing names and guide the development of new ones. Among the most important are taxonomic categories which are designated by their endings (Table 3.1). The name of a plant is a combination of genus name and specific epithet, with the first letter of the genus being capitalized and the first letter of the specific epithet being lower case and accompanied by the author’s name and appear highlighted in the text (italic and underline); when a species changes gender, the name of the author of the basionym (first name created) should be cited in brackets, followed by the name of the author who made the new combination, for example, Lycianthes pauciflora (Vahl.) Bitter. The recommendations address the less relevant aspects and indicate the preferred form of a name. Species can be further subdivided into subspecies (subsp.) or variety (var.; see Fig. 3.1). In general, the methodology adopted by taxonomists comprises: Collection of Material Collected specimens, not less than five numbers to show the population change, must present vegetative and reproductive organs and be accompanied by information recorded during the collection, such as location, frequency in the area, height of the specimens, collector name and date; and data that are lost during the process of herborization as flower color and characteristic odor. Herborization Collected specimens are placed between paper and cardboard, sealed and tied in press; the material must be dried at 70 ºC or under the sun, and the paper should be changed daily. After drying, the species is identified, recorded, and included in the herbarium. Identification The material must be identified until family, genus, or species levels, using the analytical keys. Description Vegetative and reproductive characteristics of the species should be described, comparing with other closely related species. Table 3.1  Examples of botanical nomenclature Categories Name Class Equisetopsida Subclass Magnoliidae Order Lamiales Family Lamiaceae Genus Ocimum Species Ocimum basilicum Variety Ocimum basilicum var. purpurascens

Botanical authority C. Agardh Novák ex Takht. Bromhead Martinov L. L. Benth.

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Fig. 3.1  Ocimum basilicum var. purpurascens Benth. a Habit and b flower detail with glandular trichomes

Aromatic Plants Essential oils are widely found in dicotyledonous angiosperms such as in families Apiaceae, Asteraceae, Lamiaceae, Lauraceae, Myrtaceae, Myristicaceae, Piperaceae, and Rutaceae. In monocotyledonous angiosperms, the occurrence of these metabolites is more restricted, occurring in some grasses such as species of Cymbopogon and Vetiveria, and of Zingiberaceae, such as species of Alpinia and Curcuma, inter alia. In gymnosperms, except conifers, essential oils are rarely found. The following sections summarize the main families consisting of aromatic plants. Additionally, relevant references where readers can find keys for identification of the main genres for each botanical family and geographical distribution are cited.

Apiaceae Description: herbs, rarely shrubs or trees, are often aromatic; alternate leaves, sometimes with invaginating petiole resembling a sheath, simple or complex, occasionally ripped. Inflorescence umbel type, often composed, sometimes reduced to a glomerulus; not showy flowers, bisexual or rarely unisexual, actinomorphic, diclamide or rarely monoclamide; calyx usually undeveloped and pentamer, dialisepal;

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pentamerous corolla dialipetal, valvular estivation, stamens five; ovary inferior, bicarpelar, biovular, pendulum placentation, uniovulate locule, rarely biovulate, one of them is abortive. Schizocarpic fruit with mericarpes to similar achenes. Comments: The Apiaceae have a worldwide distribution. Economically important members include a number of food, herb, and spice plants, such as dill ( Anethum); celery ( Apium); caraway ( Carum); coriander ( Coriandrum); cumin ( Cuminum); carrot ( Daucus); fennel ( Foeniculum); and parsley ( Petroselinum); some species are poisonous, such as Conium maculatum, poisonhemlock (an extract of which Socrates drank in execution); others are used as ornamental cultivars. See Plunkett et al. (1997) for a more detailed study of the Apiaceae. Citations for the family and main genera: Pimenov MG, Leonov MV (1993) The genera of the Umbelliferae. Royal Botanic Gardens, Kew Plunkett GM, Soltis DE, Soltis PS (1997) Evolutionary patterns in Apiaceae: Inferences based on matK sequence data. Syst Botany 21:477–495

Asteraceae Description: Herbs, subshrubs, shrubs, less frequently trees, or vines, latex sometimes present; spines present in some species; alternate or opposite leaves, rarely verticillate, simple or complex, without stipules, entire or serrated margin. Inflorescence type capitula, which is surrounded by bracts that form the casing flowers arranged on a large discoid receptacle; all flowers alike or different from each other in ray flowers and disk flowers; the first generally highly modified and may be sterile and have hypertrophied corolla, disk flowers are bisexual or rarely unisexual, usually actinomorphic, diclamides, or without calyxp; calyx often bristly or turned into feathery papilho; corolla usually pentamerous, gamopetal, valvular estivation; five stamens, sinanthers, epipetals, rimose anthers; inferior ovary, bicarpelar, unilocular, with a single ovule of erect placentation. Cypsela fruit with sturdy papilho, aiding in the dispersal of the fruit. Comments: The Asteraceae family occurs worldwide, with occurrences registered for different biomes and includes several plants widely used as food such as artichoke ( Cynara), chicory ( Cichorium), lettuce ( Lactuca), and sunflower ( Helianthus). Some species of the genus Artemisia are used as spices. Ambrosia is the cause of hay-fever and many species act as agricultural pests. Many genera have ornamental importance such as Helianthus, Gaillardia, Sphagneticola, Zinnia, Senecio, Tagetes, Dahlia (Dahlia), Leucanthemum (chrysanthemum), Argyranthemum, Dendranthema, and Calendula. Citations for the family and main genera: Wagenitz G (1976) Systematics and phylogeny of the Compositae ( Asteraceae). Plant Syst Evo 125(1):29–46

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Brassicaceae Description: Annual or perennial herbs, subshrubs, fusiform underground part. Rosulate basal leaves, alternate on the stem, glabrous or hairy. Androgynous flowers, actinomorphic, with yellow, violet, and lilac petals. Androecium tetradynamo (six stamens, four of which are bigger and two are smaller), with small lateral nectaries and the medianes one are large. Ovary superior, bicarpelar with two or more ovules; stigma terminal, capitate, or bilobed. Siliqua fruit. Comments: The Brassicaceae family presents a cosmopolitan distribution. Some species of this family are used for horticulture and of economic importance. The main one is Brassica oleracea, from which were obtained several horticultural varieties, especially B. oleracea var. acephala, B. oleracea var. capitata, B. oleracea var. geminifera, B. oleracea var. botrytis, and B. oleracea var. italica, through breeding. Other species of note are the radish ( Raphanus sativus), horseradish ( Armoaracia rusticana), watercress ( Rorippa nasturtium-aquaticum), and arugula ( Eruca vesicaria). Citations for the family and main genera: Hitchcook CL (1945) The South American species of Lepidium. Lilloa 11:75–134 Warwick SI, Black LD (1991) Molecular systematics of Brassica and allied genera (Subtribe Brassicinae, Brassiceae)—chloroplast genome and cytodeme congruence. Theor Appl Genet 82(1):81–92

Lamiaceae Description: Herbs, shrubs, or trees; square stems in cross section; often with iridoids and phenolic glycosides. Glandular trichomes with aromatic oils and simple, or not glandular; the latter, if present, generally multicellular and uniseriate, or a mixture of unicellular and multicellular hair. Leaves usually opposite, occasionally verticillates, simple, sometimes lobed or ripped, or pinnately compound, entire to serrated. Inflorescences with indeterminate main axis and branched as cymes, frequently congested in false verticils, terminal or axillary. Flowers bisexual, zygomorphic. Sepals usually connate, radial, or bilateral calyx, campanulate, persistent in fruit. Petals usually five, connate, usually bilabiate corolla with imbricate lobes. Four stamens, didynamos sometimes reduced to two; fillets adnats to the corolla. Carpels two, conate; ovary superior, without lobes to deeply four-lobed, bilocular, with axial placentation; style generally divided at the apex, the terminal ginobasal; stigmas two. Ovules two per carpel, inserted laterally. Nectary disk present. Fruit drupe, with one to four pits and indehiscent capsule or shizocarp. Comments: Lamiaceae family presents a cosmopolitan distribution. Due to the presence of essential oils and use of certain species as spices, the family has great economic importance, such as mint ( Mentha), lavender ( Lanvandula), Marrubium, Nepeta, basil ( Ocimum), Rosmarinus, Salvia ( Salvia), Satureja, and Thimus. Some genera are cultivated for ornamental purposes, including Ajuga, Callicarpa, Clerodendrum, Plectranthus, Holmskioldia, and Vitex. Citations for the Family and main genera:

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Epling C (1937) Synopsis of the South American Labiatae. Fedde Repert 85(1/4):1-341 Harley RM (1988) Revision of generic limits in Hyptis Jacq. (Labiatae) and its allies. Kew Bull 98:87–95 Moldenke HN (1934) Artificial key to the Species and varieties of Aegiphila. Brittonia 1(5/6):263–280

Myrtaceae Description: Trees or shrubs, rarely subshrubs, trunk usually with exfoliating bark; opposite or alternate leaves, rarely verticillate, simple, vestigial, or absent stipules, entire margin, usually coriaceous or subcoriaceous, with translucent scores, peninerve, usually with collecting marginal rib. Inflorescence usually cresting, sometimes reduced to a single flower; showy flowers, usually with predominantly white color, bisexual or rarely unisexual, actinomorphic, diclamide or very rarely monoclamide; calyx with three to six sepals generally dialisepal pre-flowering generally imbricated, sometimes forming a calyptra or opening irregularly; corolla usually three to six petals, dialipetal, imbricated pre-flowering, sometimes forming a calyptra; stamens long and showy, numerous, very rarely number equal to or double to the petals, often free or less united at the base, anthers rimose, rarely poricidal; nectary present; ovary inferior, two to numerous locules, placentation axial, bi, or numerous locules, single stylus. Fruit berry, drupe, or capsule. Comments: Family with pantropical distribution, in a large diversity of habitats; also very diverse in subtropical Australia. Includes about 144 genera and 4630 species, standing out as major genera Eucalyptus, Syzygium, Eugenia, Myrcia, Melaleuca, Corymbia, Psidium, and Calyptranthes. Eucalyptus is an important source of wood and many genera of this family include important ornamental plants with sepals, petals, and/or stamens showy. The dianthus stems of the buds of Syzygium aromaticum and the fruits of Pimenta dioica are the source of allspice. In addition, many other species provide edible fruits. Aromatic oils and antiseptics are extracted from many species of Eucalyptus. Citations for family and main genera: Johnson LAS (1976) Problems of species and genera in Eucalyptus (Myrtaceae). Plant Syst Evol 125:155–167 Landrum LR (1982) Myrceugenia (Myrtaceae). Fl Neotrop Monogr 29: 1–138 Landrum LR (1986) Campomanesia, Pimenta, Blepharocalyx, Legrandia, Acca, Myrrhinium and Luma (Myrtaceae). Fl Neotrop Monogr 45:1–180 Landrum LR, Kawasaki ML (1997) The genera of Myrtaceae in Brazil: an illustrated synoptic treatment and identification Keys. Brittonia 49(4):508–536 Legrand CD (1957) Representantes neotropicales del género Myrceugenia. Darwiniana 11(2):293–365 Legrand CD (1958) Las especies tropicales del género Gomidesia. Comm Bot Mus Hist Nat Montevideo 3(37):1–30 Legrand CD (1962) El género Calyptranthes en el Brasil Austral. Lilloa 31:183–206

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Legrand CD (1962) Sinopsis de las especies de Marlierea del Brasil. Comm Bot Mus Hist Nat Montevideo 3(40):1–39 Proença C (1990) A revision of Siphoneugena Berg. Edinb. J Bot 47 (3):239–271 Salywon AM, Landrum LR (2007) Curitiba (Myrtaceae): a new genus from the planalto of Southern Brazil. Brittonia 59(4):301–307 Schmid R (1980) Comparative anatomy and morphology of Psiloxylon and Heteropyxis and the subfamilial and tribal classification of the Myrtaceae. Taxon 29:559–595 Wilson KA (1960) The genera of Myrtaceae in the southeastern United States. J Arnold Arbor 41:270–278

Myristicaceae Description: Trees and rarely shrubs, often with reddish latex; alternate leaves, often distichous, simple, without stipules, entire margin, often aromatic. Cresting inflorescence, racemose or paniculate; little showy flowers, unisexual (monoecious plants or dioecious more often), actinomorphic, monoclamide; tepals usually three, connate, valvular estivation; two stamens to numerous, united by threads, free anthers united among themselves or occasionally rimose; superior ovary, unicarpelar, erect, uniovulate placentation. Follicle or fleshy fruit or berry subwoody, with seed surrounded by aryl fleshy, showy. Comments: Family with pantropical distribution, and very characteristic of humid lowland forest, which includes about 20 genera and 500 species. The main genera are Hosfieldia, Myristica, and Virola. Species of this group are noted for presenting economic importance as Myristica fragans Houtt., whose seeds are the source of nutmeg. Furthermore, hallucinogens are obtained from numerous species of Virola. Citations for family and main genera: Armostrong JE, Dummond BA (1986) Floral biology of Myristica fragans Houtt, the nutmeg of commerce. Biotropica 18:32–38 De Wilde WJJO (1994) Paramyristica, a new genus of Myristicaceae. Blumea 39(1–2):341–350 Sauquet H (2004) Systematic revision of myristicaceae (Magnoliales) in Madagascar, with four new species of Mauloutchia. Bot J Linn Soc 146(3):351–368 Schouten RTA (1986a) “Revision of the Genus Gymnacrathera (Myristicaceae).” Blumea 31 (2):451–486 Schouten RTA (1986a) Revision of the Genus Gymnacranthera Myristicaceae. Blumea 31(2):451–486 Smith AC, Woodhouse RP (1937) The American species of Myristicaceae. Brittonia 2:393–510 Wilson TK, Maculans LM (1967) The morphology of the Myristicaceae. I. Flowers of the Myristica fragans and M. malabarica. Amer J Bot 54:214–220

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Piperaceae Description: Herbs, shrubs, or small trees, often epiphytes or lianas; alternate leaves, verticillate or opposite less frequently, simple, with or without stipules. Inflorescence usually the type spike, rarely racemes, terminal axillary or opposed to the leaf; not showy flowers, bisexual or unisexual, aclamide, arranged in the armpit of bracts usually peltate; stamens 1–10, more often 6, rimose anthers; superior ovary, uni- or tetracarpelar, unilocular, placentation upright, usually uniovular, sessile stigma. Fruit berry or drupe. Comments: Family widely distributed in tropical and subtropical regions, including five to eight genera and about 2000 species. Peperomia and Piper stand out as major genera of the family. The fruits of Piper nigrum provide black pepper kingdom, one of the most important and oldest spices. In addition, some Piper species are used in medicine, and many species of Peperomia present an attractive foliage and are grown as ornamental plants. Citations for family and main genera: Borstein AJ (1991) The Piperaceae in the southeastern United States. J Arnold Arbor Suppl. 1:349–366 Semple KS (1974) Pollination in Piperaceae. Ann. Missouri Bot Gard 61:868– 871 Tebbs MC (1989) Revision of Piper (Piperaceae) in the New World. Review of characters and taxonomy of Piper section Merostachys. Bull Brit Mus (Nat Hist) Bot 19:118–158 Tebbs MC (1993) Revision of Piper (Piperaceae) in the New World 3. The taxonomy of Piper sections Lepianthes and Radula. Bull Brit Mus (Nat Hist) Bot 23:1–50 Thorne RF (1974) A phylogenetic classification of the Annoniflorae. Aliso 8:147–209 Wood CE (1971) The Saururaceae in the southeastern United States. J Arnold Arbor 52:479–485 Yuncker TG (1972) The Piperaceae of Brazil I: Piper—Group I, II, III, IV. Hoehnea 2:19–366 Yuncker TG (1973) The Piperaceae of Brazil II: Piper—Group V, Ottonia, Potomorphe, Sarcorhachis. Hoehnea 3:29–284 Yuncker TG (1974) The Piperaceae of Brazil III: Peperomia, taxa of urcentain status. Hoehnea 4:71–413 Yuncker TG (1975) The Piperaceae of Brazil IV. Hoehnea 5:125–145

Rutaceae Description: Shrubs or trees, rarely herbs or lianas, often with thorns; leaves alternate or less often opposite, compound or rarely simple, without stipules, entire or serrated margin, with translucent scores. Cresting inflorescence, rarely racemose, sometimes reduced to a single flower; flowers usually drab, bisexual, or unisexual,

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usually actinomorphic, diclamide; calyx usually four or five sepals, gamosepalous or dialisepal, usually imbricate estivation; corolla usually four or five petals, gamopetal or dialipetala, imbricate or valvular estivation; stamens equal to or double the petals or less frequently numerous, rarely fewer, generally free from each other, rimose anthers, staminodes sometimes present nectary, present; gynoecium gamocarpelar or dialicarpelar, uni- to pluricarpelar, superior ovary, axial or hanging placentation, the locules uni- to pluriovulate. Fruit drupe, berry, capsule, or follicle. Comments: Has predominantly pantropical distribution, including approximately 150 genera and 2000 species. The main genera of this group are Zanthoxylum, Agathosma, and Ruta. However, other genera are noted for their economic importance, such as Citrus, which has species appreciated by its edible fruit. The fruits of Fortunella and Casimiroa are also consumed. Furthermore, Zanthoxylum, Ruta, Citrus, and Casimiroa are used in medicine. Citations for family and main genera: Brizicky GK (1962) The genera of Rutaceae in the southeastern United States. J Arnold Arbor 43:1–22 Kaastra RC (1982) Pilocarpinae (Rutaceae). Fl Neotrop Monogr 33:1–198 Kallunki JA, Pirani JR (1998) Synopses of Angostura Roem. & Schult. and Conchocarpus J. C. Mikan (Rutaceae). Kew Bull 53(2):257–334 Kallunki JA (1992) A revision of Erythrochiton sensu lato (Cuspariinae, Rutaceae). Brittonia 44(2):107–139 Kallunki JA (1994) Revision of Raputia Aubl. (Cuspariinae, Rutaceae). Brittonia 46(4):279–295 Kallunki JA (1998) Andreadoxa flava (Rutaceae, Cuspariinae): a new genus and species from Bahia, Brazil. Brittonia 50(11):59–62 Kallunki JA (1998) Revision of Ticorea Aubl. Brittonia 50(4):500–513 Pirani JR (1998) A revision of Helietta and Balfourodendron (Rutaceae – Pteleinae). Brittonia 50 (3):348–380 Swingle WT (1967) The botany of Cifras and its wud raltivas. In The Citrus industry, Vol. 1, History, world distribuition, botany and varieties. In: Reuther W, Webber HJ, Batchelor LD (eds). Berkeley: Univ. of California Division of Agricultural Science, pp 190–430

Quality Control of Botanical Drugs Practically all international regulatory agencies cite the organoleptic, macroscopic, microscopic characterization, besides the presence of chemicals and class of metabolites, as principal methodologies for the identification of drugs of plant origin. Thus, trained professionals can evaluate the quality by conventional analytical methods, which together are used in determining the authenticity of a particular plant drug. Accurate identification is performed by comparison with a standard drug or a detailed description found in pharmacopoeias and specialized literature.

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Macroscopic evaluation is performed directly to the naked eye or with a magnifying glass. However, particularly when the drugs are crushed or powdered, microscopic examination is indispensable. Microscopic identification is based on the presence or absence of certain cellular structures, in particular cellular inclusions that are organic and inorganic in nature. Furthermore, it is important to observe possible differences in the organization of tissues that make up a particular plant organ. Some diagnostic structures for different organs are presented in Table 3.2.

Locations of Essential Oils Essential oils play various roles in plant, and may play the role of attracting pollinators, as in the case of osmophores, which give fragrance to the flowers, or repel insects by insecticide and deterrent action, reducing herbivory. Essential oils can be stored in various organs of a plant, such as flowers ( Citrus), leaves ( Eucalyptus), in the bark of the stems ( Cinnamomum), wood ( Santalum), roots ( Vetiveria), rhizomes ( Zingiber), fruits ( Pimpinella), or seeds ( Myristica). Although all organs of a plant can accumulate essential oils, their chemical composition, physical–chemical characters, and odors can vary depending on the location. Furthermore, the chemical composition of an essential oil extracted from the same organ of the same plant species may also vary significantly depending on the time of collection, climatic and soil conditions. In addition, depending on the taxonomic group to which they belong, essential oils can occur in specialized secretory structures such as in idioblasts, cavities, ducts, and trichomes, among others (Esau 1977). Specialized parenchyma cells that may contain various substances, different in content or form of the remaining parenchyma cells, are called idioblasts (Fahn 1979, 1988). In Lauraceae, there is the common occurrence of this type of cells storing essential oils. Essential oils can also be deposited in certain spaces in plant tissues such as cavities and ducts. These spaces can be of lysigenous or schizogenous in origin. In the first case, a group of cells, some of them accumulate secretion and its walls are ruptured, an example is cavities formed in species of Citrus L. (Rutaceae), in which the parent cell that gives rise to the cavity is shallow and migrates into the tissue dividing repeatedly. Subsequently, the most central cells begin to accumulate essential oils and finally break down its walls. The process is repeated until reaching the limit of cells that form the epithelium. In general, in lysigenous cavity remains of cell walls can be noticed. In the case of species of Baccharis L. (Asteraceae), schizogenous cavities are formed in stems and leaves, in this case, there is a spreading of cells that form an epithelium and produce a secretion that fills the space formed.

Leaf Blade Petiole

Stem Rhizome

Transverse section

Surface view Paradermal section

Radial longitudinal section

Transverse section

Radial longitudinal section

Table 3.2  Diagnostic structures of crushed or powdered herbal drugs Organ part Preparation Transverse section Root

Fibers and sclereids (wall thickening and lumen) Pith Parenchyma in the cortical region undeveloped Primary xylem external in relation to the vessel element Prismatic crystals and starch (location in the tissue) Vessel elements (Wall thickening) Fibers (length) Sclereids (cell shape) Crystals and starch (location in the tissue) Epidermis (wall contour) Stomata (type, distribution, stomatal index on the faces of the blade) Trichomes (types, shape, length, arrangement, number of cell rows) Absence of fibers Carbonate and calcium oxalate crystals (location in the tissue)

Cells/tissues Absence of collenchyma, cuticle, stomata and trichomes Epidermis or periderm (cell shape and number of layers) Fibers and sclereids (wall thickening and lumen) Parenchyma in the cortical region developed Primary xylem internal in relation to the vessel element Prismatic crystals and starch (location in the tissue) Vessel elements (wall thickening) Fibers (length) Sclereids (cell shape) Prismatic crystals and starch (location in the tissue) Epidermis or periderm (cell shape and number of layers)

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Fruit

Flower Sepals Petals Anthers Ovary

Table 3.2 (continued) Organ part

Transverse section

Paradermal section

Transverse section

Surface view Paradermal section

Preparation

Cells/tissues Collenchyma (wall thickening) Cuticle (thickening) Epidermis (cell shape) Mesophyll with palisade and spongy parenchyma Vascular Bundles (types) Epidermis (wall contour) Papillae Pollen grains (shape) Stomata (type, distribution) Trichomes (types, shape, length, arrangement, number of cell rows) Crystals (location in the tissue) Cuticle (thickening) Epidermis (cell shape) Undifferentiated parenchyma in sepal and petal Absence of collenchyma, fibers and sclereids Cuticle (thickening) Epidermis (wall contour) Stomata (type, distribution) Trichomes (types, shape, length, arrangement, number of cell rows) Crystals: drusas, prismatic and raphides (location in the tissue) Endocarp with sclerenchyma cells (cell shape and number of layers) Epicarp (cell shape and number of layers) Fibers (wall thickening and lumen) Mesocarp Oil glands Sclereids (cell shape) Secretory canals

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Seed

Table 3.2 (continued) Organ part

Paradermal section Transverse section

Preparation

Cells/tissues Vascular bundles (types) Testa (cell shape) Aleurone grains (location in the tissue) Cotyledons Endosperm (cell shape and number of layers) Fibers (wall thickening and lumen) Hemicellulose Lignified cells in palisade Mucilagens (location in the tissue) Prismatic crystals (location in the tissue) Sclereids (shape) Starch grains (location in the tissue) Testa (cell shape and number of layers)

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Glandular trichomes are involved in the secretion of various substances, including essential oils. These trichomes are formed at their end by a uni- or multicellular head, which may have a variety of shapes and sizes. The head joins the epidermis by means of a stem or stalk, unicellular or multicellular. The stalk varies in length, and is often very short that looks like a disk. The cells, which constitute the head, are secretory and usually contain numerous mitochondria and other organelles that vary according to the secreted material. When these trichomes secrete essential oils, it is stored between the walls of the secreting cell and the cuticle, and is eliminated by cuticle pores or breakdown of the cuticle. This latter process can occur one or more times, if the cuticle regenerates, and provide new subcuticular accumulation.

Classical Histochemical Methods Histochemical methods are the association of the histological techniques and physical and chemical methods for identifying, locating, and sometimes semi-quantifying compounds or groups of chemicals in cells and tissues. Histochemical tests should preferably be carried out on fresh material; however, these can be applied on fixed material without inclusion or embedded in paraffin or methacrylate (resin). In the case of the applied test for detection of essential oils, the material used must be fresh or fixed and not included. Furthermore, the realization of positive and negative controls is recommended. The negative control is performed by comparison of results with white (material without the application of reagents or dyes), because the natural color of some cells may be similar to the expected positive result for a given histochemical test or interfere with the final staining. The use of reference material to ensure the effectiveness of the reagent and the procedure is also recommended (positive control). Can be considered as a material reference one in which there is proven occurrence of a given compound. NADI reagent is used for the detection of essential oils and oleoresins in plant tissues. This reagent is a mixture of two colorless components, the α-naphthol and the dimethyl-p-phenylenediamine chloride, which gives indophenol blue by oxidation, a highly lipid-soluble compound that changes color on varying pH, allowing for differential staining essences and resin acids. Proposed method for the NADI reagent (David and Cade1964): Preparation 0.1 % α-naphthol in 40 % ethanol 1 % N,N-dimethyl-p-phenylenediamine in water Buffer Sodium phosphate 0.05 M pH 7.2

0.5 ml 0.5 ml 49.0 ml

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Procedure • Place the sections in freshly prepared reagent for 1 h at room temperature and in the dark. • Wash rapidly in buffer sodium phosphate 0.1 M pH 7.2 for about 2 min. • Note immediately because the reaction is ephemeral. Results Terpenes stain of blue (essential oils) to red (oleoresins) and acquire violet coloration in mixtures of essential oils and oleoresins. Control Terpenes extract with organic solvents or take the test in reference material.

References Bremer B, Bremer K, Chase MW et al (2009) An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG III. Bot J Linn Soc 161(2):105–121 Chase MW (2004) Monocot relationships: an overview. Am J Bot 91(10):1645–1655 David R. J.-P. Carde 1964. Coloration différentielle des inclusions lipidiques et terpéniques des pseudophylles du Pin maritime au moyen du réactif nadi. Compte-Rendu de l’Académie des Sciences, Paris 258: 1338-1340. Esau K (1977) Anatomy of seed plants. 2 edn., Wiley, New York Fahn A (1979) “Nectaries.” In secretory tissues in plants. Academic Press, New York Fahn A (1988) Secretory tissues in vascular plants. New Phytol 108(3):229–257 Judd WS, Olmstead RG (2004) A survey of tricolpate (eudicot) phylogenetic relationships. Am J Bot 91(10):1627–1644 Judd WS, Campbell CS, Kellogg EA et al (2007) Plant systematics: a phylogenetic approach. 3rd edn., SINAUER ASSOCIATESES, INC., Massachusetts McNeill J, Barrie FR, Buck WR et al (2012) International code of nomenclature for algae, fungi, and plants (Melbourne code). Koeltz Scientific Books, Königstein

Chapter 4

Essential Oils as Raw Materials in the Synthesis of Anticancer Drugs Timothy J. Brocksom, Kleber T. de Oliveira, Marco A. B. Ferreira and Bruno M. Servilha

Introduction Cancer disease is certainly one of the major human challenges of the present century, and this chapter will focus strongly on the chemical approach to solutions. It has been known for centuries that organic chemical compounds can have profound effects on human disease, and have led to traditional medicines for the cure of such infirmities (Nicolaou and Montagnon 2008; Corey et al. 2007). Cancers are of no exception to this general idea, and over the past five or six decades this is being increasingly explored. All the possible types of organic compounds are being tested for anticancer activity, with very important successes reported constantly. The pharmaceutical industry has of late turned its back on natural products, and the discipline called pharmacognosy, because of perceived inefficiencies within the intense binomial of new chemical entities versus biological/pharmacological activity profiles. The search for new bioactive compounds is really the needle in the haystack problem, amplified as never before. We will now try to explicit this scientific problem so as to justify specific natural products and their synthesis from essential oil components. Traditional pharmaceutical solutions to the creation of new drugs has been based on the coupling of well-known building blocks in different fashion, which we can call the LEGO® approach (Aggarwal 2009). Generally, these building blocks are relatively simple polyheteroaromatic structures, which can be linked together by simple chemical reactions, thus producing rapidly complex molecules of expected biological activity (Johnson and Li 2007). These molecules are usually devoid of three-dimensional stereochemistry, and can be synthesized in at the very most 10– 15 chemical reactions. These chemical entities present certain basic features which have led to immense success over the past century; relative structural simplicity, facility of production, lack of inconvenient stereochemical mixtures, and thus lower

T. J. Brocksom () · K. T. de Oliveira · M. A. B. Ferreira · B. M. Servilha Department of Chemistry, Federal University of São Carlos, São Carlos, Brazil e-mail: [email protected] © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_4

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Fig. 4.1  The erectile dysfunction drugs

cost. Figure 4.1 presents the structures of the three most popular drugs for erectile dysfunction, sildenafil, vardenafil and tadalafil. These structures demonstrate the LEGO® approach, and a certain tendency to copy that which works well, and called me-too in the industry. However, these kinds of compounds present certain disadvantages when compared with natural products, which are the lack of greater structural diversity and thus occupation of chemical space (Lachance et al. 2012), and the restrictive fixation on nitrogen atoms in aromatic heterocycles as opposed to other heteroatoms in more saturated cyclic systems. These fundamental structural differences have already been described (Feher and Schmidt 2003), and represent an important reference for the overall structural diversity of organic chemicals. In this context, combinatorial chemistry was launched some 20 years ago as the solution to all these problems, but unfortunately was a complete fiasco and has been dispensed by big pharma (the very large and traditional pharmaceutical companies). Natural products, or secondary metabolites, also have some deficiencies as potential drugs and these should be taken into account. First, as certainly has already been said, Nature is not a pharmaceutical industry and has no obligation to provide readymade quality drugs. Natural products are produced by enzymatic processes, and therefore their biosynthesis (Dewick 2009; Croteau et al. 2000) is a consequence of biological interactions with enzymes, which suggests ‘biological activity’. However this activity cannot be extrapolated from the natural occurrence to resolve questions of human diseases, as has been proposed by many phytochemists or pharmacognosists. Obviously human history has proven the fortunate exceptions to this statement. As an example, Fig. 4.2 shows the structures of vinblastine and

Fig. 4.2  Vinblastine and vincristine

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Scheme 4.1  Paclitaxel synthesized from baccatin

vincristine, used to treat a wide range of cancers, both being isolated with much difficulty from the common periwinkle ( Catharanthus roseus or Vinca rosea). Furthermore, Nature is not a pharmaceutical industry, in the sense that production on an industrial scale sufficient to attend the needs of world population is not available. The example of paclitaxel (Taxol®) is quite illustrative, in that isolation from the yew tree means killing a 60-year-old specimen for a very small weight of the drug (Jacoby 2005). On the other hand, baccatin can be isolated in much larger quantities in a recyclable way, and some relatively simple synthetic chemistry produces the desired paclitaxel (Scheme 4.1). This transformation is apparently now in execution by a microbiological process, which to our knowledge is still synthetic organic chemistry but performed with different reagents. Regardless of the fact that Nature does not provide drugs with quality, quantity, efficiency or diversity, it is remarkable how Nature provides wonderful examples of potential molecular structures that can become excellent drugs, and this has been labelled as lead structures. This point will now be clarified with more detailed explanations on the structural origin of the compound and activity, within the context of lead structures. The ideal solution would seem to be the acceptance of the immense plethora of natural product molecular structures, with excellent pharmacological activity, modified by the synthesis of structural analogues of more pronounced activity and lower toxicity. These new analogues can possess simpler structures and therefore be more accessible by synthesis. These concepts have been explored over the past 30– 40 years and have been fully delineated. Table 4.1 illustrates the classic divisions of the diverse molecular structures and origins of recognized drugs in active use and of anticancer drugs for comparison. These are basically reported by Newman, Table 4.1  The origins of all drugs and anticancer drugs

NP NP-M S S-NPb

All drugs 6 28 50 16

Anticancer drugs 12 30 36 22

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Cragg and colleagues, starting in from 1997 and being brought up to date in 2012, now covering 30 years of analysis (Cragg et al. 1997; Newman et al. 2003; Newman 2008; Cragg et al. 2009; Newman et al. 2012). Table 4.1 is based on small-molecule drugs, excluding vaccines and biological entities. Over the time scale of these reviews (1997–2012), the groups have been subdivided several times, reflecting the extreme diversity of molecular structures from totally synthetic to totally natural product, and the increasing proximity of synthetics based upon natural products. We have tried to simplify this discussion with the creation of just four groups: totally unmodified natural products (NP), natural products modified by semi-synthesis (NP-M), totally synthetic (S) and synthetics based upon natural products as models or mimics (S-NPb). The numbers are per cent values. In the specific case of anticancer activity, natural products and analogues have shown much more success than traditional big pharma molecules (Cragg et al. 2009; Newman et al. 2012). It is interesting to comment that a very similar situation exists for antibiotics, once again much more available from natural product inspiration than from classical synthetics. Over the past 8 years, several very interesting reviews have appeared dealing with aspects of natural product chemistry and application as drugs (Butler 2005; Simmons et al. 2005; Gordaliza 2007; Neidle et al. 2010; Mishra and Tiwari 2011; Montaser and Luesch 2011; Mondal et al. 2012; Dias et al. 2012; Sawadogo et al. 2013). These reviews detail the natural source, terrestrial or marine, or microorganism, and the kind of drug activity found including anticancer activity. We will try to provide convincing examples of natural product molecules and analogues, available by synthesis and demonstrating viable pharmacological properties. These properties can vary from strong but initial indications to several steps or phases, finally leading to real drugs available at the pharmacy. We have had to make a very personal selection of these examples to keep this chapter to a reasonable length, and we have already apologized to those colleagues whose excellent chemistry has not been selected. The synthesis of these molecules is directly linked to compounds found in essential oils, which are abundant, and furnish excellent starting materials.

Essential Oils Essential oils are an immense source of organic chemical feedstocks, available and renewable, as opposed to petrochemicals. The six volume tome edited by Guenther between 1948 and 1952 is probably still the best source of information available (Guenther 1948–1952). The essential oils can vary from the very abundant pine oils, produced on an extremely large industrial scale, to the precious rose oils used in specialized perfumes (Maffei et al. 2011). In this chapter we will concentrate on the most abundant essential oils, produced on a massive scale, and which provide large quantities of individual chemical compounds, suitable to be used as feedstocks and starting materials for the synthesis of more complex organic molecules with anticancer activity.

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Fig. 4.3  The abundant monoterpenes present in essential oils

The choice of essential oils in this discussion is based on availability, price, the presence of a major component that is easily purified and questions of stereochemical homogeneity. For instance, pine oils, originally known as turpentine, contain both α- and β-pinenes at variable concentrations and with variable enantiomeric purities. The separation of the pinenes is not easily executed even by distillation, and therefore methods have been developed for their direct use in chemical transformations. Citrus fruits are abundant for juice production, but the peel is very rich in limonene furnishing each enantiomer depending on the specific citrus. The 3-carene is available usually as one enantiomer, and the carvone enantiomers can be obtained from spearmint or dill. These compounds, and other readily available monoterpenes, are presented in Fig. 4.3. Terpenes have been extensively described, discussed and detailed in innumerable monographs and reviews. We cite some recent monographs that will give the reader an easier introduction to this very rich area of organic chemistry. These include a comprehensive study by Erman on monoterpenes, and textbooks on terpenes by Sell and Breitmaier (Erman 1985; Sell 2003; Breitmaier 2006). The essential oils and the isolated and purified individual compounds are available in the world market at prices ranging from a few dollars (US$ 2–5) per kilogram, with minimum orders of the metric ton, going to be perhapsUS$ 50–100 range per kilogram depending upon the purity. These considerations make the monoterpene feedstocks highly interesting for use as starting materials in the fine chemical industry. We should now make clear that although many of the major monoterpenes are readily available from Nature, they are also produced by chemical transformation on a very large scale. Obvious economic considerations decide the origin, as for example in the case of l-menthol which is produced by synthesis on a larger scale than obtained from Nature. The following schemes (Schemes 4.2, 4.3 and 4.4) illustrate several well-known chemical transformations among the monoterpenes, which thus demonstrate their ready availability on industrial scales and at very modest prices.

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a

b

c Scheme 4.2  Limonene to carvone (a), 3-carene to 2-carene (b), and the pinenes to rac-camphor and rac-α-terpineol (c)

Scheme 4.3  The iterative syntheses of geraniol and farnesol from acetone and acetylene, using the Carroll rearrangement reaction

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Scheme 4.4  β-Pinene to citronellal, menthol and other important monoterpenes

So far we have presented the monoterpene components of essential oils, and now we will introduce the phenylpropanoid components of essential oils. At this point, it is important to distinguish the biosynthetic origins of monoterpenoids, and their higher congener terpenoids, from the phenylpropanoids of C6–C3 construction (Dewick 2009; Croteau et al. 2000). These latter compounds are found as oxygenated phenyl rings (C6) coupled to an allyl group (C3). Once again these compounds are available on a large scale and at very moderate prices. We single out eugenol that is used for the degradation synthesis to vanillin (Fig. 4.4). The last compound illustrates the very frequent use of all these compounds in the food and flavour, cosmetics and toiletries industries. Having set the stage of the starting materials, we can now discuss anticancer compounds, in an increasing structural complexity sequence. Recently three reviews have detailed the organic chemist’s vision of the drug problem and its solution by synthesis (Wilson and Danishefsky 2007; Nicolaou et al. 2009; Danishefsky 2010).

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Fig. 4.4  The phenylpropanoid compounds, and vanillin

Mono- and Sesquiterpenes There are some reports in the more popular literature which suggest that the very same monoterpenes can present anticancer activities, but none have been taken very seriously so far. The essential oils themselves have been used over the centuries for many relatively simple diseases, and this causes some confusion between cause and effect. We can include a very recent example of a proven case of conjugates of the monoterpene thymoquinone (TQ), presenting enhanced activity against pancreatic cancer over TQ itself (Scheme 4.5) (Yusufi et al. 2013). On the other hand, sesquiterpenes seem to have started off the anticancer hunt among natural products, beginning in the 1960s with guaianolides and the important contributions from the Kupchan laboratory (Kupchan 1970). The guaianolides are probably the largest class of secondary metabolites, as far as the number of distinct chemical structures isolated and determined. This is certainly due in part to the expectation of finding relevant biological activity, and this has been explained by the presence of unsubstituted C–C double bonds conjugated to electron-withdrawing groups such as the carbonyl group (Ghantous et al. 2010). Arglabin is a guaianolide and helenalin is a pseudo-guaianolide, where the structural difference in carbon skeleton resides in the position of the methyl group of the five-membered ring or at the ring junction. Figure 4.5 illustrates these points, with the α-methyleneO

H X O

O

O

X

O

X

X

NaN3

N NH2

O

X

O

thymoquinone

Scheme 4.5  Thymoquinone conjugates

X

X = OH X= F

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Fig. 4.5  Arglabin and helenalin guaianolides

γ-butyrolactone unit evident as the key structural feature and acting as a Michael acceptor in enzyme ligation. Darwiche et al. have recently reviewed the relation between sesquiterpene lactones and anticancer activity (Ghantous et al. 2010), while the very large group led by Chen in China has presented an extensive study on guaianolide sesquiterpenes and leukaemia (Zhang et al. 2012). Both these publications deal with structure−activity relationships, including the reactivity of conjugated unsaturated carbonyl groups undergoing addition reactions. The synthesis of arglabin demonstrates the basic concept of hemi-synthesis (or semi-synthesis), in which a more readily available compound from Nature, with a relatively similar structure, can be transformed into a compound of enhanced value. Arglabin is being used for the treatment of breast, colon, ovarian and lung cancers, but is available in very limited quantities from Nature. The transformation of the very readily available germacranolide sesquiterpene parthenolide occurs in excellent overall yield, thus making arglabin commercially viable (Scheme 4.6). Strictly speaking, parthenolide is not an essential oil component, but is included here for the relevance of the chemistry to solve a supply problem (Zhai et al. 2012). The Ley laboratory has developed an important route to several of the thapsigargin guaianolide sesquiterpenes, which exhibit sub-nanomolar sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibition, including prostate cancer (Scheme 4.7). The starting material is ( S)-(+)-carvone, which is transformed into a related cyclopentanoid compound, before the elaboration of the seven-membered ring, and the seven different oxygenated functional groups in the correct stereochemical array. The closing of this ring with seven carbon atoms makes use of the ring-closing metathesis (RCM) reaction (using the Grubbs II catalyst), which is now very popular and applicable even on an industrial scale. Although the synthetic sequence is rather long, the Ley group has demonstrated scaleup to make these compounds more readily available (Andrews et al. 2007). Of the englerin guaiane sesquiterpenes, englerin A is much more active than other members of this small group, against renal cancer and at the nanomolar level. Since 2009, at least three different research groups have disclosed total syntheses of this molecule, but with quite different strategies and monoterpene starting materials. In chronological order, the Christmann laboratory demonstrated the use of nepetalactone as the starting material (Willot et al. 2009). This less well-known

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Scheme 4.6  Hemi-synthesis of arglabin from parthenolide

monoterpene is available by distillation of catnip oil at US $ 0.50 per gram, and has the biological activity of sexual arousal in cats. Making use of the already present five-membered ring, rather simple elaboration then allowed the construction of the seven-membered ring (RCM, Grubbs II) with all the appendages present (Scheme 4.8). In 2010, the Ma group published their synthesis of (−)-englerin A starting from ( R)-citronellal, which was elaborated into a farsenane sesquiterpene type compound

Scheme 4.7  Ley’s thapsigargin synthesis from ( S)-(+)-carvone

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Scheme 4.8  The Christmann group synthesis of (+)-englerin A

(Zhou et al. 2010). This substrate was then reacted with a gold catalyst coupled with a silver catalyst, undergoing two cyclizations to lead finally to englerin A. An important feature here is the avoidance of protecting group strategies, which is the bane of modern synthesis. In this way, the total synthesis becomes more efficient with a greater step economy (Scheme 4.9). Finally, the Metz group has synthesized englerin A from (−)-isopulegol, which undergoes a ring contraction to generate a five-membered ring intermediate (Zahel et al. 2013). Elaboration leads to a substrate for RCM reaction with the Grubbs II catalyst, furnishing the seven-membered ring, and then leading to (−)-englerin A (Scheme 4.10). To close this section, we now present artemisinin, a cadinane sesquiterpene initially launched as an antimalarial agent quite a few decades ago. This compound

Scheme 4.9  The Ma group synthesis of (−)-englerin A

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H Grubbs II OH

OAc CHO

OH

CO2Et

H OH

CO2Et

O

O

(-)-isopulegol

O Ph

O H

H

O

O H

(-)-englerin A

Scheme 4.10  The Metz group synthesis of (−)-englerin A

was then known as qinghaosu, and this led to the great worldwide interest in learning about Chinese pharmacognosy and phytochemistry. The substitution of quinines was becoming really urgent, and artemisinin arrived right on time for this really serious health problem in the southern hemisphere. Recently, it has been shown that artemisinin also has important anticancer activities (Ghantous et al. 2010). The Avery group was perhaps the first to establish a synthetic route to (+)-artemisinin, starting from ( R)-pulegone and in ten steps (Avery et al. 1992). After a classical sequence with loss of the three carbon isopropenyl groups, but which allows introduction of the other side chains, the group is reintroduced by the Claisen rearrangement. The final steps model the probable biosynthesis and lead to artemisinin (Scheme 4.11). A related synthesis was disclosed by the Constantino group, also starting with (−)-isopulegol (Constantino et al. 1996). In this synthesis, the isopropyl group is retained throughout, thus giving much more efficiency and elegance (Scheme 4.12). The Liu laboratory has demonstrated an effective synthesis of artemisinin starting from β-pinene, modified to an excellent dienophile to undergo the Diels−Alder reaction with isoprene (Liu et al. 1993). The cycloadduct is then transformed into the same substrate for biomimetic oxidation to artemisinin (Scheme 4.13).

Scheme 4.11  The Avery synthesis of (+)-artemisinin

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Scheme 4.12  The Constantino synthesis of (+)-artemisinin

Scheme 4.13  The Liu synthesis of (+)-artemisinin

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Diterpenes The diterpene section begins with the paclitaxel (Taxol®) story and the discovery of this complex natural product with very important clinical properties against several types of cancers. This topic has been extensively reviewed, because of the unusual popular interest in a molecule which seemed to promise every kind of cure (Jacoby 2005; Nicolaou et al. 1994). Paclitaxel does have a broad range of effective anticancer properties, and also some inconvenient side effects, which have led to the search and discovery of substitutes. We presented the hemi-synthesis of paclitaxel in Fig. 4.3, as an example of a natural product being made available by chemical transformation from a more readily accessible natural product. The total synthesis of paclitaxel, including from monoterpenes, is an academic exercise of the very highest quality but certainly not viable yet for large-scale production. This is due to the very many chemical reactions in sequence required to reach the synthetic goal, and therefore inefficient and very costly. We will present just one of the many excellent approaches, along this line of chemistry, choosing the Wender group as the model to be followed (Wender et al. 1997). Starting from α-pinene, easily oxidized to (+)-verbenone, the left-side ring with the dimethyl group is coupled with linkers to create the tricyclic system with appropriate functionalities, leading to paclitaxel (Scheme 4.14). As a side topic, Shing and collaborators have synthesized the right-hand sixmembered ring portion of paclitaxel, starting from ( S)-(+)-carvone (Shing et al. 2004), and this just demonstrates that a 20-carbon atom diterpene should be amenable to a very convergent approach starting from two molecules of the ten-carbon atom monoterpene precursors. The Srikrishna group has performed extensive

O

O

O

O O α-pinene

(+)-verbenone

OH

TIPSO

O OTBS

OTBS

O O O

O OH

NH O O OH

OH O O O O

paclitaxel

Scheme 4.14  The Wender group approach to paclitaxel

O

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Fig. 4.6  Paclitaxel, sarcodyctin and eleutherobin structures

synthetic investigations on sesqui- and diterpene synthesis, using carvone as the starting material, and including studies on paclitaxel as a goal (Srikrishna et al. 2005). As an important continuation of the paclitaxel story, the mechanism of microtubule interaction was extended to several other natural products, including the sarcodyctin and the eleutherobin diterpenes, and several macrolides (to be seen later). The sarcodyctins and eleutherobins (the eleutheside family) represent a quite different structural type of diterpene when compared with paclitaxel, but the same general anticancer activities have been discovered and related to the stabilization of microtubules (Ojima et al. 1999). It is noteworthy that the eleuthesides have a slightly different profile of activities when compared with paclitaxel, and are therefore being considered a second-generation substitute, especially with regard to paclitaxel-resistant cancer cell lines. Figure 4.6 permits comparison of the structures of these diterpenes. As a final comment, these eleuthesides are marine natural products found in coral species, which make their isolation difficult and in very limited quantities. Once again, synthesis must come to the rescue to provide sufficient material for the demanding biological assays and application in humans. A rather simple examination of eleutheside gross structures shows an intact para-menthane unit on the left side of the molecules. Looking at the right-hand side, the presence of a ten-membered all carbon atom ring containing an oxygen atom bridge is evident, which immediately suggests organometallic nucleophile addition to a carbonyl group or the RCM reaction as the keys to the construction of this part. As demonstrated below, these solutions have been well recognized by others. In 1998, the Nicolaou group disclosed the syntheses of two sarcodyctins starting from ( R)-(−)-carvone, as shown in Scheme 4.15. Carvone was quickly elaborated into an intermediate with the two handles necessary for extension to the ring closure substrate. In this case, a classical acetylide nucleophilic addition to an aldehyde carbonyl group was efficient, finally leading to the sarcodyctins (Nicolaou et al. 1998). The next year, the Danishefsky laboratory presented their version of an eleutherobin synthesis (Scheme 4.16), starting from ( R)-α-phellandrene. Dichloroketene cycloaddition put on the two handles, which allowed chain extension to the substrate for the Nozaki−Kishi cyclization, before final modifications (Chen et al. 1999).

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Scheme 4.15  The Nicolaou sarcodyctin syntheses

Scheme 4.16  The Danishefsky synthesis of eleutherobin

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Scheme 4.17  The Gennari approach to eleutherobin

In 2006, the Gennari laboratory presented a formal synthesis of eleutherobin starting from ( R)-carvone, which was once again elaborated to introduce the two handles (Scheme 4.17). Chain extensions furnished the substrate for a RCM reaction with the Grubbs II catalyst, for final transformation into the Danishefsky intermediate previously used for eleutherobin (Castoldi et al. 2006; Gennari et al. 2007). The ingenol group of diterpenes has provoked great interest among synthetic organic chemists, because of the important biological activities discovered and the impressive structural complexity, including a stereochemical question unique to ingenol (the in-out bridge stereochemistry of the two 7-membered rings). Recently, the FDA has approved the use of Picato®, a very simple ester of ingenol, for treatment of precancerous skin condition, actinic keratosis. The Baran laboratory has published a 14-step synthesis of ingenol, starting from (+)-3-carene, which is applicable to large-scale production. Scheme 4.18 presents the synthetic scheme. 3-Carene is elaborated into a terminal acetylene allene derivative, which undergoes a Pauson–Khand pentanelation reaction with a rhodium catalyst in the presence of carbon monoxide. A sequence of oxidative functional group modifications, with a skeletal rearrangement, then furnished ingenol (Jørgensen et al. 2013). The biosynthesis of diterpenes starts with the first cyclization of geranylgeranyl pyrophosphate to a 14-membered ring, and this type of structure is found in cembranoid diterpenes. In the second phase, the 14-membered ring is further cyclized to various groups of bicyclic, tricyclic and even tetracyclic structures, thus producing the large diversity encountered in the diterpenes (Dewick 2009; Croteau et al. 2000; Gao et al. 2012). Ellis and Crimmins have recently reviewed the synthesis of cembranoids cyclized in a way which includes the eleuthesides and related bicyclic diterpenes (Ellis and Crimmins 2008). To close this section on the diterpenes,

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Scheme 4.18  The Baran synthesis of ingenol

Maimone and Baran have reviewed the synthesis of some really relevant biologically active examples, including the compounds discussed here (Maimone and Baran 2007). Although the higher terpenes, such as the sester-, triterpenes and steroids, do present important anticancer activities, there is little total synthesis research that really starts with monoterpenes. This is certainly because of the large difference in carbon atom numbers, from 10 going up to 25 or 30, which creates even more difficulties.

Macrolides Macrolides are natural products found principally in microorganisms and marine species, with a distinct biosynthetic process from the terpenes. Generally, these compounds are biosynthesized from acetate and propionate, and usually include an ester group, thus creating the macrocyclic lactone structure (Dewick 2009; Croteau et al. 2000). These molecules possess an impressive array of stereogenic centres, involving methyl and hydroxyl groups. Figure 4.7 presents a relevant collection of these compounds. Nicolaou and collaborators have reviewed the chemistry and biology of the epothilone group of macrolides (Altmann et al. 2007). As a final point to this discussion on diterpene and macrolide structures with similar anticancer activity on microtubules, the Ojima group has presented an impressive correlation between these molecules and their active functional groups on the outer regions (Ojima et al. 1999). An overlay of the paclitaxel, nonataxel, epothilone B and eleutherobin molecules demonstrates their functional group identities, independent of their carbon skeletons.

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O O

O

R

NH

O

S OH

N

O

OH HO

Me

O

O

O

O

OH NH2 O

OH O

OH

OH O

OMe epothilone A, R = H epothilone B, R = Me

O discodermolide

migrastatin

OH HO

OH

HO O

OH

O

O

H

O

O

H

O

O

H

OH OH dictyostatin

laulimalide

Fig. 4.7  Anticancer macrolide structures

The Mulzer group’s synthesis of epithilone B was disclosed in 2001, and used (+)-citronellene as starting material. In the case of macrolide syntheses, the monoterpene is not always utilized in an integral form. After degradation, the remaining carbon atoms containing a stereogenic centre, available with the correct absolute stereochemistry, are incorporated into the product. In the Mulzer synthesis (Martin et al. 2001; Mulzer and Martin 2004), of the ten original carbon atoms of (+)-citronellene, three are lost in degradation, but seven incorporated into epothilone B (Scheme 4.19). Epothilone B shows important microtubule-binding affinities and relevant cytotoxicity against multiple drug-resistant tumour cell lines. Therefore, the role as a potential paclitaxel successor has provoked intense interest in synthesis. Other naturally occurring epothilones are also under study for their similar activities, which is certainly because of the basic macrolide core carbon skeleton. The Wessjohann laboratory has just presented a synthesis of epothilone D starting from nerol or commercially available neryl bromide, which is elaborated into a homo-farnesyl sesquiterpene derivative (Wessjohann et al. 2013). Esterification and a chromium-activated Reformatsky reaction then lead to epothilone D (Scheme 4.20). The latrunculin family of marine natural product macrolides are actin binders of the cytoskeleton and have provoked much interest in their total synthesis, especially because of the very limited availability from the natural source. The Fürstner laboratory has explored a synthetic route starting from (−)-citronellene, which by carbon chain elaboration leads to an intermediate for the ring-closing alkyne metathesis reaction (Fürstner et al. 2007a). This speciality of the Fürstner laboratory is catalysed by a molybdenum species, and permits formation of the macrocycle on

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Scheme 4.19  The Mulzer synthesis of epothilone B

the way to latrunculin A (Scheme 4.21). Other members of this macrolide group were also synthesized by this procedure. In the following publication, the Fürstner group practices ‘diverted total synthesis’, in which naturally occurring latrunculins are examined by X-ray and modelling to suggest analogues, natural product like structures, which may be easier to synthesize and yet demonstrate enhanced biological activity (Fürstner et al. 2007b).

Scheme 4.20  The Wessjohann synthesis of epothilone D

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O TBSO

O

O H

O

(S)-(-)-citronellene

H PMBN

OMe S

O

[Mo]-catalyst

O

O O

O

O H HN

O

OH S

H PMBN

O

OMe S

O

Latrunculin A

Scheme 4.21  The Fürstner synthesis of latrunculin A

The Cossy and Curran groups have established a route to the tulearin macrolactones, starting from ( S)-citronellal, and utilizing the RCM reaction (Grubbs I) as the key operation (Scheme 4.22). Tulearin A shows strong antiproliferative activity against leukaemic cell lines K562 and UT7, and is found in a marine sponge in very limited quantities (Mandel et al. 2009). The Mulzer group has also synthesized kendomycin, an ansa-macrocyclic compound with interesting structural features. The compound was isolated from an Actinomycetes species and presented potent endothelin receptor antagonist and antiosteoperotic properties, with antibacterial and cytostatic activity through proteasome inhibition. The structure of kendomycin is different from usual macrocycles, because of the quinone methide unit which is responsible in part for enzyme ligation. The synthesis starts with (+)-citronellene and leads to a precursor for the RCM reaction (Grubbs II) (Magauer et al. 2010). A final oxidation generates the quinone methide moiety (Scheme 4.23).

Nitrogen-Containing Molecules The Lycopodium alkaloids are well known as bioactive compounds, with varied activities, and two examples of the cernuane subgroup have been synthesized. Cermizine D exhibits cytotoxicity against murine lymphoma L1210 cells. The synthesis starts with citronellal, which after losing three carbons, is transformed into the

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102 I O

I

O

O OR RO

OH

H (S)-citronellal

OPMB R=TBS Grubbs I

I O

O

O

O OR RO

OH HO

OPMB

OCONH2 R=TBS

stereoisomer of tulearin A

Scheme 4.22  The Cossy and Curran approach to the tulearin A

precursor for an RCM reaction (Scheme 4.24). Relatively simple chain extensions and cyclizations furnish the two alkaloids (Nishikawa et al. 2008). Equisetin and (+)-fusarisetin A are two tetramic acid fungal metabolites, and the latter compound inhibits acinar morphogenesis, cell migration and cell invasion (MDAMB-231 cells) without showing significant cytotoxicity. The authors propose a plausible biosynthetic route, and consequently investigated a biomimetic

HO O

OH

Grubbs II O

O

OH

O

O

O (S)-(+)-citronellene MOMO

MOMO OMe

OMe

O

O H O

HO HO O kendomycin

Scheme 4.23  The Mulzer synthesis of kendomycin

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Scheme 4.24  The Takayama approach to the cernuane Lycopodium alkaloids

approach to these two compounds (Scheme 4.25). Once again, citronellal serves as starting material losing three carbon atoms, then leading to an unsaturated fatty acid intermediate (Yin et al. 2013). The Martin laboratory has formally synthesized (+)-FR900482 starting from vanillin, which by nitration permits the inclusion of the nitrogen atom. This compound is an antitumour and antibiotic agent isolated from a Streptomyces species, and together with the related analogues FK973 and FK317, displays potency against several tumour cell lines. These compounds are being prepared as substitutes for the anticancer drug mitomycin. Once again side chain terminal alkenes are prepared for the RCM reaction (Scheme 4.26), before the final cyclization to FR900482 (Fellows et al. 2000).

Phenylpropanoid-Based Molecules The relevant synthetic objectives in this section are podophyllotoxin type C6–C3 dimers (Liu et al. 2007; Nagar et al. 2011) and curcuminoid derivatives. The first group of dimeric compounds have long been of interest, while the latter group found in curry preparations have become of great interest recently (Esatbeyoglu et al. 2012). The Magedov−Kornienko laboratories have described a very interesting onestep multicomponent reaction for producing podophyllotoxin analogues. These bioisosteric molecules demonstrate antiproliferative properties in several human cancer cell lines. They also induce apoptosis in cancerous Jurkat cells after 24 h of exposure. Thus, condensation of trimethoxybenzaldehyde, amino-pyrazoles and tetronic acid produces a small library of podophyllotoxin analogues (Scheme 4.27) (Magedov et al. 2007).

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Scheme 4.25  The Gao group biomimetic synthesis of two “setins”

A large library of natural curcuminoids and analogues have been synthesized by condensation of oxygenated benzaldehydes and pentadione-1,3, including the parent curcumin (Scheme 4.28). The recent Rimbach group review (Esatbeyoglu et al. 2012) details the important biological activities of the curcuminoids, including anticancer properties. Among the benzaldehydes utilized in this investigation, vanillin and its acylated derivatives are the most studied (Pedersen et al. 1985).

OMe

OMPM

OMe OH

OBn

OTBS

OH

OHC

OHC vanilin

NO2

5-nitro-vanilin

N Boc

OBn

Grubbs I

OHC

OMPM

OCONH2

OH

OH N

O

OTBS

OBn NH

(+)-FR900482 Scheme 4.26  The Martin formal synthesis of (+)-FR900482

N OBn

Boc

4

Essential Oils as Raw Materials in the Synthesis of Anticancer Drugs R1 N N

CHO

R1

O

N N

+

NH2

+

MeO

R2

105 H N O

MCR

O

R2

OMe

O

O

OMe

MeO

OMe OMe

podophyllotoxin anagues

Scheme 4.27  The Magedov–Kornienko synthesis of podophyllotoxin analogues

MeO

O

H

O

O O B O O

+ B2O3

H2BO3-

CHO

O

H

O

HO

R

HO

OH

OMe

R

OMe

R

curcumin

Scheme 4.28  The Lawesson synthesis of curcuminoids

Lim and Parker have utilized saffrole as starting material in their synthesis of (−)-kingianin A, which is reported to bind to antiapoptotic protein Bcl-xL, considered to be an important drug target for the treatment of lymphomas, leukaemias and small lung cancers. The synthetic route chosen here is biomimetic and involves a Diels−Alder reaction as the key step (Scheme 4.29). The synthesis also requires a

OTBDPS

O

O

O

O

O

O

pinB

OTBDPS

Pd(PPh3)4 safrole

I

electrocyclization O H

O

O O

O Diels-Alder

H

O

OTBDPS

CONHEt H

CONHEt (-)-kingianin A

Scheme 4.29  The Lim and Parker synthesis of (−)-kingianin A

H

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coupling reaction followed by electrocyclization to produce the cyclobutane-annelated cyclohexadiene precursor for cycloaddition reaction (Lim and Parker 2013).

Conclusion In this chapter, we have presented an overview, based upon our personal experiences, of the importance of secondary metabolite natural products as anticancer drugs. These molecules require synthetic organic chemistry to be made readily available, and the principal focus here is upon the use of essential oil components as relevant starting materials. We feel that this approach is a viable and sustainable way for the provision of drugs to combat human cancers. In addition, we include two publications that deal with the relevance of natural products for the development of new drugs, at a moment of despair for the constant reduction of the number of new chemical entities, going into serious clinical trials prior to launching on the market (Paterson and Anderson 2005; Tietze et al. 2003). Acknowledgements The authors wish to thank the following Brazilian agencies for financial support and fellowships; FAPESP (2011/13993-2; 2013/02311-3; 2013/06532-4), CAPES and CNPq. The authors thank all former and present collaborators, for their studied input and hard work on our projects. They also acknowledge the donation of monoterpenes by Firmenich SA.

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Srikrishna A, Kumar PP, Reddy TJ (2005) Enantiospecific synthesis of B-Seco-Nortaxanes from two molecules of carvone. Indian J Chem 44B:1430–1436 Tietze LF, Bell HP, Chandrasekhar S (2003) Natural product hybrids as new leads for drug discovery. Angew Chem Int Ed 42:3996–4028 Wender PA, Badham NF, Conway SP, Floreancig PE, Glass TE, Houze JB, Krauss NE, Lee D, Marquess DG, McGrane PL, Meng W, Natchus MG, Shuker AJ, Sutton JC, Taylor RE (1997) The pinene path to Taxanes. 6. A concise stereocontrolled synthesis of taxol. J Am Chem Soc 119:2757–2758 Wessjohann L, Scheid GO, Eichelberger U, Umbreen S (2013) Total synthesis of Epothilone D: the Nerol/Macroaldolization approach. J Org Chem 78:10588–10595 Willot M, Radtke L, Konning D, Frohlich R, Gessner VH, Strohmann C, Christmann M (2009) Total synthesis and absolute configuration of the guaiane sesquiterpene englerin A. Angew Chem Int Ed 48:9105–9108 Wilson RM, Danishefsky SJ (2007) Applications of total synthesis toward the discovery of clinically useful anti-cancer agents. Chem Soc Rev 36:1207–1226 Yin J, Kong L, Wang C, Shi Y, Cai S, Gao S (2013) Biomimetic synthesis of equisetin and (+)-fusarisetin A. Chem Eur J 19:13040–13046 Yusufi M, Banerjee S, Mohammad M, Khatal S, Swamy KV, Khan EM, Aboukameel A, Sarkar FH, Padhye S (2013) Synthesis, characterization and anti-tumor activity of novel thymoquinone analogs against pancreatic cancer. Bioorg Med Chem Lett 23:3101–3104 Zahel M, Keßberg A, Metz P (2013) A short enantioselective total synthesis of (−)-englerin A. Angew Chem Int Ed 52:5390–5392 Zhai J-D, Li D, Long J, Zhang H-L, Lin J-P, Qiu C-J, Zhang Q, Chen Y (2012) Biomimetic semisynthesis of arglabin from parthenolide. J Org Chem 77:7103–7107 Zhang Q, Lu Y, Ding Y, Zhai J, Ji Q, Ma W, Yang M, Fan H, Long J, Tong Z, Shi Y, Jia Y, Han B, Zhang W, Qiu C, Ma X, Li Q, Shi Q, Zhang H, Li D, Zhang J, Lin J, Li LY, Gao Y, Chen Y (2012) Guaianolide sesquiterpene lactones, a source to discover agents that selectively inhibit acute myelogenous leukemia stem and progenitor cells. J Med Chem 55:8757–8769 Zhou Q, Chen X, Ma D (2010) Asymmetric, protecting-group-free total synthesis of (−)-englerin A. Angew Chem Int Ed 49:3513–3516

Chapter 5

Antitumor Essential Oils: Progress in Medicinal Chemistry Sócrates Cabral de Holanda Cavalcanti, Rafael dos Reis Barreto de Oliveira and Damião Pergentino de Sousa

Introduction Bioactive natural products play critical roles in medicinal chemistry. Such products are used in molecular modeling to aid the development of modern drugs, especially for antitumor, antimicrobial, and psychoactive agents. The complexity or even the simplicity of these molecules fascinates scientists and stimulates research in the isolation, characterization, and identification of biological profile of new drug candidates. In fact, the rate of growth of scientific knowledge on bioactive molecules has been so great that it increases the possibility of obtaining new drugs against incurable or partially treatable diseases. In this context, the chemistry and pharmacology of natural products is a strategic area for the development of a more efficient pharmacotherapy whose results promote well-being and recovery of patients’ health (Liang and Fang 2006). The use of medicinal chemistry tools is an effective approach to understand the bioactivity of natural products and advances in the development of new drugs. This work involves a wide range of fields, including phytochemical study focusing on the isolation of bioactive compounds, structural changes aimed at optimizing the pharmacological activity, employment of lipophilic and electronic parameters in the study, as well as total and semi-synthesis to establish the structure–activity relationships (SAR). Allied to these tools for research, the rapid progress in key areas in drug research, such as molecular biology, computational chemistry, and high

S. C. de Holanda Cavalcanti () Department of Pharmacy, Federal University of Sergipe, 49100-000, São Cristóvão, Brazil e-mail: [email protected] R. d. R. B. de Oliveira Department of Pharmacy, Federal University of Sergipe, 58051-970, São Cristóvão, Brazil D. P. de Sousa Department of Pharmaceutical Sciences, Federal University of Paraíba, 58051-970, João Pessoa, Brazil © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_5

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throughput screening (HTS) technologies, enabled the more efficient drug discovery with shorter search time (Corey et al. 2007; Liang and Fang 2006). The essential oils constituents possess several chemical characteristics, such as low molecular weight and high lipid solubility, and are commonly found in isomeric forms, when stereocenters are present, such as in mono- and sesquiterpenes. Some stereoisomers exhibit high enantiomeric purity and are useful in stereoselective syntheses. The chirality of bioactive substances is often interesting for the selectivity of drug action. Therefore, many components of essential oils are appreciable starting materials for the SAR studies and drug development. The phenylpropanoids, another chemical class found in essential oils, are also widely used in SAR studies, especially because of the presence of functional groups that are targets for chemical transformations aimed at the preparation of structurally related derivatives collections, such as eugenol, one phenylpropanoid which shows several pharmacological activities and applications in the pharmaceutical, food, and cosmetic industry (Sousa 2012). This chapter presents a survey of the most promising data published in the literature on the application of medicinal chemistry tools on constituents of essential oils. Although we found few studies reported in the scientific literature, the examples demonstrate the potential of this chemical class of natural products as starting materials and their application in molecular modeling aiming the development of bioactive substances with potential use in cancer therapy.

Monoterpenes Quinones Thymoquinone has been isolated from seeds of Nigella sativa (Mahfouz and ElDakhakhny 1960), a spice that grows in the Mediterranean region and in Western Asian countries including India, Pakistan, and Afghanistan (Banerjee et al. 2010). Its seeds exhibit moderate antitumor activity against a wide range of human tumor cells (Breyer et al. 2009). Such molecule has additionally shown to exhibit chemosensitizing effects to gemcitabine and oxaliplatin (Banerjee et al. 2009). Growth inhibition and induction of apoptosis constitute the major mechanism of action of most chemotherapeutics during cancer. However, pancreatic cancer is inherently resistant to apoptosis in all conventional cancer therapeutic agents, which poses a great challenge to clinicians for the treatment of patients diagnosed with pancreatic cancer (Banerjee et al. 2009). Consequently, novel agents are needed to overcome resistance. To study the antitumor activity of several thymoquinone derivatives against pancreatic tumor cells, Padhye and colleagues (Yusufi et al. 2013) have prepared and evaluated 29 thymoquinone derivatives synthesized either by condensation of 3-aminothymoquinone, obtained by reaction of thymoquinone

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Scheme 5.1  Synthesis of 3-amino-thymoquinone and its Schiff base analogs

Scheme 5.2  One-pot chemical synthesis of quinone derivatives

with sodium azide in acid medium, with 2,3,4-tri-hydroxy or 2,3,4-trifluoro benzaldehyde (Scheme 5.1) or by a one-pot synthesis of 2,5-bis(alkyl/aryl amino) and mercaptan 1,4-benzoquinones as depicted in Scheme 5.2 (Banerjee et al. 2009). Modifications in the structure of thymoquinone have been directed at the carbonyl sites or the benzenoid sites resulting in enhanced lipophilicity. Among these analogs, three were found to be more potent than thymoquinone in terms of their ability to inhibit cell growth and induce apoptosis in pancreatic cells. Their mechanism of action was linked to the downregulation of transcription factor NF-κB and anti-apoptotic and cell survival-related molecules such as Bcl-2, Bcl-xL, survivin, and XIAP. In addition, there are evidences of the activity of these analogs in inhibiting the production and secretion of PGE2 in high COX-2-expressing pancreatic cells (BxPC-3). (Scheme 5.1) The anticancer activity in two pancreatic MiaPaCa-2 and BxPC-3 cancer cell lines of the synthesized analogs was further evaluated. 3-Aminothymoquinone and the fluorinated derivative showed to be either similar or less potent than thymoquinone, whereas the hydroxyl derivative exhibited more potent anticancer activity against MiaPaCa-2 cell line exhibiting over 50 % enhancement in the loss of cell viability compared with parental thymoquinone (Yusufi et al. 2013). (Scheme 5.2) Docking of two synthesized compounds into the active site of COX-2 resulted in a good fit. The best fit was obtained by 2,3,4-tri-hydroxy derivative, which exhibits the best binding energy (− 8.1 kcal/mol), confirming that modification of the parent compound with additional groups does not introduce any major steric interference in the thymoquinone moiety and further allows additional hydrogen bonding interactions.

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The synthesis of thymoquinone analogs may lead to derivatives with better biological activity and without systemic toxicity than the parental thymoquinone, which may be a promising research field for better treatment outcome of patients diagnosed with pancreatic cancer.

L-Cavone, L-Carveol, and Limonene Derivatives Comparative studies of the chemoprevention of mammary carcinogenesis of d-limonene and its derivatives have been achieved (Crowell et al. 1992). The chemopreventive activity of d-limonene was compared with that of (−)-carveol and uroterpenol, two hydroxylated urinary metabolites, and that of sobrerol, a diol derivative. In all cases, hydroxylated d-limonene derivatives were more potent than d-limonene itself in the chemoprevention of mammary cancer. Sobrerol was the most potent monoterpene evaluated in this study. The monoterpene d-limonene inhibits the posttranslational isoprenylation of p21ras and other small G proteins, which may be related to the ability of this monoterpene to act as a chemopreventive agent of chemically induced rodent cancers. In view of these facts, Crowell et al. studied the SAR of a series of 26 limonene derivatives (Crowell et al. 1994). All compounds in this series were unsaturated. The series included unfunctionalized hydrophobic compounds, alcohols (monohydroxyl, diol, and triol), acids, esters, aldehydes, thiol, epoxyde, and aldehyde derivatives. Within the series of monoterpenes tested in these experiments, the most potent inhibitors of protein isoprenylation and cell proliferation had intermediate polarity. The most potent compounds were found to be perillyl alcohol, perillic acid methyl ester, and perillaldehyde, which have intermediate hydrophobicity. Either more polar or less polar compounds were less potent than compounds with intermediate polarity, which indicates that polarity of the drug, rather than ring or other structure, would seem to be the most important factor in determining its relative ability to inhibit protein isoprenylation in cells. Similarly, the SAR of l-carvone, l-carveol, and limonene derivatives have been proposed. Limonene derivatives have been obtained by synthetically modifying either limonene or l-carvone. A reasonable synthetic route to obtain analogs of mentioned compounds have been proposed by using l-carvone as a starting material via chlorination of the isopropene moiety with tert-butyl hypochlorite in n-hexane at room temperature to obtain 5-(1-chloromethyl)vinyl-2-methylcyclohex-2-enone, which is used as a starting material in a divergent synthetic pathway. Ester derivatives of this key intermediate may be obtained by reaction with substituted potassium benzoates in the presence of potassium carbonate in N,N-dimethylformamide. A piperazine moiety may be attached to the primary isopropene carbon by reaction of the key compound with N-alkylpiperazines or N-arylpiperazines in the presence of potassium carbonate. Further reduction of the resulting ketone with NaBH4 or under Wolff–Kishner conditions affords either the alcohol or the limonene derivative.

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Scheme 5.3  Synthesis of l-carvone and limonene derivatives

Additionally, reaction of the key intermediate with an aliphatic heterocyclic amine affords amine derivatives of l-carvone (Chen et al. 2006). (Scheme 5.3) The antiproliferative effect of the synthesized compounds was evaluated in human prostate cancer LNCaP cells. The IG50s of l-carvone, l-carveol, and l-limonene were higher than 100 µM. The addition of 4-substituted-benzoic acid with an electron donating group at the para position (R1 = CH3, OCH3, or NH2) significantly increased the antiproliferative activity of l-carvone. Replacing the electron donating group by an electron withdrawing group (R1 = F, Cl, or Br) or hydrogen resulted in little or no increase in the antiproliferative activity of l-carvone. Addition of substituted groups of N-alkylpiperazine or unsubstituted N-benzylpiperazine to l-carvone did not increase the antiproliferative effect of l-carvone. However, the introduction of groups at the benzyl ring significantly increased the antiproliferative effect of l-carvone. The addition of a substituted amine to l-carvone did not increase the antiproliferative effect except for the 2-thiopheneethylamine substitute. Increase in potency was accounted for by increased polarity of l-carvone derivatives. Reduction of the carbonyl in N-methylpiperazine or N-ethylpiperazine-substituted l-carvone yielded carveol derivatives. Such reduction resulted in slight increase in the antiproliferative effect compared with l-carvone derivatives. However, removal of the carbonyl in N-methylpiperazine or N-ethylpiperazine-substituted l-carvone derivatives, resulting in N-methylpiperazine or N-ethylpiperazine-substituted limonene derivatives significantly increased the antiproliferative effect of limonene (Fig. 5.1).

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Fig. 5.1  Chemical structure of methyl eugenol

Phenylpropanoids Methyl Eugenol Derivatives Methyl eugenol (Fig. 5.1) is a phenylpropanoid widely distributed in natural products which has exhibited moderate anticancer activity (Pisano et al. 2007; Groh et al. 2013). In view of these facts, the phenolic ether methyl eugenol was used as a starting material to design and synthesize 14 anti-tubulin agents using Heck and Suzuki coupling reactions (Abdel et al. 2010). Additionally, a computer-assisted approach was used to design these new derivatives using the colchicine-binding site of tubulin as the molecular target and colchicine as an active ligand. The highresolution crystal structure at 3.80 Å resolution of tubulin–colchicine–soblidotin: stathmin-like domain complex (PDB 3e22) (Cormier et al. 2008) retrieved from the Structural Bioinformatics Protein Data Bank was used in this study. Derivatives were designed by docking the compounds in the active site, which revealed two interactions with β-tubulin alanine 250 (ALA 250) and asparagine 249 (ASN 249) and one with α-tubulin asparagine 101 (ASN 101) via strong H-bonding with the 3,4-dimethoxy functionality in the methyl eugenol moiety (Fig. 5.2). The mode of binding was found to be comparable to the interaction maps of other antitubulin agents. Along with the structures in Fig. 5.2, several biaryl carbon–carbon coupling structures were docked and showed improved binding affinities at the colchicine binding site. Furthermore, selected compounds were synthesized by using Heck’s coupling reaction between the alkene group of methyl eugenol and aryl halides (Scheme 5.4). Both E and Z isomers have been produced from the Heck reaction. Higher yields of the E isomer were obtained. Suzuki coupling was more stereoselective than Heck coupling reactions resulting in derivatives with E-geometry only. Synthesized compounds were evaluated for their anti-invasive activity against the metastatic breast cancer MDA-MB-231 cells. The assay consists of a standardized in vitro model able to quantify the degree at which invasive cells penetrate a barrier consisting of basement membrane component in response to chemoattractants and/or inhibiting compounds. All compounds inhibited the invasion of MDA-MB-231 breast cancer cells in a dose-dependent manner, the compounds activity is based on 100 % invasion of the control. The Z isomers were more active as invasion inhibitors than the E isomers. The 2″-acetoxy-containing Z-isomer at 4 µM exhibited high potency, only

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Fig. 5.2  Docked structures of methyl eugenol derivatives. (Reprinted from Bioorganic & Medicinal Chemistry 18 (2010), Abdel Bar et al. 2010 Copyright (2015), with permission from Elsevier)

comparable to the positive control colchicine at the same concentration (9 % invasion). Furthermore, a trimethoxychalcone showed better activity than colchicine at 2 µM dose (8.5 % invasion vs. 12.5 % for colchicine). E-Trimethoxyphenyl and p-methoxyphenyl derivatives, exhibited high anti-invasive activity (11.0 %, 13.7 % invasion, respectively) at 4 µM. Most methyl eugenol biaryl analogs were not cytotoxic at the 10–50 µM dose range, which indicates that the anti-invasive activity of such compounds at concentration 1–4 µM is not due to a direct cytotoxic effect. In addition to the molecule design, a 3D pharmacophore mapping was proposed for the studied compounds. A methodology based on distance comparison was built for the four most active analogs. ( Z)-2-[3-(3,4-dimethoxyphenyl)prop1-enyl]phenyl acetate, ( E)-1,2-dimethoxy-4-[3-(4-methoxyphenyl)allyl]benzene,

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a

b Scheme 5.4  Heck (a), and Suzuki (b), coupling reactions between methyl eugenol and aryl halides/aryl boronic acids, respectively

( E)-5-[3-(3,4-dimethoxyphenyl)allyl]− 1,2,3-trimethoxybenzene, and ( E)-1-(3,4dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-2-propen-1-one were used and seven essential features required for high receptor binding affinity were defined, that is, two hydrophobic sites, two hydrogen bond acceptor atom, and three receptor donor sites. Additionally, the biaryl system, di- or trimethoxyphenyl moiety, and a constrained conformation features are common in all the 14 analogs and consistent with the pharmacophoric maps previously generated for colchicine site inhibitors (Nguyen et al. 2005). The docking studies revealed the structural features needed by the methyl eugenol analogs to act as breast cancer invasion inhibitors. A hydrophobic ring originally found in methyl eugenol embedded in a hydrophobic pocket, which consists of the side chains of Val 179 and Met 257, was found to be important for the activity. Additionally, the trimethoxyphenyl moiety occupies a pocket bounded by side chains of Leu 255, Ala 316, Val 318, and Ile 378. The methoxy oxygen atoms in the former aromatic ring should be located sufficiently close to make hydrogen bond with the backbone N–H of residues Asp 249 and Ala 250 at approximately 3 Å. Additional important features for the activity are an H-bond between the central methoxy and Cys 239 and the distance between the biaryl rings of 6.99 ± 1 Å.

Cinnamaldehyde Derivatives 2′-Hydroxycinnamaldehyde is a phenylpropanoid derived from the stem bark of Cinnamomum cassia. In previous studies, 2′-hydroxycinnamaldehyde and its analog 2′-benzoyloxycinnamaldehyde have been shown to induce growth arrest and apoptosis in tumor cells (Han et al. 2004; Kwon et al. 1996). In view of these facts, cinnamaldehyde derivatives have been synthesized and evaluated as antiproliferative and proapoptotic in vitro and have further shown to inhibit tumor growth in vivo (Gan et al. 2009).

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Scheme 5.5  Synthesis of Hydroxycinnamaldehyde derivatives

Three derivatives were synthesized by esterification of commercially available ( E)-2-hydroxycinnamic acid with chlorotrimethylsilane in methanol, affording methyl ( E)-2-hydroxycinnamate, followed by protection of the phenol group using the appropriate alkyl halide to give the methyl(2-alkoxy)-cinnamate. Ester reduction using Red-Al yielded final 2-alkoxycinnamaldehyde derivatives. Removal of the p-methoxybenzyl group of 2-( p-methoxybenzyl)-cinnamaldehyde using ceric ammonium nitrate gave rise to 2-hydroxycinnamaldehyde (Scheme 5.5). 2-Hydroxy-5-fluorocinnamaldehyde was further synthesized via a palladium catalyzed coupling reaction between 4-fluoro-2-iodophenol and acrolein diethyl acetal, followed by acid hydrolysis (Scheme 5.6). An SAR study was conducted on trans-cinnamaldehyde, trans-cinnamic acid, 2′-hydroxycinnamaldehyde, and the above 2′-hydroxycinnamaldehyde derivatives to study the effects on cell viability and proliferation in a number of different cell lines. Trans-cinnamaldehyde exhibited GI50 = 22 µM in the cell viability and proliferation MTT assay in HCT116 colon cancer cells. Conversely, trans-cinnamic acid was inactive, emphasizing the importance of the reactive aldehyde group. The presence of the 2-(4-methoxy-benzyl)oxy group considerably increased the potency (GI50 = 0.6 µM) compared with trans-cinnamaldehyde. Intermediary potency was observed with the ester 2-benzoyloxy (GI50 = 3.3 µM) and the ether 2-hydroxy

Scheme 5.6  Synthesis of 2-Hydroxy-5-fluorocinnamaldehyde

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(GI50 = 5.2 µM) and 2-benzyloxy (GI50 = 6.2 µM) groups. The ether groups 2-pentoxy (GI50 = 10.3 µM) and 2-methoxy (GI50 = 39 µM) conferred lower potency than trans-cinnamaldehyde. In addition to cell viability and proliferation, the effect of the analogs on cellular apoptosis by measuring caspase 3 activation in HCT116 cells was evaluated. 2-Benzoyloxycinnamaldehyde and 2-(4-methoxybenzyl)-oxy cinnamaldehyde had comparable effects on caspase 3, whereas 2-benzyloxycinnamaldehyde, 2-pentoxycinnamaldehyde, and 2-hydroxycinnamaldehyde induced caspase 3 activation with lower potency, although 2-methoxycinnamaldehyde exhibited no effect. Very good correlation between cell viability and proliferation results and apoptosis induction was observed. The propenal in cinnamaldehyde is believed to form a Michael acceptor group that may interact with cellular proteins containing sulfhydryl groups. Therefore, cinnamaldehyde derivatives may target proteins with cysteine amino acids in their receptor (Gan et al. 2009). Unlike the above-mentioned 2-hydroxycinnamaldehyde derivatives, the fluorinated derivative 2-hydroxy-5-fluorocinnamaldehyde reduces cell viability with higher potency compared with 2-hydroxycinnamaldehyde in HCT116 (colon cancer), MCF-7 (breast cancer), MDA468 (breast cancer) cells, HT29 (colon cancer cells), and A549 (lung cancer cells) although the two later cancer cells exhibit a certain resistance to both compounds (56.2 and 70.8 µM, respectively as compared with 3.3–7.2 µM of the other compounds). Additionally, 2-hydroxy-5-fluorocinnamaldehyde induces cell death faster than 2-hydroxycinnamaldehyde and induced only very little cleavage of caspase 3 or PARP in HCT116 control cells. Both 2-hydroxycinnamaldehyde and 2-hydroxy-5-fluorocinnamaldehyde were unable to induce apoptosis in cancer cells via reactive oxygen species.

Sesquiterpenes and Diterpenes Few examples of medicinal chemistry studies of antitumor sesqui- and diterpenes are found in the literature. In this topic, some approaches using these terpenes in the reactions will be discussed. α-Bisabolol glycosides as antitumor on a broad panel of tumor cell lines. α-Bisabolol is a sesquiterpene alcohol isolated from the essential oil of a variety of plants, shrubs, and trees. For example the essential oil of Matricaria chamomilla contains α-bisabolol, which was found to have selective cytotoxic effect on human and rat glioma cells over normal rat glial cells (Cavalieri et al. 2004). It originally exists in plants as a glycosidically bound compound. α-Bisabolol β-dfucopyranoside has been isolated in large amounts from the Mediterranean weed Carthamus glaucus (Taglialatela-Scafati et al. 2012) and has exhibited cytotoxic activity by the assay using Artemia salina (Mikhova et al. 2004). In view of these facts, Piochon et al. (Piochon et al. 2009) synthesized O-glicoside derivatives of α-Bisabolol. Six α-bisabolol glycosides were synthesized, for example, α-d-glicopyranose was fully protected by benzoyl groups using BzCl/DMAP/pyridine followed by

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selective deprotection of the anomeric position by bromination with HBr/HOAc followed by basic hydrolysis in the presence of silver carbonate. d-Fucose 2,3,4-triO-benzoyl-α-d-fucopyranose was reacted with trichloroacetonitrile and cesium carbonate as catalyst to convert the anomeric hydroxyl into the trichloroacetimidate leaving group which was useful at the glycosidation reaction. Most trichloroacetimidate products resulted in β-configuration at C1. Glycosidation was accomplished by using a catalytic amount of TMSOTf and α-bisabolol mixed before dropwise addition of 2,3,4-tri-O-benzoyl-α-d-glicopyranosyl trichloroacetimidate. Fully protected α-bisabolol glycosides were obtained at low temperatures (− 78 to − 20 °C) in high yields. Subsequently, deprotection of the benzoyl groups in basic medium (NaOH/MeOH/THF/H2O) afforded α-bisabolol β-d-fucopyranoside (Scheme 5.7). α-Bisabolol and six synthesized α-bisabolol glycosides (fucoside, rhamnoside, xyloside, glucoside, galactoside, and mannoside) were evaluated for their antitumor activity against human lung carcinoma (A549), colon adenocarcinoma (DLD-1), breast adenocarcinoma (MCF-7), melanoma (SK-MEL-2), ovary teratocarcinoma (PA-1), prostate adenocarcinoma (PC-3), pancreas adenocarcinoma (PANC 05.04), glioma (U-251), glioblastoma (U-87), and murine glioma (GL-261). Cytotoxicity was also carried out on normal human skin fibroblasts (WS1). Although α-bisabolol was found to be weakly cytotoxic against U-87 with an IC50 of 130 μM (29 μg/ml),

Scheme 5.7  Synthesis of α-bisabolol β-d-fucopyranoside

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α-bisabolol glycosides exhibited stronger activity than the aglycone, except for α-bisabolol β-d-galactopyranoside, which exhibited IC50 > 100 μM. α-Bisabolol α-l-rhamnopyranoside was the most potent glycoside, exhibiting pronounced cytotoxic activity observed for all tumor cell lines, particularly against A549 (IC50 40 μM) and PA-1 (IC50 40 μM). Other synthesized compounds exhibited intermediate potencies. Furthermore, in silico prediction of pharmacokinetic parameters was performed. One of the goals of these in silico parameters is to predict the ability of compounds to pass through the blood–brain barrier (BBB) based on its physicochemical properties. Important parameters and its limits are molecular weight (MW < 450 Da), hydrogen bond donor (HBD, 0 to 1), polar surface area (PSA 3; PSA > 90 Å) suggesting that, although, the lipophilicity of α-bisabolol is decreased by the addition of a rhamnoside it does not alter its capacity to pass through biological barriers especially the BBB.

Conclusion The studies presented in this chapter were carefully selected from SAR publications using secondary metabolites found in essential oils as starting materials. The experimental results described show the therapeutic potential of essential oils derivatives for the development of drugs with application in the treatment of cancer.

References Abdel Bar FM, Khanfar MA, Elnagar AY, Badria FA, Zaghloul AM, Ahmad KF, Sylvester PW, El Sayed KA (2010) Design and pharmacophore modeling of biaryl methyl eugenol analogs as breast cancer invasion inhibitors. Bioorgan Med Chem 18(2):496–507. doi:10.1016/j. bmc.2009.12.019 Banerjee S, Kaseb AO, Wang ZW, Kong DJ, Mohammad M, Padhye S, Sarkar FH, Mohammad RM (2009) Antitumor activity of gemcitabine and oxaliplatin is augmented by thymoquinone in pancreatic cancer. Cancer Res 69(13):5575–5583. doi:10.1158/0008-5472.Can-08-4235

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Banerjee S, Padhye S, Azmi A, Wang ZW, Philip PA, Kucuk O, Sarkar FH, Mohammad RM (2010) Review on molecular and therapeutic potential of thymoquinone in cancer. Nutr Cancer 62(7):938–946. doi 10.1080/01635581.2010.509832 Breyer S, Effenberger K, Schobert R (2009) Effects of thymoquinone-fatty acid conjugates on cancer cells. ChemMedChem 4(5):761–768. doi:10.1002/cmdc.200800430 Cavalieri E, Mariotto S, Fabrizi C, de Prati AC, Gottardo R, Leone S, Berra LV, Lauro GM, Ciampa AR, Suzuki H (2004) α-bisabolol, a nontoxic natural compound, strongly induces apoptosis in glioma cells. Biochem Biophys Res Commun 315(3):589–594. doi: 10.1016/j. bbrc.2004.01.088 Chen JJ, Lu M, Jing YK, Dong JH (2006) The synthesis of L-carvone and limonene derivatives with increased antiproliferative effect and activation of ERK pathway in prostate cancer cells. Bioorgan Med Chem 14(19):6539–6547. doi: 10.1016/j.bmc.2006.06.013 Corey EJ, Czakó B, Kürti L (2007) Molecules and medicine. Wiley, New Jersey, p. 272 Cormier A, Marchand M, Ravelli RB, Knossow M, Gigant B (2008) Structural insight into the inhibition of tubulin by vinca domain peptide ligands. EMBO Rep 9(11):1101–1106. doi:10.1038/ embor.2008.171 Crowell PL, Kennan WS, Haag JD, Ahmad S, Vedejs E, Gould MN (1992) Chemoprevention of mammary carcinogenesis by hydroxylated derivatives of d-limonene. Carcinogenesis 13(7):1261–1264 Crowell PL, Ren ZB, Lin SZ, Vedejs E, Gould MN (1994) Structure–activity-relationships among monoterpene, inhibitors of protein isoprenylation and cell-proliferation. Biochem Pharmacol 47(8):1405–1415. doi: 10.1016/0006-2952(94)90341-7 De Sousa DP (2012) Medicinal essential oils: chemical, pharmacological and therapeutic aspects. Nova Science, New York, p. 236 Gan FF, Chua YS, Scarmagnani S, Palaniappan P, Franks M, Poobalasingam T, Bradshaw TD, Westwell AD, Hagen T (2009) Structure–activity analysis of 2’-modified cinnamaldehyde analogues as potential anticancer agents. Biochem Biophys Res Commun 387(4):741–747. doi:10.1016/j.bbrc.2009.07.104 Groh IAM, Chen C, Lüske C, Cartus AT, Esselen M (2013) Plant polyphenols and oxidative metabolites of the herbal alkenylbenzene methyleugenol suppress histone deacetylase activity in human colon carcinoma cells. J Nutr Metab 2013:1–10 Han DC, Lee MY, Shin KD, Jeon SB, Kim JM, Son KH, Kim HC, Kim HM, Kwon BM (2004) 2’-benzoyloxycinnamaldehyde induces apoptosis in human carcinoma via reactive oxygen species. J Biol Chem 279(8):6911–6920. doi:10.1074/jbc.M309708200 Hitchcock SA, Pennington LD (2006) Structure–brain exposure relationships. J Med Chem 49(26):7559–7583. doi:10.1021/jm060642i Kwon BM, Cho YK, Lee SH, Nam JY, Bok SH, Chun SK, Kim JA, Lee IR (1996) 2’-Hydroxycinnamaldehyde from stem bark of Cinnamomum cassia. Planta Med 62(2):183–184. doi:10.1055/s-2006-957851 Liang X-T, Fang W-S (2006) Medicinal chemistry of bioactive natural products. Wiley, New Jersey, p. 480 Mahfouz M, El-Dakhakhny M (1960) The isolation of a crystalline active principle from Nigella sativa seeds. J Pharm Sci 1(1):9–19 Mikhova B, Duddeck H, Taskova R, Mitova M, Alipieva K (2004) Oxigenated bisabolane fucosides from Carthamus lanatus L. Z Naturforsch 59c:244–248 Nguyen TL, McGrath C, Hermone AR, Burnett JC, Zaharevitz DW, Day BW, Wipf P, Hamel E, Gussio R (2005) A common pharmacophore for a diverse set of colchicine site inhibitors using a structure-based approach. J Med Chem 48(24):6107–6116. doi: 10.1021/Jm058275i Piochon M, Legault J, Gauthier C, Pichette A (2009) Synthesis and cytotoxicity evaluation of natural alpha-bisabolol beta-D-fucopyranoside and analogues. Phytochemistry 70(2):228–236. doi:10.1016/j.phytochem.2008.11.013

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Pisano M, Pagnan G, Loi M, Mura ME, Tilocca MG, Palmieri G, Fabbri D, Dettori MA, Delogu G, Ponzoni M, Rozzo C (2007) Antiproliferative and pro-apoptotic activity of eugenol-related biphenyls on malignant melanoma cells. Mol Cancer 6:8. doi:10.1186/1476-4598-6-8 Taglialatela-Scafati O, Pollastro F, Cicione L, Chianese G, Bellido ML, Munoz E, Ozen HC, Toker Z, Appendino G (2012) STAT-3 Inhibitory Bisabolanes from Carthamus glaucus. J Nat Prod 75(3):453–458. doi: 10.1021/Np2008973 Yusufi M, Banerjee S, Mohammad M, Khatal S, Swamy KV, Khan EM, Aboukameel A, Sarkar FH, Padhye S (2013) Synthesis, characterization and anti-tumor activity of novel thymoquinone analogs against pancreatic cancer. Bioorg Med Chem Lett 23(10):3101–3104. doi: 10.1016/j.bmcl.2013.03.003

Chapter 6

Clinical Advances in Anticancer Essential Oils Ammad Ahmad Farooqi, Rubina Sohail, Sundas Fayyaz and Iryna Shatynska-Mytsyk

Introduction Cancer chemoprevention refers to the use of natural or synthetic agents to inhibit, delay, or reverse the multifaceted and genomically complex process of carcinogenesis. Herbal extracts and bioactive ingredients have emerged as impressive natural agents because of their notable anticancer effects in cancer cell lines and preclinical cancer models. Data obtained through high throughput technologies provide evidence that cancers differ considerably from one another. Genetic, genomic, and proteomic studies have increased our knowledge of the molecular oncology of cancers, and it is now known that genetic/epigenetic mutations contribute to the malignant transformation of cancer progenitor cells. Increasingly, it is clear that cancers are dysregulated with respect to the spatio-temporal organization of the biological mechanisms that modulate normal growth and tissue homeostasis. Apoptosis is a form of programmed cell death that cancer cells manage to escape from. In vitro studies show that some cancer cells are resistant to tumor necrosis factor-related apoptosisinducing ligand (TRAIL), one of the most widely used apoptosis-inducing ligands. More interestingly, the autophagy induced by chemotherapeutic drug treatment, a cytoprotective mechanism in cancer cells, has received considerable attention. Therefore, a substantial proportion of research has focused on natural agents that can maximize or restore the apoptotic response in cancer cells (Farooqi et al. 2012).

A. A. Farooqi () · S. Fayyaz Laboratory for Translational Oncology and Personalized Medicine, Rashid Latif Medical College, Lahore, Pakistan e-mail: [email protected] R. Sohail Services Institute of Medical Sciences, Lahore, Pakistan I. Shatynska-Mytsyk Diagnostic Imaging and Radiation Therapy Department, Lviv National Medical University, Lviv, Ukraine © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_6

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Genomic instability and oncogenic fusion proteins also present a challenge to the standardization of therapy. Breakpoint Cluster Region (BCR)-Abelson (ABL) is a fusion oncoprotein that makes leukemic cells difficult to target (Farooqi et al. 2013). TMPRSS2-ERG, another fusion transcript identified in prostate cancer cells, was recently the focus of in vitro and in vivo studies to identify factors that target its protein product (Farooqi et al. 2014). In addition to the increasing list of intrinsic factors that determine tumor development and progression, the tumor microenvironment is now recognized as playing a significant role in oncogenesis and in host cancer cell communication. In this chapter, we provide an overview of the efficacy of elemene, limonene, and perillyl alcohol (POH) in clinical trials of cancer patients.

Elemene Emulsion Injection (EEI) Forty elemene emulsion injection (EEI)-treated patients showing a considerable regression in tumor size from 6.70 cm3 (pretreatment) to 2.67 cm3 (posttreatment) were enlisted in a trial (Tan et al. 2000). Based upon the treatment response, patients were categorized into two groups: a complete response (4 patients) group and a partial response group (26 patients). The Karnofsky performance status (KPS) was notable in EEI-treated patients and declined from 94.7 to 88.2 after treatment. The dose of EEI administered was 0.4‒1.2 g/d via an intravenous drip or intravenous infusion. An emulsion of EEI was also injected into the carotid artery or infused through a carotid artery catheter using a pump. Data obtained through a multicenter phase III clinical trial for the management of malignant pleural and peritoneal effusion have also deepened our understanding of the efficacy of elemene (Fig. 6.1). A study was conducted to evaluate the efficacy of elemene in 313 patients with pleural effusion and 171 patients with peritoneal effusion. The drug (200 mg/m2) was administered as a single dose by intraperitoneal injection for 1‒2 weeks. A higher dose was also administered twice per week for 2 weeks. Patients with malignant pleural effusion showed a response rate of 77.6 %. Moreover, the response rate observed in patients with peritoneal effusion was 66.1 % (Wang et al. 1996).

Fig. 6.1  Chemical structures of anticancer terpenes found in essential oils

OH

Elemene

D-Limonene

Perillyl alcohol

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D-Limonene D-limonene (Fig. 6.1) is another naturally occurring cyclic monoterpene with chemotherapeutic activity and relatively low toxicity. It is commercially obtained from citrus fruits through centrifugal separation or steam distillation. D-limonene was administered orally for 21 days at a dose of 0.5–12 g/m2 in 32 patients with refractory solid tumors. Patients completed 99 courses of D-limonene. Moreover, breast cancer patients were also included in the study and given 15 cycles of D-limonene at 8 g/m2 daily. The maximum tolerated dose reported was 8 g/m2 and one breast cancer patient partially responded to the drug during a period of 11 months. Three colorectal carcinoma patients experienced prolonged stable disease after treatment. LC/MS-based analysis revealed that circulating metabolites of D-limonene included dihydroperillic acid (DHPA), limonene1,2 diol, perillic acid (PA), uroterpenal, and a PA isomer. It is increasingly recognized that substantial intra-tumoral accumulation of D-limonene and uroterpenol occurs in human tissues (Vigushin et al. 1998). Extensive studies show that limonene accumulates in breast cancer tissue, and one contemporary study showed that D-limonene was well tolerated when administered orally (Poon et al. 1996). There was a 22 % reduction in the expression of cyclin D1 when 43 breast cancer patients, programmed for a surgical intervention, were given 2 g of limonene for 2‒6 weeks (Miller et al. 2013). There is circumstantial evidence showing that limonene metabolites only appear after 4 h in blood, and not immediately, in patients who ingested 100 mg/kg limonene in custard (Crowell et al. 1994). Citrus peel is rich in limonene and decreases cancer risk; however, there are major issues that remain to be addressed concerning the association between the injection of citrus peel and cancer risk. Inter-/intra-patient variability is a major problem when assessing limonene as a risk-lowering agent (Hakim et al. 2000).

Perillyl Alcohol POH (Fig. 6.1) is a monoterpene isolated from the essential oils of a number of plants, including cherries, lavandin, peppermint, celery seeds, spearmint, sage, and cranberries. POH possesses potent antineoplastic activity against numerous cancers; for instance, POH administration results in the regression of mammary, pancreatic, prostate, colon and hepatic cancers. POH may also prevent lung, skin, and colon cancers. Over the past decade, preliminary trials have evaluated the treatment response, pharmacokinetics, pharmacodynamics, and toxicity of various formulations of POH in patients with various types of human cancers. In most of these trials, POH was predominantly administered orally and formulated in soft gelatin capsules or other formulations with a soybean oil base. Two major metabolites were detectable in serum following administration: PA and DHPA. The dosage of POH varied across clinical trials. In patients with advanced colorectal carcinoma, 1200‒1600 mg/m2 of POH did not show clinical efficacy (Meadows et al. 2002). In a phase I clinical

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trial, the dose of POH used was 4800‒11,200 mg/m2; however, this dose did not show antitumor efficacy. Although there was an increase in the blood concentration of POH metabolites, the concentrations showed considerable intra- and inter-patient variability. The maximum tolerated dose (MTD) for oral administration was determined to be 8400 mg/m2 (Azzoli et al. 2003). In phase II clinical trial, oral treatment with POH was given to patients of advanced ovarian cancer. Dosage used was 1200 mg/m2and drug-induced side effects including gastrointestinal toxicity and fatigue limited the dose escalation in patients. Results revealed 17 % progression-free survival in 6 months. Complete or partial response was not detected in any patient (Bailey et al. 2002). In a phase I clinical trial, 21 patients were given dosage ranging from 1.6 g/m2 to 2.1 g/m2 monoterpene POH using soybean oil base for the drug formulation. Metabolites of POH noted after dosage of POH were PA and DHPA. A notable side effect was gastrointestinal adverse effect, attributed to the soybean oil base of the drug formulation (Murren et al. 2002). In a phase I clinical trial, 17 patients were given POH, at a dose of 1600 mg/m2. Subsequent cohorts received escalated dosages at 2100 and 2800 mg/m2. Escalated dosage-related side effects included chronic nausea, fatigue, and (grade 1–2) hypokalemia. POH at a dose of 1600 mg/m2 was well tolerated as evidenced by laboratory assays (Hudes et al. 2000). A dose escalation study was conducted to evaluate clinical efficacy of Perillyl Alcohol (POH). It was given 4 times daily, at dosages 800, 1200, 1600 mg/m2. Moreover, POH formulations were designed consisting of 250 mg of POH and 250 mg of soybean oil. It was observed that a patient with advanced colorectal cancer had an ongoing near complete response of > 2 years duration. Stable disease was noted in several patients receiving POH for > or = 6 months (Ripple et al. 2000). There is an exciting piece of evidence suggesting antitumor activity of POH in mouse model of pancreatic cancer. It was shown that POH and mda-7/Il-24 expressing adenovirus synergistically inhibited tumor progression in mice xenografted with pancreatic cancer cells (Lebedeva et al. 2008). A study has been conducted to assess the topical application of POH cream. In the phase 2 of the study it was noted that topically administered POH at 0.76 % twice daily for 3 months has a modest effect in reducing nuclear chromatin abnormality in moderately to severely sun-damaged skin (Stratton et al. 2010). POH has also been investigated for efficiency in glioblastoma multiform (GBM). POH was administered through the intranasal route. For a better understanding of POH-induced effects, plasma proteins were studied using the differential gel electrophoresis proteomic approach. The results indicated that POH administration resulted in the suppression of antithrombin in the patients (Fischer et al. 2008). Immune cell counts of patients undergoing chemotherapy and/or radiotherapy decrease considerably. Therefore, it is essential to improve the count of immune cells so that immunological responses can be maximized. A study was conducted on 105 cancer patients and results revealed that the Chinese medicinal herb complex (CCMH) substantially improved the immune cell count. The study was conducted for 6 weeks and CCMH improved the leukocyte and neutrophil counts in patients (Zhuang et al. 20092009). POH has also been tested in 67 patients of recurrent malignant gliomas. Notable therapeutic response was observed in patients. There was a decrease in tumor size and peritumoral brain edema (PTBE). Another important

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Table 6.1  Showing perillyl alcohol-mediated effects in patients with relapsed malignant gliomas in a phase I/II clinical trial PR Stable disease Progressive course Progression-free survival GBM 1 (3.4) 13 (44.8) 15 (51.7) 48.2 AA – 3 (60) 2 (40) 60 AO 1 (33.3) 1 (33.3) 1 (33.3) 66.6 GBM glioblastoma multiforme, AA astrocytoma, AO anaplastic oligodendroglioma, PR partial response

finding of this study was longer survival rate of patients with tumor in basal ganglia (Da Fonseca et al. 2009). Intranasal administration of POH has previously been tested in GBM, astrocytoma (AA), and anaplastic oligodendroglioma (AO) patients. It was concluded that POH induced regression of tumor size with no notable toxicological effects (Da Fonseca et al. 2008). A multi-institutional phase II clinical trial was conducted for the treatment of refractory metastatic breast cancer patients who were selected for the study. However, results obtained were disappointing as evidenced by the lack of response and poor tolerance. An important conclusion drawn from the study was zero freedom from progression rate, thus highlighting the fact that POH was ineffective in treating breast cancer (Bailey et al. 2008). Table 6.1 gives information related to POHmediated effects on patients in clinical trials. POH efficacy was tested in 37 breast neoplasia patients. Five were given single dose (1.5 g/m2) for 2 days before surgery and 16 were given an escalating dose (1.2 g/m2–4.8 g/m2 per day) for 2 days. TGFβ1, IGF1, IGF2, ER, PR, apoptosis, and proliferation were studied using different methodologies. It was concluded that the preoperative POH administration was well tolerated in patients; however, it did not interfere with surgical management (Stearns et al. 2004). POH was tested for efficacy in patients with advanced malignancies. POH was administered orally (500 mg capsules of 250 mg POH) to 20 patients, four times a day, regularly for 14 days. Between two cycles there was 14 days of rest period. However, there was no evidence of POH-mediated effects on TGFβ or Ras proteins. Moreover, it was concluded that the interrupted administration of POH did not prove to be advantageous (Bailey et al. 2002). POH-mediated biological activity has been tested in eight pancreatic cancer patients. There was an improved in the survival time of patients and greater apoptotic rate. It is relevant to mention that tumor size and CA19-9 levels were unchanged (Matos et al. 2008). There was a 6-month phase I/II trial conducted and 37 patients were enrolled.

POH and Genotype: Personalized Treatment It has recently been reported that the genotype of Epidermal Growth Factor (EGF) has a relationship with glioma progression. Data obtained through Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) revealed

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that the higher frequency of AA genotype and A allele correlated with primary glioblastoma. It was indicated that patients having AA, AG, or AA + AG genotypes had higher EGF levels. Moreover, EGF levels below 250 pg/ml were related with increased survival. Better response to POH-based therapy was observed in patients with lower EGF levels (Da Silveira et al. 2012). In one relatively large study, 95 patients with malignant glioma and 100 matched healthy controls were evaluated to identify the influence of GSTM1 and GSTT1 polymorphisms, which are crucial enzymes for detoxification of various carcinogens. GSTM1 and GSTT1 polymorphism-mediated effects on the survival rate of patients with malignant glioma with intranasal POH-based therapy had recently been reported. It was indicated that a deletion of GSTT1 (28 weeks) and the lack of GSTM1 deletion (31 weeks) correlated with a longer survival rate. The lack of a GSTT1 deletion (19 weeks) and GSTM1 deletion (23 weeks) correlated with a poor survival rate. GSTT1 deletion was frequently lower in glioma patients compared to healthy subjects. The results suggested that GSTT1 deletion exerted its inhibitory effects thus playing a protective role against gliomagenesis, impact response to intranasal POH treatment, and increased overall survival (Silva et al. 2013). There is one case report of a patient with a recurrence of progressive anaplastic oligodendroglioma despite the applied complex treatment involving surgery, radiotherapy, and chemotherapy. Favorable response to intranasal delivery of 0.3% concentration of perillyl alcohol 4 times daily was noted as evidenced by reduction in size of the enhancing lesion on followup MRI scan after 5 months of treatment was registered (Da Fonseca et al. 2006).

Curcuma Aromatic Oil Promising treatment outcomes were obtained in another clinical trial using an infusion of embolized curcuma (turmeric oil) aromatic oil into the hepatic artery in combination with conventional chemotherapy in primary liver cancer (Cheng et al. 2001). Thirty-two patients treated by infusion of embolized turmeric oil into the hepatic artery were compared to a matched control group treated with transcatheter artery chemoembolization (TACE). In the main study group, one patient achieved complete remission and 13 out of 32 showed partial remission. The alpha fetal protein (AFP) level, a major oncologic marker, returned to the normal reference range in seven cases and reliably decreased in seven other patients, while in the control group, ten patients achieved partial remission, and the AFP level returned to normal in five patients and decreased in two patients. Thus, there was no reliable difference between the two groups with respect to the treatment response. The incidence of post-embolism syndrome was comparable between the two groups. Myelosuppression was no higher in the main group than in the control group ( P 29 %), cedrol (> 17 %), 6,10,14-trimethyl-2-pentadecanone (> 9 %), 9-cis-octadecadienoic acid (> 8 %).

Antitumor Activity of Pyrolae herba Essential Oil Research assessing the in vitro cytotoxic activity of P. herba essential oil (PHVO), by MTT assay showed the ability of the chemical constituents present in the oil to inhibit the proliferation and viability of SW1353—human chondrosarcoma cells (Cai et al. 2013). Knowing that cell proliferation is essentially regulated by activities occurring in the cell cycle, Sherr (1996) and Cai et al. (2013), investigating total PHVO activity on SW1353 cells using a flow cytometry technique, showed that when incubated with 50, 100, and 150 µg/ml of PHVO for 48 h, a reduction of cells in the synthesis stage (S) and an accumulation of cells in the G1 stage, interfering in cell cycle progression, and inhibiting cell proliferation was induced.

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The transition phase, the checkpoint between G1/S stages within the cell cycle, is considered one of the main verification points responsible for the starting and the finishing of DNA strand doubling (Nurse 1994). It is important to note that the process of cell cycle progression is an event regulated positively by cyclin-dependent kinases such as CDK4/CDK6, and subunits of regulatory cyclins such as cyclin D1, that regulate CDK4 or CDK6 complexes controlling cell cycle progression and the evolution from the G1 to the S phase (Zhang et al. 2009). Using Western blot analysis, Cai et al. (2013) demonstrated that PHVO treatment decreased the expression of important cell cycle proteins, such as cyclin D1, CDK4, and CDK6, and increased p21 marker expression in a dose-dependent manner. One of the cardinal markers of malignancy (changes in overexpression of the cyclin D1) may induce hyperactivity of the CDK4 complex, inducing exacerbated cell division (Kessel and Luo 2000; Zafonte et al. 2000; Haraken et al. 2008). It is worth pointing out that the p21 protein inhibitor of cyclin/CDK complexes regulates the cycle through inhibition of cyclin/CDK complex activity (Harper et al. 1993; Chen et al. 1994; Goubin and Ducommun 1995).

General Considerations Concerning Pyrolae herba Essential Oil In vitro assays with P. herba essential oil (PHVO) show antiproliferative activity against SW1353 cells. This activity correlates to the combination of chemical components present in the oil, inhibiting cell cycle progression (Fig. 7.7). This is

Fig. 7.7  Interaction possible of essential oil of P. herba (PHVO) with proteins of the cell cycle, based on research (Cai et al. 2013)

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important to understand when evaluating in vivo antitumor activity. The oil’s low toxicity is also a distinct advantage (Cai et al. 2013).

Salvia libanotica For thousands of years the plants have been used for the treatment of diseases, this is due to the presence of bioactive compounds in these species (Taylor 2000; Balunas and Kinghorn 2005). Salvia libanotica belongs to the family Laminaceae (Labiateae), having approximately 900 species worldwide, and is endemic to the Mediterranean (Al-Kalaldeh et al. 2010). The essential oil of S. libanotica (EOSL) has been used for centuries to combat numerous diseases such as headaches, abdominal pain, coronary disorders and even microbial infection, and inflammation, such as therapeutic activities seem to be related to terpenoid compounds present in the oil (Hilan et al. 1997; Gali-Muhtasib et al. 2000; Cha et al. 2005; Wan et al. 2006). Phytochemical study of EOSL has revealed a rich oil composition of more than ten constituents, highlighting α-terpineol (Te), ( p-menth-1-en-8-ol), linalyl acetate (Ly), (3,7-dimethyl-1,6-octadien-3-yl acetate), and camphor (Ca) (7.7 trimethylbicyclo [2.2.1] heptan-2-one) (Itani et al. 2008).

Antitumor Activity of Salvia libanotica Essential Oil The components present in S. libanotica essential oil were found to be synergistically effective to inhibit growth (< 60 %, 48 h incubation), and to cause cell cycle arrest, and apoptosis in colon cancer HCT116 (p53 +/+ and p53 −/−) cells. However, this cytotoxicity was not observed in strains with normal FHs74Int intestinal epithelial cells. Tumor cell growth inhibition is related to the activity of Ly, Te, and Ca compounds, and combinations thereof (Itani et al. 2008). The cytotoxic activity of EOSL was confirmed by flow cytometry; combinations of Ly, Ca, and Te in p53 +/+ cells induced accumulation of the population of cells in the stage Pre-G1 dependent on time (64 % in 48 h), and with reduced time in the phases G0/G1 and G2/M (Itani et al. 2008). Cytometry assays were confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique analyzing death by apoptosis (caspase expression, both initiator and effector, especially caspase-3), and using pretreatment of the cells for 2 h with the caspase-3 inhibitors (Z-DEVD-FMK), caspase-8 (Z-IETD-FMK), caspase-9 (Z-LEHD-FMK), and pan-caspase inhibitor (Boc-D-FMK) as well. Tumor cells treated with Ly + Te, Ly + Ca, and Ly + Ca + Te confirmed EOSL cytotoxicity as contributing to change in the membrane potential Δψm by 55, 24 and 60 %, respectively, to the compounds tested alone. It is also worth pointing out that expression of cytochrome c, Bax, Bcl-2, and Bcl-XL were confirmed by Western blot (Itani et al. 2008). EOSL was also able to inhibit fibrosarcoma

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L929Sa in concentrations of 180 µg/ml. Other work has shown that against MDA-MB 231 metastatic human breast carcinoma cells, cytotoxicity of EOSL was achieved with a concentration of 290 μg/ml (Kaileh et al. 2007). Itani et al. (2008) showed inhibition of Bcl-XL expression induced by Ly + Te + Ca in HCT116 p53 +/+ cells, and confirmed that Ly + Te + Ca acts mainly through the mitochondrial pathway, the antiapoptotic protein Bcl-XL suppresses mitochondrial cytochrome c release (Bras et al. 2005). Treatment of HCT116 (p53 −/−) cells with EOSL components, and a specific caspase-3 inhibitor was unable to reverse the antineoplastic effects of Ca + Ly + Te which explains the inactivation of caspase-3. Selective activation of caspases or reduced activation thresholds may help stimulate apoptosis in tumor cells (Philchenkov 2004). Treatment with Ca + Ly + Te induced release of cytochrome c from mitochondria due to Bax activation, inhibition of Bcl-XL, and subsequent caspase-3 activation, leading to apoptosis (Itani et al. 2008).

General Considerations Concerning Salvia libanotica Essential Oil The study of the S. libanotica essential oil (EOSL) revealed the presence of three bioactive components, Ly, Te, and Ca, which act synergistically by inducing cell cycle arrest and apoptosis in human colon cancer cells HCT 116 (p53 +/+ p53 −/−). These components induce a higher number of apoptotic events in the tumor cells than in healthy cells. The apoptosis occurs with release of mitochondrial cytochrome c into the cytoplasm, caspase-3 activation, and resulting PARP cleavage. Caspase dependency was confirmed through pan-caspase inhibitors of caspase-3 (Itani et al. 2008). In vitro studies with EOSL were important for the design of in vivo in antitumor activity models, and they also contribute to the development of new therapeutic proposals based on this species.

The Contribution of Essential Oils for the Development of New Therapeutic Tools Against Cancer The compounds originating from natural sources (such as plants) have been applied to treat many diseases, especially in the twentieth century, after World War II, when the isolation techniques, characterization and synthesis of compounds have gained more strength and boosted drug development and most of the medicines sold to a variety of conditions including cancer originated from natural sources. Despite the search for molecules from natural sources with potential antitumor activity such as animals (vertebrates and terrestrial invertebrates) and marine organisms, the phytocompounds continue to represent the largest source for obtaining biologically active substances, due among other causes through their structural diversity and chemical simplicity of some molecules, as well as the

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availability of some fresh and local plant species to study the performance in natura or in loco. According to Newman et al. (2003) and Brandão et al. (2010) drugs that have active principles derived from plants or from chemical basis flora relate to the treatment of more than 85 % of human disease, demonstrating the importance of plants for the development of new therapeutic strategies. The essential oils obtained from a wide variety of plants have been used for decades in various applications, including activities in alternative medicine such as aromatherapy. The popular use, as based on scientific evidence, has shown that essential oils present broad therapeutic potential, including for the treatment of cancer in the medium and long term (Fig. 7.8). Table 7.14 shows the antitumor activities of the plants discussed in this chapter.

Conclusion Scientific studies discussed in this section show the therapeutic potential of essential oils for the development of drugs with application in the treatment of cancer.

Eucalyptus benthamii

Cymbopogon flexuosus

Croton regelianus

Abies balsamea

Plant

1,8-Cineole Aromadendrene Epiglobulol Globulol Terpinen-4-ol Viridiflorol α-Pinenea γ-Terpinene

Chemical constituent of essential oils (active principle) α-Humulenea ɤ-Caryophyllene p-Cymene Ascaridolea Cânfora α-Terpinene Geraniol Geranyl acetate α-Bisabolol Isointermedeola Limonene Borneol

Mitochondrial disfunction

Kumar et al. (2008) Sharma et al. (2009)

Mitochondrial disfunction

Antitumoral in sarcoma 180 and Ehrlich ascite carcinosarcoma models

Not described by the mentioned authors

Nakase et al. (2008) Bezerra et al. (2009)

Not described by the mentioned authors Antitumoral in sarcoma 180 model

Cytotoxicity against lines of leukemia cancer cell and solid tumor cell Cytotoxicity against lines of leukemia cancer cell and solid tumor cell Increase in ROS intracellular production In HL-60 cells Production of H2O2 In ⇩ Bcl-2 protein levels, an ⇧ in Bax protein levels, and caspase-9, -8, and -3 activation query ⇩ (Δψm) Transmembrane potential mitochondrial Decreased expression of NFkβ Cytotoxicity against lines of leukemia cancer cell and solid tumor cell Decresead expression of NFkβ

Legault et al. (2003)

Mitochondrial disfunction Mitochondrial disfunction

In vivo activity

In vitro activity

Possible mechanism of References action

Table 7.14  Chemical constituents present in the plant species discussed in the chapter and its main biological activities

7 Antitumor Essential Oils 163

Lindera umbellata

α-Pinene Limonene 1,8-Cineole Geraniol Geranyl acetate Linaloola

Guatteria pogono- α- and β-Pinene pus (Annonaceae) ɤ-Patchoulene (E)-Caryophyllenea Germacrene D Bicyclogermacrene Thymola Lippia gracilis Schauer p-Cymene (Verbenaceae) ɤ-Terpinene Myrcene

Table 7.14 (continued) Plant Chemical constituent of essential oils (active principle) Guatteria friesiana α-Eudesmol β-Eudesmol γ-Eudesmol

Antitumoral in sarcoma 180

generation In ⇩ Bcl-2 protein levels, an ⇧ in Bax protein levels, and caspase-9, -8, and -3 activation. Cell cycle arrest (G1 stage) in HepG2 cells Cytotoxicity against lines of leukemia cancer cell and solid tumor cell Increase in ROS intracellular production Regulation of expression of differentiation genes (retinoid X receptor (RXR) and nuclear retinoic acid receptors (RARs) Inhibition of cyclin-dependent kinase pathway upregulation of the p53 gene in cell cycle

Not described by the mentioned authors

Antitumoral in sarcoma 180

Deb et al. (2011) Ferraz et al. (2013)

Induction of apoptosis Linney (1992) by upregulation of p53 Robertson et al. (1992) Chiang et al. (2003) Ravizza et al. (2008) Loizzo et al. (2008) Paik et al. (2005) Gu et al. (2010) Goto et al. (2010) Maeda et al. (2012)

Induce caspase-independent apoptosis

Britto et al. (2012) ERK pathway blockade (extracellular signal by regulated kinase) and CREB (cAMP binding protein response element) and inhibition in vitro of angiogenesis Tundis et al. (2009) Not described by the mentioned authors Fontes et al. (2013)

Antitumoral in sarcoma 180 and H22 (hepatocarcinoma transplanted in BALB/c mice) model

Cytotoxicity against lines of leukemia cancer cell and solid tumor cell Antiangiogenic

Cytotoxicity against lines of solid tumor cell

Possible mechanism of References action

In vivo activity

In vitro activity

164 H. I. F. Magalhães and É. B. V. de Sousa

b

a

Chemical constituent of essential oils (active principle) Hexadecanoic acid Cedrol 6,10,14-Trimethyl2-pentdecanone cis-9-Octadecadienoic acid α-Terpineol (Te)b, ( p-Menth-1-en8-ol), Linalyl acetate (Ly)b, (3,7-Dimethyl1,6-octadien3-yl acetate), Camphor (Ca)b (7,7-Trimethylbicyclo [2.2.1] heptan-2-one)

Majority compound Bioactive compound

Salvia libanotica

Pyrolae herba

Plant

Table 7.14 (continued) In vivo activity

Cytotoxicity against lines of solid tumor cell Variation in membrane potential Δψm and release of cytochrome c Decreased Bcl-2 protein levels, an increased Bax protein levels, and caspase-3, -8, and -9 activation

Not described by the mentioned authors

Not described by the menCytotoxicity against SW1353— tioned authors human chondrosarcoma cells Upregulation of p21 protein Downregulation of cyclin D1, CDK4, and CDK6

In vitro activity

Mitochondrial disfunction

Cell cycle arrest in G1 phase

Itani et al. (2008)

Cai et al. (2013)

Possible mechanism of References action

7 Antitumor Essential Oils 165

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Chapter 8

Antitumor Monoterpenes Janaina Fernandes

Introduction Natural products are sources of new candidate compounds for antitumor drugs used to treat or prevent the development of cancer. They are also used as model molecules for the discovery of synthetic chemicals used in drug production (Ramshankar and Krishnamurthy 2014). In the past few years, natural products such as terpenes have emerged as alternatives in the treatment of cancer and inflammation (Shanmugam et al. 2012). Monoterpenes are a subclass of terpenoids that may be found in essential oils. They are highly volatile and are found in the essential oil of several plant species that have been shown to have several biological activities under investigation including anticancer activities. Monoterpenes have presented various pharmacological activities such as analgesic, anxiolytic, antimicrobial, and antitumor activities (De Sousa 2012). It has already been shown that several compounds used in anticancer chemotherapy are able to induce cell death by either the activation of key elements of the apoptotic program or downregulation of the survival pathways (Chen et al. 2012). Apoptosis is characterized by distinct morphological features such as plasma membrane blebbing, cell shrinkage, chromatin condensation, DNA fragmentation, and the breakdown of the cell into apoptotic bodies (Westhoff et al. 2014). Monoterpenes can induce apoptosis by both the extrinsic (Fas-mediated) (Rajesh and Howard 2003) and intrinsic (mitochondrial pathway) pathways (Yeruva et al. 2010) by upregulating proapoptotic BcL-2 family members and/or downregulating the antiapoptotic members of the same family (Verhaegen et al. 2014). Several reports show that monoterpenes are able to induce cell arrest (Kim et al. 2012) and can act as radiosensitizers (Rajesh et al. 2003). Another important feature of monoterpenes is their capacity to act synergistically, thus enhancing the activity of other J. Fernandes () NUMPEX-BIO—Pólo Xerém, Universidade Federal do Rio de Janeiro and National Institute for Translational Research on Health and Environment in the Amazon Region—INPETAM Centro de Ciências da Saúde, Bloco B17, Cidade Universitária, Rio de Janeiro, RJ 21949-900, Brazil e-mail: [email protected] © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_8

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monoterpenes (Russo et al. 2013) and drugs used in clinical treatment (Miyashita and Sadzuka 2013) to promote the accumulation of drugs in resistant tumors (Eid et al. 2012). The latter effect has a strong impact on clinical outcomes and patient survival because drug-resistant tumors are the main challenge in cancer treatment. Monoterpenes have been effective on antitumor tests in vitro, in vivo, and in clinical trials against breast, brain, colon, and lung cancers, and the molecular mechanisms that underlie its effects have been elucidated. In this chapter, we present the current and emerging cancer molecular targets affected by monoterpenes, the main pathways activated by them and the physicochemical properties that contribute to their treatment of both sensitive and resistant tumors.

Cancer and the Main Death Pathways Activated by Monoterpenes Induction of tumor death is the main objective of cancer chemotherapy and refers to not only the tumor death but also the type of tumor cell death. Monoterpenes can induce cell death in several types of tumors through different mechanisms. Apoptosis is one of the most common types of cell death induced by natural or synthetic compounds (Chen et al. 2010; Wani et al. 2013). Apoptosis signaling and execution count on several proteins that can occur in both the extrinsic and intrinsic pathways, where the latter is the major route used by several chemotherapeutic drugs (Chen et al. 2010). Activation of the mitochondrial permeability transition pore leads to the dissipation of the mitochondrial membrane potential and to apoptosome formation in the cytosol, which is the complex formed by cytochrome c, Apaf-1, and procaspase-9, leading to the morphological features of apoptosis (membrane blebbing and DNA fragmentation) (Brinkmann and Kashkar 2014). Autophagy also plays an important role in cellular homeostasis in apoptosis. In autophagy, the autophagosome fuses with lysosomes to become autolysosomes, and their contents are degraded to be recycled elsewhere in the cell. The autophagic response can be activated by several stimuli such as nutrient deprivation, high temperatures, and hypoxia. The molecular mechanism of autophagy involves autophagy-related genes (Atgs) (Ge et al. 2014). Among them, Beclin1 is responsible for the nucleation and expansion of the autophagosome. The microtubule-associated protein light chain 3, LC3, is recruited to the autophagosome double membrane through an Atg5-dependent mechanism (Papackova and Cahova 2014). Autophagy has been implicated in tumorigenesis and the rescue of tumors from cell death as well as in cell death because of the overactivation of the autophagic machinery (Leng et al. 2013). It has been well established by studies in model organisms that autophagy upregulation extends the life span whereas autophagy downregulation may lead to neurodegeneration. The role of the autophagy increase in non-tumoral tissues has a beneficial effect in both in vitro and in vivo models, and it is an important pharmacological approach to reduce the symptoms of neurodegenerative diseases (Silva

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et al. 2014). On the other hand, there is some controversy about whether autophagy has a beneficial or a deleterious effect on tumor cells, and thus the role of autophagic pathways as targets for cancer treatment is still under debate (Katayama et al. 2007; Carew et al. 2007; Schmukler et al. 2013). Even studies using the same compound have found different results and reached different conclusions. Leng et al. 2013 showed that ursolic acid, a triterpene, was able to promote cell death by autophagy in cervical cancer; on the other hand, Shin et al. 2012, also using ursolic acid, found that the inhibition of autophagy enhanced the apoptosis induced by ursolic acid in prostate cancer and that the induction of autophagy had a protective effect on the tumor cell used in the study.

Autophagy Terpinen-4-ol and Geraniol Despite the controversial role of autophagy in tumor cell death, the activities of monoterpenes also include the induction of autophagy. There are only a few studies involving monoterpenes and autophagy; however, the emerging studies present consistent data. The work of Banjerdpongchai and Khaw-On 2013 showed that terpinen-4-ol (Fig. 8.1) induced autophagic cell death in the leukemia cell line HL-60 through the induction of Beclin1, LC3I, LC3II, and Atg5 without signs of apoptosis, despite the raised tBid and slight activation of caspase 8, but without altering the Bcl-2 family members. Geraniol (Fig. 8.1) induced both apoptosis and autophagy in PC3 prostate cancer cells. The mechanism involves raising the expression of LC3II, Atg5, and Bax, autophagosome formation, activation of caspase 3, and reduction of Bcl-2 and Bcl-xL (Kim et al. 2012). Menthol, cineol, and linalool were also tested for the expression of LC3, but menthol showed weak staining compared with geraniol whereas cineol and linalool showed no staining for LC3, one of the main markers of autophagy (Tanida et al. 2005). Monoterpenes can either act through the general mechanisms of cell death induction, as discussed above, or through specific target pathways that induce cell growth impairment and cell death. The main pathways targeted by monoterpenes are discussed in the following section.

Fig. 8.1  Terpinen-4-ol and geraniol OH

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Main Molecular Targets of Monoterpenes The Ras–RAF–ERK Pathway Mitogen-activated protein kinase (MAPK) mediates survival and proliferation signals transduced by several membrane receptors. Upon activation of the membrane receptors, RAS proteins are activated, which bind and activate the RAF kinase, and RAF is recruited to the cell membrane and activated, triggering the sequential phosphorylation of MEK 1/2 and ERK, which translocate to the nucleus and induce the expression of several genes involved in survival and proliferation (Santarpia et al. 2012). Another way to engage constitutive activation of this pathway is through the increased expression or mutational activation of proteins that constitute the pathway, and the most frequently observed mutations are those arising in the three human RAS genes ( HRAS, NRAS, and KRAS) (Malumbres and Barbacid 2003) and also BRAF (Yuan et al. 2013). The frequency with which this pathway is deregulated in cancer makes it an attractive target for directed therapy. Inhibitory agents with low molecular weight have been developed to target (1) the various enzymes that catalyze translational modifications of the Ras proteins, (2) the kinase activity of the RAF, MEK, and ERK Ser/Thr kinases, and (3) the molecules upstream and downstream of the Ras pathway (EGFR, PDGFR, mTOR, and PKC) (Chappell et al. 2011; Santarpia et al. 2012). Several of these agents have shown promising clinical activity toward renal cell carcinoma, hepatocellular carcinoma, non-small cell lung cancer (NSCLC), and colorectal cancer (Chappell et al. 2011). Sorafenib was the first and only RAF kinase inhibitor to be approved for clinical trial by the Food and Drug Administration (FDA) for the treatment of advanced renal cell carcinoma and hepatocellular carcinoma. However, another RAF inhibitor, Dabrafenib, was recently discovered and is thought to be a potent, selective, and efficacious inhibitor of B-Raf bearing the V600E mutation (Rheault et al. 2013).

Perillyl Alcohol and d-Limonene Perillyl alcohol (POH) is a naturally occurring hydroxylated product of d-limonene (Fig. 8.2) formed by the condensation of two isoprene units. It is found in the essential oil of mints, cherries, lavenders, lemongrass, sage, cranberries, perilla, wild bergamot, gingergrass, savin, caraway, and celery seed. POH readily metabolizes perillic acid (PA) and dihydroperillic acid (DHPA) (Bailey et al. 2004). The capacity of POH to reduce the activation and expression of proteins in the RAS–RAF–MEK–ERK cascade has been demonstrated (Holstein and Hohl 2003). POH was able to inhibit the prenylation of RAS, which is dependent on mevalonate synthesis, and it is one of the activating steps for this pathway (Hardcastle et al. 1999; Cerda et al. 1999). POH inhibited RAS, RAF activation, and ERK 1/2 phosphorylation, with the consequent regulation of the intrinsic apoptotic pathway:

8 Antitumor Monoterpenes Fig. 8.2  Perillyl alcohol and d-limonene

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Raised BAX expression and the reduction of Bcl-2 (Chaudhary et al. 2009). The same effect was observed for a different model of d-limonene (Chaudhary et al. 2012). POH inhibited Mek and Erk kinase activities in transformed leukemia cells; however, in this study, POH was unable to inhibit Ras even though the POH concentrations were sufficient to induce growth arrest and apoptosis as a consequence of blocking the Erk signaling pathway at the level of Mek, making it suitable for the treatment of leukemia (Clark et al. 2003). d-Limonene exhibited the same pattern of activity, and its activity in experimental hepatocarcinogenesis was not related to RAS activation (Kaji et al. 2001). There are quite a few recent studies on the activity of monoterpenes in the Ras pathway in vitro and in vivo. This could be related to the development of specific inhibitors of this pathway after 2005. Other monoterpenes also reduced the expression of RAS without mevalonate depletion as ( R)-perillyl alcohol and ( S)-perillyl alcohol, whereas ( S)-perillaldehyde, myrtenol, myrtanol, (−)-menthol, isomenthol, neomenthol, carveol, dihydrocarveol, and carvone exerted activity under mevalonate depletion (Holstein and Hohl 2003). NF-κB Activity Nuclear factor-κB (NF-κB) is a transcription factor that is very frequently activated in tumors and involved in tumor growth, progression, and resistance to chemotherapy (Erstad and Cusack 2013). NF-κB is regulated by many posttranslational modifications such as methylation, acetylation, phosphorylation, and ubiquitination. NFκB is located in the cytoplasm associated with IκB, its inhibitory subunit. Under phosphorylation by IKK kinase, IκB is targeted for E2- and E3-ligase-mediated polyubiquitination and subsequent 26S proteasomal-mediated degradation. The IκB subunit is degraded, thus releasing the activated NF-κB that accumulates in the nucleus (Gupta et al. 2010). In the nucleus, NF-κB binds to DNA and activates the transcription of several genes such as EGFR (Chun and Kim 2013), MDR1 (Wang et al. 2013), and cyclins D1 and D2 (Oida et al. 2014), making NF-κB a potential drug target in hematological malignancies and solid tumors. Inhibition of NF-κB activity can be achieved by the blockage of its translocation through the inhibition of IκB phosphorylation (IKK kinase inhibition). Some

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natural compounds that are able to prevent IκB phosphorylation such as B-carboline (Yoon et al. 2005) have been identified. However, the mechanism of action is known only for a few IKK inhibitors (Gupta et al. 2010). Monoterpenes can affect the NF-κB pathway in several ways.

Cineole 1,8-Cineole, cineole, or eucalyptol (Fig. 8.3) is the major constituent of the essential oil of Eucalyptus globus leafs. Its presence has been detected in several plant species, and it can also be synthetized by the isomerization of α-terpineol (Leão Lana et al. 2006). The inhibition of NF-κB activity occurs in the interplay between inflammation and antitumor activities. Several drugs with anti-inflammatory activities also have antitumor activities. Grainer et al. (2013) first described that 1,8-cineole significantly reduced cell viability in the human cancer cell lines U373 (glioblastoma) and HeLa. The activity of NF-κB was also reduced by 1,8-cineole even after lipopolysaccharide (LPS)-dependent stimulation of NF-κB activity. After 90 min, cineol reduced the expression of IκB and the target genes of NF-κB. This activity was directly related to the antitumoral effect of 1,8-cineol. Linalyl Acetate and α-Terpineol A combination of linalyl acetate and α-terpineol (Fig. 8.4) dose dependently reduced the NF-κB signaling in HCT-116 colon cancer cells and thus their viability as demonstrated by Deeb et al. 2011. The induction of apoptosis reached 60 %, Fig. 8.3  1,8-Cineole O

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Fig. 8.4  Linalyl acetate and α-terpineol

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Fig. 8.5  α-Pinene

α-Pinene and the DNA binding assays revealed that the combination treatment suppressed p65 nuclear translocation and IκB-alpha degradation. Additionally, a combination of linalyl acetate and α-terpineol downregulated the expression of NF-κB-regulated antiapoptotic and proliferative gene products. α-Pinene α-Pinene (Fig. 8.5) is richly present in conifer trees. It is a bicyclic monoterpene that inhibits the translocation of NF-κB into the nucleus in THP-1 LPS-stimulated cells. The work of Zhou et al. (2004) showed that in THP-1 cells pretreated with α-pinene, the translocation of NF-κB was strongly reduced. Even though it is a powerful inhibitor of NF-κB, at the concentrations used to inhibit translocation, α-pinene did not reduce the viability of these cells. Another effect on this pathway is the blockage of the LPS-mediated degradation of IκBα, the inhibitory protein. In THP-1 cells, the presence of α-pinene had the same effect as TLCK (tosyl-lysinechloromethylketone), a serine protease inhibitor that antagonizes the translocation of NF-κB by blocking the phosphorylation of IκBα (Heyen et al. 2000). Further studies are necessary to assess the effect of α-pinene on the phosphorylation of members of the NF-κB pathway.

Limonene and Perilyl Alcohol Limonene possesses both antitumoral and anti-inflammatory activities, as do some of its derivatives including POH, PA, and menthol. These compounds inhibit NFκB signaling in lymphoma cells and induce NF-κB dependent apoptosis (Berchtold et al. 2005). Most of the monoterpenes studied for NF-κB pathway inhibition have dose-dependent effects and act by inhibiting the translocation of active NF-κB through the blockage of IκB proteasomal degradation and downregulating its expression, but the final fate of whether the inhibition of NF-κB is accompanied by apoptotic death seems to be dependent on the cell line.

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Telomerase Activity (Through mTOR) Telomerase is a ribonucleoprotein complex responsible for adding six base pair (bp) repeats to the end of a chromosome to prevent the loss of DNA during replication. This action is necessary because of the fundamental limitation of polymerization at chromosome ends. Human telomerase reverse transcriptase (hTERT), the ratelimiting enzymatic portion of telomerase, is a potential candidate for cancer therapy because of its absence in most normal somatic cells and its reactivation in many tumor cells. Importantly, telomerase activation is an early and key event in the creation of tumor cells and, as such, is an important target in cancer prevention. Kawauchi et al. 2005 showed that both mTOR (mechanistic target of rapamycin) and S6K (S6 kinase) were found to co-immunoprecipitate with hTERT, heat shock protein 90 (Hsp90), and Akt, suggesting that these proteins form a physical and functional complex. mTOR forms a complex with RAPTOR (regulatory associated protein of mTOR), the so-called mTORC1 complex, which associates with hTERT. Thus, the regulation of mTOR and its cascade may interfere with telomerase activity.

Perillyl Alcohol Through a series of papers, Sundin and collaborators have characterized the influence of POH in telomerase activity. This series of papers is the only (to date) work dealing with monoterpenes and telomerase activity. POH (400 µM for 16 h) suppressed telomerase activity in the prostate cancer cell lines PC3 and DU145, ranging from 65 to > 95 %, as determined by a real-time quantitative telomerase repeat amplification protocol and confirmed by polyacrylamide gel analysis. It was also shown that POH did not reduce hTERT mRNA levels, even though it reduced the hTERT protein levels through proteasome activity as indicated by the proteasome inhibitor MG123 (Sundin et al. 2012). The additional data provided by Sundin et al. 2013a and Sundin et al. 2013b showed that POH was able to disrupt the hTERT–mTOR–RAPTOR complex, leading to the reduction of telomerase activity in DUT145 prostate cancer cells. Furthermore, another consequence of the POH impairment of mTOR signaling is the reduction of protein synthesis. Under cell quiescence, eIF4E (the rate-limiting cap-binding protein) is sequestered by 4E-BP1 (eIF4E-binding protein 1) to prevent eIF4E from promoting translation initiation. As mTOR signaling leads to the phosphorylation of 4E-BP1, it promotes protein synthesis through the release of eIF4E. Overexpression of eIF4E can reduce tumor responses to POH (Sundin et al. 2013b). Collectively, this series of papers showed that POH reduces protein levels of hTERT without reducing mRNA levels and that the low levels of hTERT protein are related to proteasome activity and the reduction of protein synthesis via the inhibition of mTOR signaling through the disruption of the mTOR complex formation with members of the cascade. Furthermore, this study also showed that POH af-

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fected telomerase activity under the overexpression of elF4E. By targeting mTOR, POH affects protein activity that is essential for tumor initiation and progression and proteins that are overexpressed in tumors.

Cell Cycle Arrest Among other features, tumor malignancy is dependent on cell cycle progression, a collection of events that, when over activated, ultimately lead to cell division and tumor proliferation. The process is dependent on the expression and activity of cyclin-dependent kinases (CDKs; Canavese et al. 2012) and activation that can be achieved by mitogenic signaling from several pathways that are overexpressed in tumors (Pruitt and Der 2001). Cyclins and CDKs, in association with each other, promote the transition from the G1 phase to the S phase of the cell cycle by phosphorylating the tumor-suppressor retinoblastoma protein (RB) (Macdonald and Dick 2012). The complex cyclin– CDK is negatively regulated by CDK inhibitor (CKI) families of proteins: CIP/ kip (p21, p27, and p57) and INK4 (p16, p15, p18, and p19). The former negatively regulates cyclin E/A–CDK2 and cyclin D–CDK4/6 complexes, whereas the latter specifically inhibits cyclin D–CDK4/6 complexes (Bruyère and Meijer 2013). The capacity of monoterpenes to inhibit cell cycle progression may be accompanied by cell death or not; in the latter case, they can act as cytostatic compounds.

Perillyl Alcohol, Perilic Acid, and Linalool POH and PA (Fig. 8.6) induced cell cycle arrest and apoptosis in lung cancer cells (Koyama et al. 2013). These authors observed an increased expression of Bcl2, bax, and p21 and increased caspase-3 activity in cells treated with POH and PA. Part of these data was already shown by Wiseman et al. 2007. Linalool (Fig. 8.6) was able to upregulate the expression of cell cycle inhibitors in Molt-4 leukemia cells after 12 h. A time-dependent increase in the mRNA of a variety of cyclin-dependent kinase inhibitors, such as p21Waf1/CIP1 (31.4-fold), p27Kip1 (43.0-fold), P57Kip2 (32.46-fold), and p15 (4.0-fold), was observed. AdFig. 8.6  Perilic acid and linalool

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Fig. 8.7  Menthol

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ditionally, increased p53 expression and apoptosis related to p53 expression were observed. In this work, linalool does not affect the growth of normal hematopoietic cells at the concentrations that killed tumor cells (Gu et al. 2010).

Menthol Menthol (Fig. 8.7) is a monoterpene that has been widely used in cosmetics and pharmaceutical products and also as flavoring in food. The mechanisms by which menthol induces a cooling sensation were finally elucidated in 2002 by the works of McKemy et al. 2002 and Peier et al. 2002. These studies showed that the transient receptor potential melastatin 8 (TRPM8), a Ca2+-permeable cation channel, is known to regulate [Ca2+]i in response to cold temperatures or pharmacological stimuli. TRPM8 is a protein that is upregulated in prostate and some other types of cancer cells (Tsavaler et al. 2001). TRPM8 was recognized as a molecular target of menthol by several groups, but the involvement of TRPM8 in menthol-induced cell death is still under discussion. It was found that menthol can induce both mediated (Yamamura et al. 2008) and non-mediated (Kim et al. 2009) cell death by TRPM8 and is also correlated with TRPM8 expression (Kijpornyongpan et al. 2014). Menthol primarily affects the expression of cell cycle-related genes in PC-3 prostate cancer cells, as revealed by DNA microarray analyses. Menthol induced G2/M arrest and downregulated polo-like kinase 1 (PLK1), a key regulator of G2/M phase progression, and inhibited its downstream signaling (Kim et al. 2012). In another prostate cancer cell line, DUT145, menthol also induced cell arrest but in the G0/G1 phase and without apoptosis (Wang et al. 2012). However, the relevance of TRPM8 for prostate cells is still under debate because the inhibition of the expression or function of the channel reduces the proliferation rates and proliferative fraction in tumor cells but not in non-tumor prostate cells (Valero et al. 2012). The effect of menthol in the TRPM8 system is not restricted to prostate cancer but is also present in melanoma (Yamamura et al. 2008) and leukemia (Lu et al. 2006) in which the effect was necrosis.

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Fig. 8.8  Thymol and citral O OH

Thymol

Citral

Miscellaneous For some isolated monoterpenes, there are few studies on the capacity of these compounds to induce cell death or the information at the molecular level is limited. Thymol (Fig. 8.8) may be protective at low concentrations (Archana et al. 2011; Shettigar et al. 2014), but at elevated concentrations, it is able to induce Ca influx and a reduction of viability in glioma cells (Hsu et al. 2011) and melanoma (Satooka and Kubo 2012). Citral (Fig. 8.8) induces cell arrest in the G2 phase in MCF7 breast cancer cells. α-Pinene was able to induce apoptosis in melanoma through the mitochondrial pathway, ROS production, caspase-3 activity, and DNA fragmentation (Matsuo et al. 2011).

Physicochemical Properties of Monoterpenes and Antitumor Efficiency Monoterpenes and Brain Tumors—Intranasal Administration Gliomas are by far the most common type of intrinsic brain tumor in adults, affecting 5–10 individuals/100,000/year, and account for more than 50 % of all intrinsic brain tumors. The standard protocol to treat glioma patients includes radio- and chemotherapy with temozolomide (TMZ) as a concomitant and later as adjuvant chemotherapy. This treatment results in the current prognosis of a median survival of 14.6 months from diagnosis. (TMZ)-resistant malignant gliomas represent an additional challenge to treatment outcomes and patient survival (Grimm and Pfiffiner 2013). Several drugs used to treat brain diseases, including tumors, induce severe side effects because of the necessary dosage to reach efficiency. This drug delivery challenge to the brain is the blood–brain barrier (BBB), which only permits the penetration of highly lipid-soluble molecules under a threshold of 400–600 Da. The BBB maintains more than 98 % of low-molecular-weight drugs and almost 100 % of high-molecular-weight drugs (Pajouhesh and Lenz 2005).

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Table 8.1  Physicochemical properties of monoterpenes with antitumor activity CID TPSA Reference x-Log P MW 31253 Myrcene 0 4.3 136,234 Mitić-Culafić et al. 2009a 7463 Cymene 0 4.1 134,218 Kljun et al. 2011b 440917 Limonene 0 3.4 136,234 Russo et al. 2013a 6654 Pinene 0 2.8 136,234 Döll-Boscardin et al. 2012a 11463 Terpinolene 0 2.8 136,234 Okumura et al. 2012a 2758 Cineole 9.2 2.5 154,249 Greiner et al. 2013a 7794 Citronellal 17.1 3.0 154,249 Osato 1965a 638011 Citral 17.1 3.0 152,233 Chaouki net al. 2009a 16441 Perillaldehyde 17.1 2.6 150,218 Elegbede et al. 2003a 16724 Carvone 17.1 2.4 150,218 Hussain et al. 2010b 2537 Camphor 17.1 2.2 152,233 Itani et al. 2008b 10545 Ascaridole 18.5 2.3 168,232 Bezerra et al. 2009a 6989 Thymol 20.2 3.3 150,218 Satooka et al. 2012a 8842 Citronellol 20.2 3.2 156,265 Yoshida 2005a 10364 Carvacrol 20.2 3.1 150,218 He et al. 1997a 16666 Menthol 20.2 3.0 156,265 Yamamura et al. 2008a 369312 Perillyl alcohol 20.2 2.7 152,233 Fernandes et al. 2005a 637566 Geraniol 20.2 2.7 154,249 Kim et al. 2014a 6549 Linalool 20.2 2.7 154,249 Gu et al. 2010a 64685 Borneol 20.2 2.7 154,249 Su et al. 2013b 17100 Alpha-terpineol 20.2 1.8 154,249 Hassan et al. 2010a 9017 Citronellyl acetate 26.3 3.8 198,302 Paik et al. 2005b 8294 Linalyl acetate 26.3 3.3 196,286 Deeb et al. 2011a 1256 Perillic acid 37.3 2.7 166,217 Yeruva et al. 2007a a Antitumor activity of the monoterpene b Antitumor activity of the essential oil containing the monoterpene, a complex containing monoterpene or synergistic effect CID compound ID in PubChem Compound Database, TPSA topological polar surface area, MW molecular weight

Drug delivery to the brain relies on the brain-to-plasma concentration rate (equilibrium rate) and the time to reach that equilibrium. These two parameters are dependent on several drug physicochemical properties such as lipophilicity, the tendency to form more than six hydrogen bonds, the molecular size, and the polar surface area (PSA) (Pajouhesh and Lenz 2005). Monoterpenes are derived from the mevalonate pathway and are formed by two isoprene units; therefore, most of them possess low molecular weights. Many of the monoterpenes with antitumor activity possess a topological polar surface area as low as zero and have low molecular weights (Table 8.1), making this compound class suitable to treat not only brain tumors but also useful to enhance drug availability for other tumors. Because of the problems with drug availability leading to severe side effects, alternative methods have emerged to raise the efficiency of the treatment through higher drug availability and reduction of side effects (Hwang and Kim 2014). Intranasal administration is one of the approaches that meets those requirements to

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Fig. 8.9  Borneol HO

Borneol

improve treatment and patient’s quality of life (van Woensel et al. 2013). Because of their physicochemical properties, monoterpenes may be suitable for intranasal administration to reach tumors of the central nervous system such as gliomas. The presence of gliomas can affect the integrity of the BBB. Although this could help drug delivery to the brain, it may allow the entry of neurotoxic molecules and thereby raise inflammation that promotes glioma progression and resistance (Lee et al. 2009).

Borneol Borneol (Fig. 8.9) is a monoterpenoid found in medicinal plants and, like POH, it possesses a PSA of 20.2 Å2 and an x-Log P of 2.7 (Table 8.1). It is widely used in traditional Chinese medicine combined with gardenia for stroke treatment (Xu et al. 2014). Some studies have shown that borneol can improve the nasal (Lu et al. 2011) and gastrointestinal bioavailability of drugs (Shen et al. 2011), thus accelerating the opening of the BBB (Yu et al. 2013) and enhancing the distribution of drugs in the brain tissue (Li et al. 2012; Cai et al. 2008; Dai et al. 2009). Furthermore, an in vitro model of the BBB has also indicated that borneol increased geniposide transport in the BBB, and this effect may be attributed to the disassembly effect on tight junction integrity (Chen et al. 2013).

Perillyl Alcohol The clinical application of POH in several chemotherapeutic agents against glioma is hampered by adverse effects following oral administration (Azzoli et al. 2003). Recurrent tumors are usually resistant to TMZ. Once GBMs become resistant to TMZ, there are very limited treatment options available. With a PSA of 20.2 Å2 xLog P of 2.7 (Table 8.1), intranasal POH has been very effective and well tolerated with minimal systemic side effects and only local irritation to the nasal mucosa (Da Fonseca et al. 2006; Da Fonseca et al. 2009; Da Fonseca et al. 2011). POH presents antitumoral activity against gliomas (Fernandes et al. 2005), including several TMZ-resistant ones, without significantly affecting normal cells. POH also acted as a chemosensitizing agent, enhancing the cytotoxicity of TMZ (Cho et al. 2012). The successful association of both compounds led researchers to develop a novel conjugated compound, called TMZ–POH (T–P), with promising in vivo and in vitro results (Chen et al. 2014).

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Monoterpenes, Efflux Pumps, and Tumor Resistance Tumor resistance to chemotherapy is a multifactorial condition involving several molecular pathways, and one of the most well-characterized resistance effectors is the overexpression of efflux pumps such as MDR-1 (Pgp) and MRP1 (ABCC1) (Juliano and Ling 1976; Cole et al. 1992). These transmembrane glycoproteins transport a wide array of xenobiotics and drugs in both tumor and normal tissues (Holland et al. 2003). Once present in tumor cells such as gliomas (de Faria et al. 2008) and the BBB (Wanek et al. 2013), the transmembrane glycoproteins constitute a double challenge to brain tumor chemotherapy. De Faria et al. 2008 investigated the expression of Pgp and MRP1 in 50 gliomas using immunohistochemistry. Compared with Pgp, MRP1 positivity was observed in the highest percentage of grade IV glioma samples, whereas grade II gliomas exhibited a greater number of Pgp-positive samples compared with grades III and IV. The hydrophilic nature of several anticancer drugs makes them suitable for transport by the efflux pumps, and this established the correlation between the overexpression of efflux pumps and tumor resistance to chemotherapy (Wong et al. 2014). In contrast, it was already shown that several terpenes such as triterpenes and diterpenes are not substrates for these pumps and, in fact, can even partially block their activities (Wink et al. 2012). However, to date, there are quite a few studies on the direct interaction of monoterpenes and efflux pumps. α-Pinene, Citronellol, Citronellal, and 1,8-cineole α-Pinene is not a substrate for Pgp in Caco-2 cells overexpressing this protein (Green et al. 2006). The same was observed by Yoshida et al. 2006. Citronellol, citronellal (Fig. 8.10), geraniol, and 1,8-cineole isolated from Zanthoxyli Fructus were able to inhibit the efflux of digoxin mediated by Pgp in Caco cells in a dosedependent manner (Yoshida et al. 2005). In 2006, Yoshida expanded this work to evaluate other monoterpenes such as (−)-β-pinene, terpinolene, α-terpinene, ( S)(−)-β-citronellol, DL-citronellol, ( R)-(+)-citronellal, and citronellal. These monoterpenes exhibited activity similar to PSC-833, a potent and specific inhibitor of Pgp (Boesch et al. 1991). It is important to elucidate whether these monoterpenes interact directly with the pump or if there is competitive inhibition. Fig. 8.10  Citronellal and citronellol

O

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Regarding MRP-1, among several factors, the PSA of monoterpenes may avoid their transport by these proteins (Fernandes and Gattass 2009). Molecules with a PSA greater than 140 Å2 are believed to have a low capacity for penetrating cell membranes, whereas those with a PSA ≤ 60 Å2 are easily absorbed. The TPSAs of several monoterpenes with antitumor activities are below 60 Å2 (Table 8.1), theoretically prompting them to pass the BBB and avoid tumor resistance mediated by efflux pumps. ( R)-(+)-Citronellal, ( S)-(−)-β-citronellol, α-terpinene, terpinolene, and (−)-β-pinene did not interfere with other efflux pumps as MRP2 and BCRP did (Yoshida et al. 2008). Even though there are few studies on this subject, it is known that these pumps generally transport hydrophilic compounds, and the physicochemical properties of monoterpenes are one of the features that make them unsuitable for transport by resistance proteins in tumors and a valuable choice to treat multidrug-resistant tumors.

Enhancement of Antineoplastic Activity of Drugs by Monoterpenes Even when not possessing antitumor activity by themselves, monoterpenes can enhance or promote the activity of other monoterpenes or the activity of other compound classes in several types of cancer. This synergic effect has been shown to be consistent in several studies for antitumor properties and has also been simulated in silico (El-Shemy et al. 2013). The study of synergism among monoterpenes generally involves an evaluation of essential oils with known compositions, while this interaction among isolated monoterpenes is less frequently assessed. One of the few examples is the synergism between limonene and linalyl acetate, which separately do not affect neuroblastoma cells, but their combination induces apoptosis with an approximately 50 % enhancement of activity (Russo et al. 2013). Another association of isolated linalyl acetate with camphor and terpeniol enhances growth inhibition and induced apoptosis in the human colon cancer cell lines HCT-116 (p53 + / + and p53−/−) and had no effect on the growth of the normal human cell line; furthermore, this combination was able to induce cell cycle arrest (Itani et al. 2008).

Synergism and Complex Synthesis Involving Monoterpenes and other Compound Classes The efficiency of several of the anticancer drugs already approved for clinical use is hampered by the high toxicity of these drugs, which imposes dose limits on the treatment and leads to poor patient quality of life, mainly because of the strong side

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effects of the antitumor drugs (Tallarida 2011). The use of the synergic interactions in compounds is an important strategy to improve treatment efficiency without increasing the dose.

Monoterpenes and Metal Derivatives Membrane transporters are responsible for both cisplatin influx [copper transporter-1 (Ctr1), copper transporter-2 (Ctr2), organic cation transporter-2 (OCT2)] and efflux (p-type adenosine triphosphatases—ATP7A and ATP7B). Once inside the tumor cell, the molecular basis of the cisplatin mode of action involves induction of the DNA damage response, which leads to apoptosis following cell cycle arrest (Jamieson and Lippard 1999; Cohen and Lippard 2001). Cisplatin-resistant cells often have reduced intracellular platinum accumulation following drug exposure (Lu and Chao 2012). The generation of conjugated metal-monoterpene complexes is an interesting way to take advantage of this physicochemical property, similar to the pharmacological properties of monoterpenes, by adding another route to achieve tumor cell death. A strategy to overcome resistance to platin derivatives involves association with monoterpenes to raise the platin intracellular concentration. Menthol was reported to partition from the aqueous phase into various types of membranes resulting in their expansion and increased fluidity and permeability, which may enhance the transmembrane transport of drugs. Additionally, it is known that menthol activates the Ca2+-permeable channel TRPM8, which may induce tumor cell death. Schobert et al. 2007 described the generation of menthol–platin complexes that take advantage of the activities of both, and in fact, the conjugation enhanced the platin uptake by resistant tumors and cell death. Another study characterized the antitumoral effect of the cisplatin–monoterpene conjugate and found that the complex was more effective in the treatment of cisplatin-resistant tumors through the induction of apoptosis involving caspase-8 and independent of p53 (Biersack et al. 2011). Another monoterpene–metal combination already evaluated is the ruthenium complexes that possess anticancer and antimetastatic properties; furthermore, depending on the combination, they may be less toxic to normal cells (Grau-Campistany et al. 2013). Complexed with the monoterpene cymene ([Ru(η6-p-cymene) Cl2{Ph2P(CH2)nS(O)xPh-κP}]), cytotoxic activity was observed against several tumor cell lines including melanoma, anaplastic thyroid tumors, head and neck tumors, breast, and colon cell lines. These complexes possessing the ruthenium(II)cymene nucleus exhibited activity higher than cisplatin in MCF-7 breast cancer (Ludwig et al. 2012). Despite the effectiveness of these complexes, it was only recently shown that, in addition to targeting the DNA of chromatin, they can also preferentially form adducts on the histone proteins (Adhireksan et al. 2014). Monoterpenes are also able to interact with platin derivatives to synergistically enhance antitumoral activity without complex formation. The monoterpenes POH (Yeruva et al. 2007; Yeruva et al. 2010) and PA (Yeruva et al. 2007) synergize with

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cisplatin to induce cell cycle arrest and apoptosis through bax, p21, and caspase-3 activity.

Monoterpenes and Anthracyclins Anthracyclines, such as doxorubicin, have been widely used to treat hematological malignancies, so as solid tumors. However, its cardiotoxic effects ranging from alterations of the myocardial structure and function to severe cardiomyopathy and heart failure have limited its clinical use (Gammella et al. 2014). Although there is no clinical study on the combination of monoterpenes and anthracyclines, the presence of the monoterpene can enhance the activity of doxorubicin without raising the dose. Linalool promoted doxorubicin influx in tumor cells and increased the doxorubicin concentration in cells, and it is expected that linalool enhances the antitumor activity of doxorubicin (Miyashita and Sadzuka 2013). Another report showed that linalool as a single agent has weak antiproliferative effects on MCF WT (breast cancer) and its resistant counterpart; however, the apoptosis induced by doxorubicin is enhanced when sublethal concentrations of linalool are added. Interestingly, the doxorubicin concentration was fixed while the synergistic effect was dose-dependently related to the concentrations of linalool (Ravizza et al. 2008). Other combinations involving monoterpenes and doxorubicin include thymol and menthol. Thymol combinations with doxorubicin resulted in a synergistic increase in sensitivity, which were 2.56-fold and 2.19-fold in Caco-2 and CEM/ADR5000 cells, respectively, whereas menthol induced a synergistic increase in sensitivity of 2.07fold and 1.42-fold, respectively, in the same model (Eid et al. 2012). The synergistic effects involving efflux pumps may be related to competitive inhibition of the ABC transporter activity with the monoterpene acting as a substrate for the efflux pump (Wink 2012). However, as previously described, because of the lipophilic properties of monoterpenes, they can influence the permeability of the cell membrane, thus increasing the bioavailability of doxorubicin.

Monoterpenes as Radiosensitizers Radiotherapy is one of the treatment options for locally or regionally advanced glioma, prostate, breast, and a variety of solid tumors, but radioresistance of several of them is a practical limitation of radiotherapy (Harada 2011). Radiotherapy induces double strand breaks in DNA, raises the levels of reactive oxygen species, cell arrest, and apoptosis (Han et al. 2007; Begg et al. 2011). An important feature of several classes of compounds, including monoterpenes, is the capacity to enhance the effectiveness of radiotherapy either exacerbating the effects of radiation or activating additional routes to achieve tumor regression (Samaila et al. 2004). There are

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quite few studies on the combination of monoterpenes with radiotherapy, and POH is the major subject of study. The presence of POH enhanced a dose dependent upregulation of the membrane bound form of the Fas ligand in prostate cancer (PC3 and DU145) cell lines. POH treatment did not alter the level of expression of the Fas receptor in both the cell lines (Rajesh and Howard 2003), furthermore, POH induced the same response in glioma cell lines (Rajesh et al. 2003). The efficiency of POH was also tested against radioresistant tumor cells, and although the cell lines were resistant to chemotherapic agents as cisplatin, melphalan, and doxorubicin, POH was able to reduce the clonogenic potential (Russell and Ling 2003). Thus, POH not only can be effective against radioresistant tumors but also may provide an additional route for achieving cell death after tumor irradiation.

Conclusion Even though there are few studies on monoterpenes in cancer, it is unequivocal that monoterpenes have a strong potential to impact public health by either acting in cancer prevention compounds (Crowell 1999; Miller et al. 2013) or alleviating side effects of radiotherapy in vivo and in vitro (Pratheeshkumar et al. 2010; Archana et al. 2011), as well as in the clinical treatment of the disease (Da Fonseca et al. 2011). The antitumor properties of this class involve the activation of apoptotic pathways, both intrinsic and extrinsic, the reduction of the pro-survival signals through disruption of the MAPK cascade at several levels, the impairment of cell cycle progression and the increased effectiveness of radiotherapy. Even when not possessing antitumor effects by themselves, this class can promote and enhance the effectiveness of other drugs and natural products by facilitating BBB penetration, enhancing the intracellular concentration of the drug, and enhancing the effect of other compound classes, with or without the generation of complexes. The growing knowledge of the mode of action of monoterpenes, in addition to their low toxicity, may allow the development of strategies that take advantage of its physicochemical properties, and because of the variety of pathways that monopterpenes can engage in with tumor cells, its future widespread clinical use seems quite promising and probably inevitable.

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Chapter 9

Sesquiterpenes from Essential Oils with Promising Antitumor Properties Fayaz Malik and Suresh Kumar

Introduction Cancer, a Global Health Threat Cancer is a dreadful disease and claims millions of deaths worldwide. It is the second largest killer after cardiovascular diseases. An estimate of 7.6 million deaths were caused because of cancer worldwide, accounting for 13 % of total deaths in 2008. Lung, stomach, liver, colon, and breast cancers are leading causes of mortality with, lung cancer remaining the most frequent among all cancers and accounts for about 17 % of all cancers with 23 % of total cancer deaths. Breast cancer is most frequently diagnosed cancer in women accounting for 23 % of all cancer cases and 13 % of cancer related deaths. Most of the cancer related deaths (70%) occur in developing countries (Jemal et al. 2011). According to the latest report, about 1.6 million new cancer cases and 0.58 million cancer deaths were projected in the USA in the year 2013 (Siegel et al. 2013). Cancer could be prevented by applying the knowledge and implementing various programs for cancer control. Different behavioral activities can also play an important role in the prevention of cancer as about 30 % of cancer deaths are because of five leading behavioral and dietary risks: high body mass index, low fruit and vegetable intake, lack of physical activities, consumption of tobacco, and use of alcohol. Based on the GLOBOCAN 2008 estimates, the death toll from cancer will rise to about 13.1 million by 2030 (Jemal et al. 2011). Although there is a decline in cancer deaths for the past few years, during the year 2005–2009, the cancer death rate has declined by 1.8 % per year in men and 1.5 % per year in women, and overall cancer deaths have been declined by 20 % from 1991 to 2009. Over the past 10 years (2000–2009) the largest decline in death rates

F. Malik () · S. Kumar Department of Cancer Pharmacology, CSIR-Indian Institute of Integrative Medicine, Jammu, India e-mail: [email protected] © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_9

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was reported for chronic myeloid leukemia which is 8.4 % per year, followed by stomach cancer (3.4 %), colorectal cancer (3 %), and non-Hodgkin lymphoma (3 %) (Siegel et al. 2013). Although some success has been achieved because of various efforts for the past few decades, further progress can be accelerated by applying existing cancer control knowledge across all segments of the population, with an emphasis on discovering new anticancer agents from various natural resources. There are many chemotherapeutic agents for different types of cancers, although many of these are with side effects leading to different types of health problems. So there is a need for discovering new chemotherapeutic agents with minimum side effects and health concerns. Essential oils have been found to possess anticancer activity against various cancers. They are also used as home remedies for cancer in developing countries. Here we will discuss about the anticancer properties of sesquiterpenes from essential oils.

Natural Product Essential Oils and Cancer Therapeutics Natural products have continuously been exploited for their activity against different diseases including cancer. About 60 % of anticancer drug candidates approved during 1981–2002 are either natural products or their synthetic counterparts (Torres et al. 2011). Most of the existing chemotherapies target rapidly dividing cells and cannot specifically distinguish cancer from other rapidly dividing cells. So these drugs also affect gastrointestinal and blood cells that have a high proliferative rate than other cells in the body (Edris 2007). Therefore, there is a need to develop anticancer agents that have specific targets in cancer, thereby targeting only cancerous cells without affecting the normal function of the body. Target-based therapy could be of great significance in this context. Pharmaceutical properties of many aromatic plants are partially because of essential oils (Torres et al. 2011). Essential oils are natural products having terpenes as their main constituent in addition to other non-terpene components (Edris 2007). Several essential oils from different sources have been found to possess antitumor activity against a variety of tumors. Efforts are currently made by researchers to identify the anticancer potential of different essential oils and their active constituents. Although essential oils are continuously coming up with their antitumor activity in vitro and in vivo (Misharina et al. 2013; Kpoviessi et al. 2014), most of them have not yet made it to clinical trials. So it is necessary to identify active constituents from essential oils and explore their anticancer potential and exploit the same in cancer therapy.

Role of Sesquiterpenes from Essential Oil in Cancer Sesquiterpenes from essential oils have been found to have promising anticancer potential. Different sesquiterpenes have been shown to have antitumor activity against a variety of tumors in vitro and in vivo. Despite of having antitumor

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O

Caryophyllene

Caryophyllene oxide O HO

Humulene

Zerumbone

H

Bisabolol

Fig. 9.1  Chemical structures of antitumor sesquiterpenes found in essential oils

activities against a variety of tumors and targeting important signaling pathways that play crucial roles in cancer development, most of the sesquiterpenes have not reached clinical trials probably because of the lack of detailed studies and molecular mechanisms behind their anti cancer properties. A detailed preclinical studies are required to uncover their therapeutic potential against cancer. Here we will review different sesquiterpenes from essential oils with reference to their anticancer potential. ɑ-Bisabolol is a sesquiterpene alcohol present in essential oils and has been found to have antitumor activity against different tumors. β-Caryophyllene oxide is another sesquiterpene isolated from natural medicinal plants (Ryu et al. 2012) such as guava ( Psidium guajava) and has been found to have antitumor activity against various tumors (Kim et al. 2013) ɑ-Bisabolol (Fig. 9.1) has been shown to inhibit pancreatic cancer cell lines proliferation by the inhibition of AKT signaling; ɑ-bisabolol treatments triggered the activation of early growth factor which was responsible for antiproliferative activity as the inhibition of EGR through siRNA reversed ɑ-bisabolol-induced cell death in pancreatic cancer cell lines (Seki et al. 2011). ɑ-Bisabolol has also been reported to induce apoptosis in the preclinical model of primary acute human leukemia cells by disrupting the mitochondrial pathway (Cavalieri et al. 2011). Several other studies suggested the anticancer potential of ɑ-bisabolol against different tumors in vitro and in vivo (Costarelli et al. 2010; Chen et al. 2010; Cavalieri et al. 2004). β-Caryophyllene (Fig. 9.1) is a sesquiterpene isolated primarily from the essential oils of medicinal plants such as oregano ( Origanum vulgare L.) and has been reported to show antitumor activity against a number of tumors. β-Caryophyllene has been shown to inhibit important pathways including STAT signaling (Kim et al.

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2013) and PI3K/AKT signaling (Park et al. 2011) in cancer. γ-Humulene (Fig. 9.1) is another important sesquiterpene from essential oils of Emilia sonchifolia (L.). It has also been reported for its antiproliferative potential against colorectal carcinoma by targeting different pathways (Lan et al. 2011; Satsu et al. 2004). Zerumbone (Fig. 9.1), a sesquiterpene from the edible plant Zingiber zerumbet Smith, is another sesquiterpene and has antiproliferative potential against a number of cancer types. It has been found to induce apoptosis in different tumors through the activation of caspases and by inhibiting the AKT pathway (Muhammad et al. 2013; Sobhan et al. 2013). It has also been reported to induce apoptosis in breast tumor in vitro and in vivo (Sehrawat et al. 2012). Zerumbone has been found to induce apoptosis in various tumors which, in turn, inhibit angiogenesis through the inhibition of nuclear factor κB (NFκB) pathway (Takada et al. 2005). Recent reports revealed that zerumbone can inhibit tumor angiogenesis in pancreatic and gastric tumors through the inhibition of NFκB pathway (Shamoto et al. 2014; Tsuboi et al. 2014). A variety of sesquiterpenes from essential oils have been reported for their anti-cancer activities, however a detailed studies are needed to dentify the clinical efficacy of these small molecules against cancer.

Biological Role of Various Sesquiterpenes from Essential Oils Several sesquiterpenes inhibit cancer cell proliferation by affecting different biological targets. Most of them induce cell death by targeting pathways that are frequently hyper-activated in cancer. Here we will discuss the role of some biological pathways involved in the progression of cancer that are targeted by sesquiterpenes to inhibit tumor growth.

Angiogenesis and Cancer Angiogenesis is one of the most important features of solid tumors and targeting angiogenesis is one of the promising strategies in cancer therapy. Angiogenesis involves cell proliferation, migration, matrix degradation, tube formation, and vessel maturation (Kumar et al. 2013a; Carmeliet and Jain 2000). The absence or inhibition of angiogenesis is considered as a rate-limiting step for cancer progression (Carmeliet and Jain 2000). Tumor volume was attenuated in transformed mice treated with angiogenic inhibitors; the apoptotic index was doubled in treated tumors as compared with untreated tumors (Parangi et al. 1996). Vascular endothelial growth factor receptors play a vital role in angiogenesis progression and their inhibition has been directly associated with the inhibition of angiogenesis in vitro and in vivo (Kumar et al. 2013a). There are several anti-angiogenic inhibitors that have either been approved by the FDA or are in the final stage of clinical trial (Bonifacio et al. 2012).

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Role of Sesquiterpenes in Targeting Angiogenesis Sesquiterpenes have been reported to inhibit angiogenesis which, in turn, can lead to cancer cell death (Fig. 9.2). Zerumbone has been found to inhibit tumor angiogenesis in different tumors. It has been found to inhibit the expression of antiapoptotic  and  pro-angiogenic  protein  through  the  downregulation  of  NFκB  signaling  in different cancer cell lines (Takada et al. 2005). A couple of recent studies also suggested the role of zerumbone in angiogenesis inhibition in pancreatic and gastric cancers (Shamoto et al. 2014; Tsuboi et al. 2014). ɑ-Bisabolol has been found to  have antiangiogenic properties (Magnelli et al. 2010).  β-Caryophyllene  oxide,  a  sesquiterpene isolated primarily from the essential oils of medicinal plants such as  guava ( P. guajava) and oregano ( O. vulgare L.), has also been reported for its antiangiogenic activities. It has been shown to induce apoptosis through the inhibition of angiogenesis by targeting the PI3K pathway (Park et al. 2011). A recent study  also  revealed  the  role  of  β-caryophyllene  oxide  in  the  inhibition  of  angiogenesis  and apoptotic induction, through the inhibition of the p65 subunit of NFκB leading  to anti-antiangiogenic and pro-apoptotic activities of β-caryophyllene oxide (Kim  et al. 2014).

Sesquiterpenes

NFkB PI3k/Akt/mTOR

Angiogenesis

Autophagy Apoptosis

Cell death Fig. 9.2 Sesquiterpenes inhibit the major pathways that are hyperactivated in different types of  cancers

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Miscellaneous Despite many studies about the sesquiterpenes from essential oils revealed the role of NFκB in the inhibition of angiogenesis. NFκB is a relatively old target and is itself controlled by several upstream factors like PI3K/AKT signaling pathway (Dan et al. 2008; Kloo et al. 2011), which has been found to have more control over angiogenesis than NFκB (Jiang and Liu 2009; Karar and Maity 2011; Shiojima and Walsh 2002). PI3K pathway inhibition has also been associated with angiogenesis inhibition (Harfouche et al. 2009; Okumura et al. 2012; Li et al. 2012). Therefore, identifying the role of sesquiterpenes that targets the hyper-activated kinase pathways like PI3K in the angiogenesis inhibition could be of clinical importance.

Apoptosis and Cancer Apoptosis is the characteristic of normal cells which undergo apoptosis after completion of certain life span, and normal cells also have certain checkpoints that control their proliferation, therefore it is termed as programmed cell death type 1 and other being autophagy which is termed as programmed cell death type 2. However, cancer cells do not undergo apoptosis and can proliferate continuously without any control. Therefore, apoptosis induction in cancer is considered as one of the promising strategies in the treatment of different cancer types. Apoptosis is associated with different parameters involving DNA damage, mitochondrial membrane disruption, and many more. The biochemical parameters involve activation of caspases and different death receptors. There are intrinsic and extrinsic pathways for apoptosis, the extrinsic pathway involves receptor-mediated apoptosis that involves different death receptors including TNFR1, FAS, DR4, and DR5, whereas the intrinsic pathway involves receptor-independent apoptosis that involves mitochondrial pathway and caspases (Elmore 2007).

Sesquiterpenes and Apoptosis Many of the sesquiterpenes from essential oils are known for their apoptotic potential. They induce apoptosis through the inhibition of important pathways such as PI3K, ERK, and NFκB in cancer (Fig. 9.2). ɑ-Bisabolol has been shown to induce apoptosis in a variety of tumors by targeting different pathways (Chen et al. 2010; Cavalieri et al. 2009). α-Bisabolol has also been found to exhibit synergistic effect with tyrosine kinase inhibitors, imatinib and nilotinib. The combination of bisabolol and imatinib displayed the dose reduction of each compound by up to 7.2 and 9.4 folds, respectively, while the combination of ɑ-bisabolol and nilotinib allowed the dose reduction of up to 6.7 and 5-folds, respectively (Bonifacio et al. 2012). β-Caryophyllene is a sesquiterpene isolated primarily from the essential oils of medicinal plants and is known for its proapoptotic potential against dif-

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ferent types of tumors (Amiel et al. 2012). A recent study suggested apoptotic induction by β-caryophyllene oxide, an analog of the β-caryophyllene, through the modulation of NFκB signaling pathway (Kim et al. 2014). The study demonstrated that β-caryophyllene oxide potentiated the apoptosis induced by tumor necrosis factor-α (TNF-α) and the potentiation was reversed in p65 null cells suggesting the role of NFκB in the induction of apoptosis. The PI3K/AKT pathway is one of the most frequently mutated pathways in cancer and is currently one of the most important targets in cancer therapy (Engelman 2009). β-Caryophyllene oxide has been reported to induce apoptosis through the inhibition of PI3K pathway (Park et al. 2011). γ-Humulene is a sesquiterpene from essential oils of E. sonchifolia (L.) DC, and has been identified for its pro-apoptotic activities mainly against colorectal carcinoma. γ-Humulene has been shown to activate both extrinsic and intrinsic pathways of apoptosis in colorectal cancer (Lan et al. 2011; Lan et al. 2012). Zerumbone has been shown to have promising anticancer activities against a variety of cancers. Various studies revealed the role of zerumbone as a potent apoptotic inducer in cancer. Zerumbone induces apoptosis in pancreatic and cervical cancers by modulating p53 signaling (Zhang et al. 2012). Reactive oxygen species (ROS) generation has long been associated with the induction of apoptosis (Malik et al. 2007). Zerumbone-potentiated TRAIL (TNF-related apoptosis-inducing ligand) induced apoptosis through the induction of ROS generation in different cancer cell lines while antioxidants pretreatment reversed the zerumbone-potentiated apoptosis (Yodkeeree et al. 2009). Zerumbone has also been reported to induce apoptosis in skin, colon, and lung tumors in vivo (Kim et al. 2009; Murakami et al. 2004).

Miscellaneous Taken together all these evidences suggest that sesquiterpenes from essential oils have the ability to induce apoptosis in a variety of tumors. Moreover, some of these can potentiate or synergize the pro-apoptotic potential of various chemotherapeutic agents. A detailed per-clinical studies are required to explore the individual or combinatorial anticancer potential of these molecules for their therapeutic value.

Autophagy, Cancer and Sesquiterpenes Autophagy refers to the self-digestive process which involves lysosomal degradation of cytoplasmic organelles and proteins, and by doing this autophagy maintains cellular homeostasis by contributing toward the organelle and protein turnover. Autophagy is also termed as programmed cell death type 2. Two different modes of autophagy have been identified depending upon their pathways, macroautophagy and microautophagy. In microautophagy, there occurs engulfment of cytoplasmic organelles on lysosomal surface by invagination, protrusion, and separation of lysosomal-limiting membrane. On the other hand, macroautophagy involves for-

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mation of double membrane vesicles that sequester surface of the cytoplasm. Recently a third type of autophagy namely “chaperon-mediated autophagy” has also been recognized, which utilizes Hsc-70 or other chaperones to get recognized by lysosomal-associated membrane protein (Kaushik and Cuervo 2008). In chaperonmediated autophagy, substrate proteins cross the lysosomal membrane and reach the lumen for their degradation. However, the role of autophagy in cancer is still very much complex whether autophagy is protective or exacerbating the disease. The relationship between autophagy and cancer depends upon tumor type, stage, and genetic context (Kimmelman 2011). There are examples where autophagy promotes the tumor progression (Carew et al. 2007; Park et al. 2008; Thorburn et al. 2009), although several other studies suggested that autophagy play an important role in killing tumor cells (Levine and Yuan 2005; Kumar et al. 2013b; Zhang et al. 2011). There are also evidences that suggest the supportive role of autophagy in apoptosis. Here we will discuss the role of sesquiterpenes from essential oils in autophagy. Autophagy activation can inhibit the process of apoptosis, and thus leads to survival of cancer cells (Kimmelman 2011). Therefore, chemotherapies that can trigger both apoptotic and autophagic cell deaths could be of great significance. The role of sesquiterpenes in autophagy is relatively less explored. Very few studies have been carried out so far to see the effect of sesquiterpenes on autophagy. A recent study on zerumbone suggested the evidence that the sesquiterpene can induce autophagy (Ohnishi et al. 2013) in cells. However there are not sufficient studies related to the induction autophagy by different sesquiterpenes from essential oils against different cancer cell lines. Therefore, studying the autophagic potential of sesquiterpenes will definetly add into the current knowledge of their therapeutic potential against apoptosis resistant cancers.

Sesquiterpenes from Essential Oils and Critical Pathways in Cancer The progression of cancer is regulated by biological different pathways. PI3K/AKT/ mTOR, NFκB, RAS/ERK/MEK, and Wnt signaling pathways are some of the important ones that regulate cancer. All of these pathways are found hyperactivated in most of the cancers. Here, we will discuss the role of sesquiterpenes from essential oils in the regulation of PI3K/AKT and NFκB signaling pathways.

Sesquiterpenes Targeting the PI3K/AKT/mTOR Pathway Currently substantial efforts are being made to identify the optimal target for specific cancer. Target-based studies have been helpful in finding new drugs against some types of cancers, for example, imatinib has been identified as a potent drug candidate against chronic myelogenous leukemia, trastazumab against breast

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cancer, and erlotinib and gefitinib against lung cancer expressing mutant epethilial growth factor receptor (Engelman 2009). Existing genetic and cancer biology studies reveal a prominent role of the PI3K/AKT/mTOR signaling pathway in many types of cancers. Many of the important proteins of this pathway are mutated in different types of cancers. For example, PTEN, an important negative regulator of the PI3K signaling, is mutated in many types of cancers, including breast cancer, melanoma, prostate, and ovarian cancers. PI3KCA and different isoforms of AKT are also found mutated in various cancers (Engelman 2009). Therefore, this pathway may be called as one of the most frequently mutated pathways in across different types of cancers an cancers and therefore is currently considered as one of the promising drug-able targets in cancer therapy. Moreover, this pathway negatively regulates both types of programmed cell death, autophagy and apoptosis. AKT is the central protein of this pathway and has been found to control many of the cellular processes, including cellular growth, differentiation, metabolism, and death of the cell. Therefore, efforts are being made to target this pathway in different types of cancers; currently many inhibitors targeting this pathway are in clinical trials for different types of malignancies. Some of the sesquiterpenes from essential oils have been found to inhibit PI3K/ AKT signaling. α-Bisabolol has been reported to inhibit pancreatic cancer cell proliferation through the inhibition of AKT signaling (Seki et al. 2011). β-Caryophyllene oxide has also been shown to inhibit PI3K/AKT signaling (Park et al. 2011). Recently Weng et al. explored the antitumor role of Zerumbone. This sesquiterpene was also bioactive against AKT in GBM8401 cells (Weng et al. 2012). There are very less studies that highlight the inhibitory effect of different sesquiterpenes on the PI3K/AKT pathway. As evidenced from many reports sesquiterpenes have great potential against the PI3k/AKT pathway and therefore, more studies are needed to explore the detailed role of sesquiterpenes in this direction.

Sesquiterpenes Targeting NFκB Signaling NFκB is a transcription factor that controls transcription and is involved in a number of cellular processes including developmental processes, immunomodulation, and cellular growth. NFκB has also been reported to play an important role in the regulation of apoptosis in cancer cells, and there are many biochemical, genetic, and clinical evidences that suggest the role of NFκB in cancer (Baud and Karin 2009). Inhibition of NFκB can lead to reduced expression of many antiapoptotic proteins, including Bcl-xL, XIAP, and cIAP1, and can promote the expression of many proliferative proteins such as cyclin D1 and IL6 (Bharti et al. 2003; Sanda et al. 2005; Dai et al. 2004; Mitsiades et al. 2002). Different studies suggested that the inhibitory activity of bortezomib is partially because of the inhibition of NFκB (Hideshima et al. 2001a; Hideshima et al. 2001b; Mitsiades et al. 2003). Sesquiterpenes have been reported to inhibit NFκB signaling in different types of cancers. NFκB perhaps is the most studied signaling pathway in case of

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sesquiterpenes-induced cell death. Different sesquiterpenes from essential oils have been found to inhibit cancer cell proliferation by targeting the NFκB pathway (Kim et al. 2013; Kim et al. 2014).

Conclusion Although a decline in cancer inflicted deaths and new cases have been observed over the past two decades but still millions of new cases and deaths are reported annually worldwide. Chemotherapy is one of the promising ways to treat cancer but it has several side effects. Therefore, efforts are made to discover target based therapeutic agents with minimum side effects. Natural products and their derivatives have been found to have anticancer potential against various cancers. Sesquiterpenes from essential oils have been found to have antitumor activities against different tumors. Sesquiterpenes have been found to inhibit the pathways that are drug-able targets in different types of cancers. Sesquiterpenes have been found to induce both apoptosis (type I programmed cell death) and autophagy (type 2 programmed cell death) through their inhibitory potential of tumor driving signaling pathways. But till date sesquiterpenes from essential oils have not reached clinical trials because of lack of detailed per-clinical studies. Sesquiterpenes from essential oils have been shown to have a combinatorial effect with other chemotherapeutic agents but again insufficient studies have been carried out in this context. Different types of cancers become resistant to chemotherapeutic drugs by adopting alternate pathways; therefore, combination of these drugs with sesquiterpenes that can overcome the resistance acquired could also be of great clinical importance in cancer therapy. Conclusively, despite of evidences of sesquiterpenes from essential oils having anticancer potential, these small molecules have not yet reached clinical trials, probably because not much attention has been given to explore their comprehensive therapeutic potential. Therefore, if studied in detail these molecules from essential oils could be of clinical significance either individually or in combination with other chemotherapeutic agents.

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Chapter 10

Antitumor Phenylpropanoids Miriam Teresa Paz Lopes, Dalton Dittz Júnior and Fernanda de Oliveira Lemos

Introduction Cancer treatment, especially for solid tumors, is carried out with interventions such as surgery, radio-, chemo-, hormone-, and immunotherapies, which can be used separately or combined, depending on the tumor stage. Surgery and radiation are regarded as local therapies, and for well-delimited tumors, the first one is considered curative. With respect to systemic treatment in an attempt to eliminate tumor cells that are adjacent to the primary tumor (invasion), in the circulation or proliferating at remote sites (metastasis), cytotoxic chemotherapy are mainly used. Immunotherapy techniques have been developed, for which own defense stimulus such as interferon-α, interleukin-2, or even transfer of cytotoxic T lymphocytes (LTCD8) that can reduce tumor cells are used, decreasing the residual risk of recurrence and metastasis. Despite promising treatment, immunotherapy is still a co-adjuvant therapy, especially used to destroy residual cancer cells after surgery or other treatment (Kruger et al. 2007; Banchereau 2008). Also as an alternative, hormones/antihormones (e.g., tamoxifen and raloxifene or flutamide and cyproterone, competitive inhibitors of estrogen or androgen receptors, respectively) are used when the tumor is derived from hormone-sensitive tissues and they are dependent for growth and survival, as breast and prostate tumors (Rau et al. 2005). In cytotoxic chemotherapy there are synthetic substances that act as antimetabolites or alkylating agents, as well as natural substances, such as cytotoxic antibiotics—anthracyclines and mitomycin-C, derived from plants, represented by the vinca alkaloids, taxols, and podophyllotoxins (Almeida et al. 2005). Antitumor activities of several compounds isolated from nature, such as from marine organisms (Sawadogo et al. 2013) and microorganisms (Zhang 2002), have

M. T. Paz Lopes () · D. Dittz Júnior · F. d. O. Lemos Department of Pharmacology, Federal University of Minas Gerais, Belo Horizonte, Brazil e-mail: [email protected] © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_10

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been studied. However, the prevalence of molecules derived from plants for this purpose is unquestionable. Irrespective of origin, the different classes of chemotherapy drugs affect the cell proliferation by different mechanisms of action (Almeida et al. 2005). There is a great difficulty in finding targets of selective action because of similarities between tumor and normal cells. This treatment, which is commonly accomplished by association of different chemotherapeutic agents, provides primary tumor elimination. However, it does not show satisfactory anchoring effects of tumor cells to distant tissues and metastases development (Zhang 2002). In that perspective, knowledge of tumor invasion and metastatic processes has been of great value to better define targets of chemotherapeutic agents. The tumor invasion involves (i) the adhesion of tumor cells to basement membrane (extracellular matrix—ECM) by specific receptors (integrins) to membrane elements such as fibronectin, laminin, collagen, and vitronectin, (ii) the ECM digestion by proteolytic enzymes, especially metalloproteinases family, and (iii) migration of tumor cells through the digested ECM (Liotta 1986). The metastatic capacity of tumor cells depends on angiogenesis, the process by which the tumor induces the formation of new blood vessels by increasing vascular endothelial growth factor (VEGF) and other cytokines, as well as cell motility of endothelial cells, for angiogenesis, and of tumor cells (Friedl and Gilmour 2009). The inflammatory process, characterized by modulation of mediators, in the same way, is directly linked to the metastatic process (Vendramini and Carvalho 2012). Therefore, the search for drugs that may overcome one or more failures of the current chemotherapeutic arsenal is incessant. Few are the drugs, or drug candidates, which are emerging as antimetastatic agents. There are basically inhibitors of angiogenesis (Thalidomide and some azole) and of metalloproteases, such as marimastat (Wong et al. 2013). Another important therapeutic approach is the prevention of cellular changes that can lead to malignant transformation and, consequently, tumor formation. Carcinogenesis is a multistep process, accompanied by accumulated genetic alterations in somatic cells. These alterations (mutations) include the up-regulation of proto-oncogenes and down-regulation of tumor suppressor genes (Kahlos et al. 2010). The result of these mutations can be observed in the cell transformation by an increase of cellular turnover, loss of DNA repair enzymes, and impairment of antioxidant signaling network. It is extensively described in the literature that reactive oxygen species (ROS) production is also among the major causes and/ or mediators of carcinogenesis, such as that occurring in chronic inflammation. In general, oxidative stress contributes to the initiation of cell malignancy and cancer progression, causing genomic instability (Weinberg and Chandel 2009; Weyemi and Dupuy 2012). Considering the diversity of compounds in the plant kingdom, and that many of them participate in the processes of plant protection and defence against insects and other pests, there is a search for compounds that are able to exercise any of these activities in mammalian cells. Among these, we have the phenylpropanoids that are

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found throughout the plant kingdom and serve as precursors for a series of natural polymers, which provide protection against ultraviolet light, defence against herbivores and pathogens, and mediate plant−pollinator interactions by pigmentation and floral aroma compounds (Hahlbrock and Scheel 1989). Phenylpropanoids are a class of organic compounds derived from the junction of the phenyl group (aromatic ring) and a three-carbon side chain (propyl group), which are synthesized from phenylalanine in the first step of biosynthesis. In the scientific literature reports, different phenylpropanoids with diverse biological and/ or pharmacological properties can be found. Among these may be mentioned cytotoxic or antitumorigenic effects. In the following section, information related to the cytotoxic and antitumor activities, including the action against invasion or angiogenesis, will be discussed. On the other hand, some of these phenylpropanoids (Fig. 10.1) can act as anticarcinogenic and chemopreventive agents.

OAc CH2

MeO

OMe

OMe

OH

Eugenol

Methylchavicol

AcO

Anethole

1'-Acetoxychavicol acetate

OMe

O MeO

H

O

OMe

O

MeO OMe

β -Asarone

O

MeO

Asaraldehyde

O

O

MeO

O MeO

Cinnamic acid

O

O OH

Cinnamaldehyde

Safrole

O

Dillapiole

Fig. 10.1  Chemical structures of phenylpropanoids reported in this chapter

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Anethole Anethole (1-methoxy-4-(1E)-1-propenylbenzene) occurs naturally as a major component of essential oils in fennel and star anise, and is also present in numerous plants such as dill, basil, and tarragon. The trans isomer is, by far, more abundant (> 99 %) than the cis isomer in natural oils. It is widely used as a popular aniseed flavoring agent in a variety of confectioneries and in alcoholic and nonalcoholic beverages, and has been used as an important ingredient of herbal medicines for thousands of years. Anethole was not found to be potently toxic in genotoxic, mutagenic, and immunotoxic studies and in some short- and/or long-term dietary studies (Nakagawa and Suzuki 2003). Anethole is metabolized along the three pathways, namely o-demethylation, o-hydroxylation followed by side-chain oxidation, and epoxidation of the 1,2-double bond (Marshall and Caldwell 1992). The cytotoxicity and estrogen-like effects have been described through biotransformation of anethole in the hydroxylated intermediate (4OHPB). A comparative analysis of anethole and its metabolites showed that 4OHPB (4-propenylphenol) was more toxic than the parent compound and 4MCA (4-methoxycinnamic acid) when MCF-7 cells were cultured with 10−4 M of each compound for 6 days (Nakagawa and Suzuki 2003). The metabolic intermediate anethole epoxide is responsible for the cytotoxicity of anethole in isolated hepatocytes in suspension. It presents mutagenic effect, evaluated by Ames test, and induces hepatoma and/or skin papillomas (Nakagawa and Suzuki 2003; Kim et al. 1999).

Asarone and Asaraldehyde Asarone (2,4,5-trimethoxy-1-allyl phenyl) is an ether present in α( trans) and β( cis) forms, being found as the major component in plant rhizomes of some Acorun and Asarum species. Acorus gramineus (Araceae), which contains large amounts of βasarone, has ethnopharmacological use in different symptoms most related to the central nervous system, such as learning and memory improvement, neuroprotection, sedation, and analgesia. Besides this, it is used for stomach disorder treatment, as an antibacterial agent, and for the extermination of insects (Park et al. 2011). Few data are available on the cytotoxic activity of asarone forms in tumor cell lines, such as possible antitumor activity. In addition, there are reports showing that these phenylpropanoids can be considered mutagenic, genotoxic and, therefore, carcinogenic (Hasheminejad and Caldwell 1994; Kim et al. 1999). One of these research showed that isolated β-asarone and asaraldehyde from the MeOH extract of A. gramineus rhizome, among other compounds, have low cytotoxicity with IC50 > 30 µM tested against four human tumor cells (A549, SK-OV-3, SK-MEL-2, and HCT15; Park et al. 2011). Another study found high cytotoxicity, on HepG2 cells, to α- and β-asarone, being the first compound more toxic than the latter. On the other hand, investigation of the genotoxicity using the micronucleus

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assay in the HepG2-cell system showed that after metabolic activation by a liver microsomal preparation, only β-asarones was able to induce micronuclei at concentrations higher than 50 μg/mL (Unger and Melzig 2012). By mechanisms distinct from those which promote cell death, asarone forms may favor increased cytotoxic activity of chemotherapeutic agents. Both α- and β-asarones increased vincristine cytotoxic activity on Caco-2 cells, detected by the increase of uptake and the decrease of efflux of rhodamine, in a concentrationdependent manner. A possible action mode is described: inhibition of activity and expression of P glycoprotein, a known efflux pump, responsible for resistance to various chemotherapeutic agents (Meng et al. 2014).

Acetoxychavicol Acetate and Methylchavicol Methylchavicol (estragole) and acetoxychavicol acetate forms are found in oils of species Ocimum basilicum and Languas galanga, respectively, plants whose parts are used as condiments. The authors describe acetoxychavicol acetate as a potential antitumor agent, since this compound obtained by in vitro assay, but not some derivatives, was able to inhibit the activation of tumor promoter-induced Epstein– Barr virus, at a concentration of 1.3 µM for 50 % inhibition (Kondo et al. 1993). Furthermore, this phenylpropanoid is an inhibitor of xanthine oxidase (Noro et al. 1988), indicating that it may exhibit antitumor activity by inhibiting the generation of anions during tumor promotion (Kensler et al. 1989). In a comparative study among phenols, furan derivatives, and oxides, isolated from essential oils, methylchavicol was not cytotoxic to HeLa cells (Stojcev et al. 1967). Nevertheless, methylchavicol has been described and registered with the regulatory organs and scientific committees as both genotoxic and carcinogenic agents (Berg et al. 2011). It is noted that the toxic effects are achieved with amounts found in food supplements based on plant extracts.

Cinnamaldehyde Cinnamaldehyde is a principle and a bioactive compound isolated from leaves of Cinnamomum cassia Presl. (Lauraceae) and is also the main precursor of phenolics metabolism pathway (Ng and Wu 2011; Bemani et al. 2012; Chuang et al. 2012). Studies have demonstrated that cinnamaldehyde has anti-tumorigenic effects (Ka et al. 2003; Shin et al. 2006). In human hepatoma cells (HepG2 and Hep3B), cinnamaldehyde has an IC50 of 0.3 mM (Stammati et al. 1999) and modulates the inhibitory effect on growth and proliferation (Chuang et al. 2012; Ng and Wu 2011), partly by promoting apoptosis. In addition, the ability of cinnamaldehyde to induce growth arrest was verified by the observation that it significantly decreased the protein levels of cyclin D1 and PCNA but increased the protein levels of p27Kip1

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and p21Waf1/Cip1. Activation of the JAK2–STAT3/STAT5 signaling pathway was markedly blocked by cinnamaldehyde. On the other hand, the specific JAK2 inhibitor significantly enhanced the effect of cinnamaldehyde, inhibited cellular mitogenesis (Chuang et al. 2012). Cinnamaldehyde derivatives, 2′-hydroxycinnamaldehyde (HCA) and 2′-benzoyloxycinnamaldehyde (BCA), are reported as antiangiogenic and immunomodulatory substances. When BCA or HCA was administered to C57BL/6 J mice, from 3.5 months of age to 6 months of age (10 weeks), hepatocellular carcinoma formation was delayed. Results suggest that BCA could be a chemopreventive agent against hepatocellular carcinoma, especially H-ras induced by immunostimulatory function of BCA. The reason why HCA did not show a significant chemopreventive effect in vivo compared to BCA could be explained by its smaller effect on T-cell proliferation and inhibitory effect on B cells under our experimental conditions (Moon et al. 2006). In human promyelocytic leukemia HL-60 cells, cinnamaldehyde induces depletion of intracellular thiols, unbalances the redox status, and then allows ROS to mediate a mitochondrial permeability transition, cytochrome c release into the cytosol, caspase-9 processing, activation of caspase-3, and DNA fragmentation (Ka et al. 2003). However, in HCT 116 cells, cinnamaldehyde inhibited thioredoxin reductase and induced Nrf2 (Transcription factor NF-E2-related factor 2), which plays a pivotal role in coordinating the phase II response through its binding and activation of the common antioxidant responsive element (Chew et al 2010). In K562 cells (human myelogenous leukemia line), cinnamaldehyde induces apoptosis of human myeloid leukemia K562 cells, upregulates Fas/CD95 expression, decreases mitochondrial transmembrane potential (∆ψm) in treated K562 cells, and synergizes the cytotoxicity of CIK cells to K562 cells. These data suggest cinnamaldehyde may trigger the extrinsic or Fas-mediated apoptotic pathway in K562 cells as well as the intrinsic or mitochondrial-mediated apoptotic pathway (Zhang et al. 2010). In human PLC/PRF/5 cells, cinnamaldehyde treatment inhibited PLC/PRF/5 cell proliferation and caused cell cycle arrest in the S phase. Cells treated with cinnamaldehyde exhibited an induction of Bax, a decrease in Bcl-2 and Mcl-1 proteins, an activation of caspase-8, and cleavage of Bid. These events consequently led to cell death. Recent evidence indicates that the MAPK family protein kinases, JNK and p38, are important mediators of apoptosis induced by a variety of stress-related stimuli. These mediators are also activated and phosphorylated after cinnamaldehyde treatment in a time-dependent pattern (Wu et al. 2005).

Cinnamic Acid Cinnamic acid, a major constituent of Cinnamomum cassia, has been shown to possess antioxidant, anti-inflammatory, anticancer, and other activities (Ng and Wu 2011; Hoi-Seon et al. 2004; Ekmekcioglu et al. 1998). In HepG2 cells, hepacel-

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lular carcinoma, cinnamic acid exhibits inhibitory effects on the growth with an IC50 value of 34.20 +/− 0.99 μM, an intermediate value among others derived from Cinnamomum cassia (Ng and Wu 2011). Cinnamic acid also inhibits proliferation and DNA synthesis of Caco-2 cells (colorectal adenocarcinoma). The inhibitory concentration of cinnamic acid causing a 50 % reduction in Caco-2 cell proliferation (IC50) was between 4.0 and 5.0 mM. This compound seems to affect carcinoma cells and not normal cells like fibroblasts, where 20.0 mM CINN was necessary to cause an IC50 (Ekmekcioglu et al. 1998). Although it has cytotoxic activity against Hep G2 and Caco-2 cells, cinnamic acid did not exhibit cytotoxic activity against SK-OV-3, XF-498, and HCT-15 (ovarian adenocarcinoma, glioma, and human colorectal adenocarcinoma, respectively) tumor cells, as its precursor, cinnamaldehyde (Hoi-Seon et al. 2004).

Dillapiole Dillapiole is described as the major essential oil component (74–88.4 % yield) from Piper aduncum L. var aduncum and cordulatum (Gottlieb et al. 1981), besides being found in other species of Piperaceae. Leaf alcoholic extracts of these Piperaceae show interesting insecticidal properties against Aedes atropalpus L., Aetalion sp., or other agricultural pests (Bernard et al. 1995). However, the P. aduncum oil is considered, among the class of xenobiotic agents, to be less toxic (Sousa et al. 2008). In the single report on the cytotoxic activity of pure dillapiole (Parise-Filho et al. 2012), this compound shows low toxic effects on normal 3T3 fibroblast cells at lower concentrations (6–12.5 µM), but at higher concentrations it can induce significant cytotoxic effects (IC50 = 22 µM).

Eugenol Eugenol (4-allyl-2-methoxyphenol) is a biologically active phenolic component extracted specially from cloves species ( Syzigium aromaticum, Eugenia aromaticum, or Eugenia caryophyllata). Further aromatic plants like Cinnamomum tamala, Myristica fragrans, Melissa officinalis, Ocimum basilicum, Ocimum tenuiflorum, Illicium anisatum, and Cinnamomum verum also contain eugenol (Lee and Shibamoto 2001). It appears as a clear to pale yellow oily liquid that is generally well soluble in organic solvents and sparingly soluble in water. Different pharmacological activities have been described for eugenol, for example, antiseptic, antibacterial, analgesic, antiviral, apoptosis-inducing, cytotoxic, antioxidant, antiangiogenic, antiproliferative, anti-inflammatory, and antiinvasive properties (Fujisawa et al. 2002; Rasheed et al. 1984; Kaur et al. 2010; Park et al. 2005; Okada et al. 2005; Hemaiswarya and Doble 2009).

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Regarding the antitumor activity, eugenol shows cytotoxic and antiproliferative activities in several tumor lines (Jaganathan and Supriyanto 2012). In P-815 (murine mastocytoma), K-562 (human chronic myelogenous leukemia), CEM (acute T lymphoblastoid leukemia), MCF-7 (human breast adenocarcinoma), and its counterpart resistant to gemcitabine (MCF-7 gem), the IC50 values range from 0.09 to 0.87 uM, measured by MTT metabolism. However, this component did not alter the cell cycle (Jaafari et al. 2012) as opposed to that observed in different melanoma lineages, that eugenol blocked cell cycle progression in the S phase and, because of it, induced apoptosis (Ghosh et al. 2005). Studies performed in the LNCaP cell line (human prostate cancer androgen responsive) and PC-3 (androgen independent) also demonstrated that eugenol promoted cell arrest in G1 and G2/M (Ghosh et al. 2009). In human hepatoma (HepG2—cell with some P-450 activity) and fibroblast lineage (HFF—cells which lack P-450 xenobiotic metabolizing capacity), eugenol promoted approximately equivalent cytotoxicity in a micromolar order (IC50 0.26 mM), measured by Neutral Red assay. At the same time, when a hepatic S-9 microssomal fraction is added to the test system, the cytotoxicity was increased, especially in HFF cells. It suggests that eugenol metabolites are more toxic than itself (Babich et al. 1993). These observations agree with the literature that shows eugenol metabolism leading to the formation of cytotoxic compounds. These metabolisms are especially carried out by two pathways, a peroxidation reaction and a reaction catalyzed by P-450 microsomal enzymes (Thompson et al. 1990; Thompson et al. 1989). A selective cytotoxic activity of eugenol is also described in the literature. In a study, eugenol inhibited the viability of HeLa cells (human cervical carcinoma cell line) in a concentration-dependent manner, with IC50 of 500 µM. Additionally, the microscopic examination of cells exposed to eugenol showed characteristic rounding off of dying cells, indicating that the cell death induced by eugenol is through the apoptotic pathway. On the other hand, the researchers observed that eugenol treatment showed no significant effect on cell viability of normal lymphocytes (Hussain et al. 2011). Considering the antioxidative activity of eugenol, more studies were performed to elucidate its mechanism of action. For it, HSG cell lineage (human adenocarcinoma submandibular gland cell line) was exposed to eugenol and evaluated as cytotoxicity and reactive oxygen species (ROS) production. An IC50 value was determined, but it was observed that this concentration did not produce ROS (Fujisawa et al. 2004; Atsumi et al. 2005). However, eugenol cytotoxicity was enhanced when this compound was exposed together with stress oxidative agents (H2O2 or visible light irradiation). Under these conditions, an ROS increase was observed when the cells were exposed at a concentration less than 500 µM, while the ROS amount decreased at high concentration (> 500 µM) (Atsumi et al. 2005). On the other hand, this ROS generation was not related to glutathione depletion, since eugenol did not alter this one content in all concentrations tested after 1 h exposure (until 1 mM; Atsumi et al. 2005). The ROS increase in cells treated with eugenol (in the presence of stress oxidative agents) was also observed in other cell lineage, for example HGF (primary culture of human gingival fibroblast). However, these ROS gain did not alter the cell viability (Atsumi et al. 2001).

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The anticarcinogenic activity is also well described for eugenol. In a skin tumor promoted by DMBA-TPA, eugenol reduced the percentage of animals with it, as well as the weight of tumor. Besides this, the survival was increased with the treatment. This eugenol anticarcinogenic activity is related to a decrease in the overexpression of H-ras and c-Myc, two products of oncogenes. Eugenol treatment also significantly increased the number of apoptotic cells when compared with the control. This effect is explained by a decrease in transcription and expression of Bcl-2 (an antiapoptotic protein) as well as an increase in the levels of p53 and caspase-3 (proapoptotics; Pal et al. 2010). In another anticarcinogenic evaluation of eugenol, it was shown that the prophylactic treatment with this compound led to a reduction in tumor multiplicity (average number of skin tumors per mouse) during initiation and promotion stage. Furthermore, eugenol pretreatment significantly delayed the onset of tumors, lowered the incidence and tumor size. The ornithine decarboxylase activity, a hallmark of tumor promotion and greatly induced during tumorigenesis, was downregulated in the eugenol pretreatment group. Besides this, glutatione and the activity of antioxidant enzymes were also recovered by eugenol, as well as the pretreatment significantly suppressed the enhancement of lipid peroxidation. Regarding the cellular proliferation, eugenol suppressed TPA-induced mitosis, which was shown by [3H]-thymidine incorporation and PCNA staining. Tumor samples from mice that received eugenol pretreatment showed an intense increase in apoptosis indexes (1.5-fold increment). This effect can be explained by an increase of p53 and p21WAF1 (cyclin-dependent kinase inhibitor) expression. Pretreatment with eugenol significantly attenuated TPAinduced expression of COX-2 and iNOS and the levels of TNF-a, IL-6, and PGE2 (proinflammatory cytokines) secreted (Liotta 1986). The last result shows the importance of eugenol anti-inflamatory activity to prevent tumor growth, which was also observed in different cell lineages, for example, HeLa (adenocarcinoma) cells. It was observed that eugenol significantly reduced COX-2 expression as well as downregulated IL-1β (a proinflammatory cytokine; Hussain et al. 2012). Eugenol also presented antitumor activity in different experimental models. For example, in Ehrlich carcinoma, 100 mg/kg of eugenol was found to be potent against ascites and solid tumors (Jaganathan et al. 2010). Studies performed with melanoma showed that eugenol inhibited anchorage-dependent and -independent growth cells that represent different disease progression stage (Sbcl2-primary melanoma; WM3211-radial growth phase; WM98-1-primary radial growth phase, radial and vertical growth phase; WM1205Lu-primary radial growth phase and vertical growth phase, metastatic melanoma). In in vivo antitumor evaluation, eugenol (125 mg/kg, intraperitoneal) resulted in significant tumor growth delay, decreased tumor size, and prevented tumor metastasis in a B16F10 xenograft model. Tumor sections of treated animals showed intense apoptosis induction. The expression of E2F family members was modulated by eugenol. This substance inhibited E2F1 transcriptional activity (a key regulator of genes involved in cell cycle progression; Ghosh et al. 2005). Considering the eugenol chemopreventive effect and accumulating reports that indicate that concurrent use of chemotherapy with chemopreventive agents may potentiate the efficacy of chemotherapy at lower doses, thus minimizing chemotherapy-induced toxicity, some studies pointed to this application. For example, a synergism was described in the cytotoxic effect with eugenol and conventional anticancer

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drugs, such as Methotrexate or Cisplatin, when tumor cells (P-815 (murine mastocytoma), K-562 (human chronic myelogenous leukemia), CEM (acute T lymphoblastoid leukemia), MCF-7 (human breast adenocarcinoma) and its counterpart resistant to gemcitabine (MCF-7 gem) were exposed to both in low concentrations (Jaafari et al. 2012). In the LNCaP cell line (human prostate cancer androgen responsive) and PC-3 (androgen independent), eugenol combined with 2-methoxyestradiol (2ME2) also presented a cytotoxic synergism, both in sublethal concentrations (Ghosh et al. 2009). Eugenol combined with gemcitabine resulted in a significant decrease in HeLa cell viability compared with either of the compounds alone (Hussain et al. 2011). Another study found increased sensitivity of cisplatin-induced cytotoxicity in the presence of eugenol by decreasing the expression of multidrug-resistance protein 2 (MRP2) (Mashima and Tsuruo 2005). Also, eugenol in combination with gammaradiation induced radiosensitization of various tumors by initiation of membrane oxidative damage and intracellular ROS generation (Baell and Huang 2002).

Myristicin Myristicin or methoxy safrole is a natural organic compound present in small amounts in the essential oil of nutmeg ( M. fragrans) and to a lesser extent in other spices such as parsley ( Petroselinum crispum) and dill ( Drymus angustifolia). This phenylpropanoid is similar to safrole and dilapiolle, presenting a methoxyl and a methylenedioxy group bonded to an aromatic ring. Myristicin is known to induce psychedelic effects like visual distortions and a state of semiconsciousness, by antagonistic action on serotonin receptor and weak inhibition of monoamine oxidase, MAO. The dosage required to achieve such effects is variable (0.15–0.25 g of powdered walnut fresh/kg of body weight), and these hallucinogenic effects may occur to 7 h after ingestion, but up to 72 h are still perceived. Myristicin has low toxicity (1–2 mg/kg body weight and LD50 > 1000 mg/ kg; Hallstrom and Thuvander 1997); however, there are several reports of its antitumor or anticarcinogenic activity. Regarding the determination of the anticarcinogenic activity, several models and methodologies have been used for this purpose. In the screening of substances or monitoring the activity of extract fractions or isolated compounds, measurement of glutathione-5′-transferase (GST) or NAD(P)H: quinone oxidoreductase (QR) activity has shown predictive value. These enzymes that belong to the detoxifying phase II enzyme system are responsible for many xenobiotic metabolisms (Jakoby and Habig 1980). Thus, any compound that promotes an increase in the activity of these enzymes may be considered a potential inhibitor of chemically induced tumorigenesis. Accordingly, the effect of myristicin (four doses of 20 mg over 1 week) on the induction of GST and QR activities for four strains of rats was described. The GST and QR activities are significantly increased in the livers, intestines, lungs, and stomachs of at least three out four strains. The study also revealed increased GST activity in the presence of myristicin, against both 1-chloro-2,4-dinitrobenzene

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(CDNB) and 4-nitroquinoline-1-oxide (4NQ), compounds with carcinogenic potential (Ahmad et al. 2009). Regarding phytochemical aspects (Zheng et al. 1992a; 1992b), myristicin appears to be a major chemical constituent of Parsley leaf oil and is responsible for the high GST-inducing activity. In this extract, myristicin and dihydromyristicin are found in fraction C. Both the compounds are two to four times more potent than the controls and did not differ in the ability to induce GSH activity in different tissues evaluated. Thus, the plant, myristicin and its derivatives show promises as useful chemopreventive agents. Still about myristicin, it has been shown to be cytotoxic and apoptosis-promoting in the human neuroblastoma SK-N-SH cells. A dose-dependent reduction in cell viability at ≥ 0.5 mM of myristicin was accompanied by apoptotic cell morphology, typical DNA fragmentation, accumulation of cytochrome c, and activation of caspase-3. This apoptosis induction provides further insight into the molecular mechanisms of myristicin cytotoxicity and its possible antitumor activity (Lee et al. 2005).

Safrole Safrole is one of the important food-borne phytotoxins found in many natural products such as oil of sassafras and spices, for example, anise, basil, nutmeg, pepper (Yu et al. 2012), and presents structural similarity to dillapiole, previously described (Gottlieb et al. 1981). Besides this, safrole is found in the essential oil of commonly used Chinese medicine. In Taiwan, about 2 million people (10 % of the population) chew betel quid, a mild stimulant comprising areca nut, slaked lime, and piper betel inflorescence. Safrole was banned by the United States Food and Drug Administration for use as flavorings and food additives in 1960 because it caused hepatocarcinoma (Chiang et al. 2011). In vivo studies have shown that safrole is metabolized by the cytochrome P-450 pathway to metabolites that are carcinogenic and ROS (Martati et al. 2012; Borchert 1973a; Borchert 1973b; Dietz and Bolton 2011). Although safrole and its metabolites are known to be carcinogenic (Daimon et al. 1998; Chen et al. 1999), some studies show the contrary picture. In human tongue squamous carcinoma SCC-4 cells, safrole induced apoptosis at 75 µM, which was accompanied with up-regulation of the protein expression of Bax and Bid and down-regulation of the protein levels of Bcl-2 (up-regulation of the ratio of Bax/Bcl-2), resulting in cytochrome c release, increased Apaf-1 level, and sequential activation of caspases-9 and -3 in a time-dependent manner (Yu et al. 2012). As in SCC-4 cells, safrole also induces a typical apoptosis by caspase-3, -8, and -9 activation in A549 human lung cancer cells (Du et al. 2006). When BALB/c mice were treated with safrole (4–16 mg/kg), the weights of body, spleen, and liver were not affected when compared with the normal mice group (Fan et al. 2012). The major metabolite of safrole, safrole-2′,3′-oxide (SAFO), causes significant dose-dependent increases in cytotoxicity, mean Comet tail moment, and micronucleated binucleated cells in human HepG2 cells. SAFO exhibited a dose- and

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time-dependent cytotoxic effect in HepG2 cells with IC50 values of 361.9 and 193.2 μM after 24 and 48 h exposure, respectively (Chiang et al. 2011).

Conclusion According to our proposal, in this chapter, we show how some of the phenylpropanoids can have cytotoxic/antitumor activity. These phenylpropanoids act by inhibiting or modulating events that occur during or after the tumor development, such as proliferation, angiogenesis, invasion, or cellular death associated or not associated with apoptosis. In the same way, some of these phenylpropanoids (e.g., eugenol, myristicin, and cinnamic acid derivatives) also exert stimulatory action on enzymes responsible for metabolizing mutagenic agents or abolish routes of oxidative stress, conditions directly involved in cell transformation (Figs. 10.1 and 10.2). There is no doubt that this natural products class deserves more effort to elucidate their potential characteristics as antitumor agent, and as a chemotherapeutic, in preventing the tumor development.

Fig. 10.2  Antitumor action mechanisms of phenylpropanoids. a Action on enzymes responsible for modulating oxidative stress and other conditions directly involved in cell transformation; and b action on events that occur during or after the tumor development

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Chapter 11

Antitumor Essential Oils: Synergy and Chemotherapeutic Interactions Rogerio Correa Peres, Carolina Foot Gomes de Moura, Flavia Andressa Pidone Ribeiro and Daniel Araki Ribeiro

Introduction The term “essential oil” was used for the first time in the sixteenth century by Paracelsus von Hohenheim, who named the effective component of a drug “quinta essential” (Guenther 1950). Currently, approximately 3000 essential oils are known, 300 of which are commercially important, especially in the pharmaceutical, agronomic, food, sanitary, cosmetic, and perfume industries (Clarke and Mullin 2008; Prabuseenivasan et al. 2006). Many studies have demonstrated the potential antitumor effects of essential oils. Tumor may be eliminated by surgery, cytotoxic drugs (chemotherapy), or penetrating irradiation (radiotherapy), or by a combination of light and phototoxic drugs (phototherapy; Korkina et al. 2009). Despite the importance of chemotherapeutic anticancer drugs, these xenobiotics lack tumor selectivity, provoking adverse effects in healthy non-tumor tissues. Among the known chemical models, prodrugs released biochemically in the tumor remain a valid solution, but have had limited success so far. A potential reason for this failure is likely related to the insufficient penetration of the drug into the tumor tissue (Korkina et al. 2009). Penetration could be improved by sensitizing the tumor tissue to the drug. Some studies demonstrate the potentiation of the chemotherapeutic effect of some drugs by essential oils. This is particularly important because it would make possible the use of lower doses of toxic agents. Several studies show antitumor activity in drug-resistant cells. Thus, the results seem very promising (Lai and Roy 2004). To the best of our knowledge, few studies have addressed the therapeutic outcomes of combinations of chemotherapeutic agents and antitumor oils and the possibility synergy between them.

D. A. Ribeiro () · R. C. Peres · F. A. Pidone Ribeiro Department of Biosciences, Federal University of São Paulo, São Paulo, Brazil e-mail: [email protected] C. Foot Gomes de Moura · D. A. Ribeiro Departament of Pathology, Federal University of São Paulo, São Paulo, Brazil © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_11

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In this chapter, we discuss the role of essential oils with antitumor properties, while focusing on synergy and chemotherapeutic interactions.

Synergy and Chemotherapeutic Interactions Induced by Essential Oils M’Barek et al. (2007) observed that the volatile essential oil of thyme was cytotoxic to a human ovarian adenocarcinoma cell line resistant to vincristine and cisplatin but not to adriamicyn. The essential oil of thyme has low cytotoxicity in normal human peripheral blood mononuclear cells. An in vivo study with tumor-bearing DBA-2 (H2d) mice showed that the injection of essential oil of thyme inhibited tumor proliferation in a dose-dependent manner while the control mice exhibited increasing tumor volumes (M’Barek et al. 2007). Thyme is the most popular medicinal plant in Morocco, and it has been used in traditional medicine for thousands of years in African and European countries, particularly in the Mediterranean basin (Lai and Roy 2004; M’Barek et al. 2007). It is reported that the essential oil of Thymus vulgaris, the most studied species of thyme, exerts antibacterial, antifungal, and antioxidant activities (M’Barek et al. 2007). Perillyl alcohol (POH) is a monoterpene alcohol present in several aromatic plants. POH is cytotoxic to a variety of experimental cancer cells in vitro and in vivo (Ahn et al. 2003). Studies demonstrate that exposing non-small-cell lung cancer cells (NSCLC, A549, and H520) to the IC50 concentrations of POH or perillic acid (PA), the major circulating metabolite of POH, sensitized them to cisplatin and radiation in a dose-dependent manner. Head and neck cancer cells can be similarly sensitized to radiation (Yeruva et al. 2007; Samaila et al. 2004). Combination of POH and methyl jasmonate (MJ), a chemical compound produced by plants in response to many biotic and abiotic stresses, was toxic to breast cancer cells in the presence of cisplatin (Yeruva et al. 2010). Similarly, lower doses of MJ sensitized cervical cancer cells to conventional X-ray and cisplatin (Milrot et al. 2013). β-Ionone (βI) is a cyclic isoprenoid and product of β-carotene degradation present mainly in grapes and wine aromatizers. Geraniol, an acyclic monoterpene and important constituent of the essential oils of ginger, lemon, lime, orange, and nutmeg, suppresses cell proliferation and/or trigger apoptosis in several tumor lines, including human Caco-2 colon adenocarcinoma cells (Mo and Elson 2004; Carnesecchi et al. 2001). Geraniol suppresses prostate cancer growth and increases docetaxel chemosensitivity in cultured cells (Kim et al. 2012). However, combinations of isoprenoids do not represent an effective chemopreventive strategy (Vieira et al. 2011). Linalol, another monoterpene alcohol found in the essential oils from many aromatic plants, potentiates doxorubicin-induced cytotoxicity and apoptosis in two human breast adenocarcinoma cell lines at subtoxic concentrations (Liu et al. 2008). Other terpenoids, such as thymol, menthol, and aromadendrene, showed synergism with doxorubicin in treating multidrug-resistant human colorectal adenocarcinoma and leukemia cell lines (Eid et al. 2012).

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The Rhizoma curcumae oil, an essential oil used in traditional Chinese medicine, induces apoptosis. Studies show that a combination of tamoxifen and R. curcumae oil markedly decreased the volume of ectopic endometrial implants in rat endometriosis models, suggesting clear synergy (Kong and Zhang 2006). Eugenol, a potential chemopreventive agent, is a component of clove present in several other spices, such as basil, cinnamon, and bay leaves. A study of cervical cancer showed that a combination of eugenol and gemcitabine induced growth inhibition and apoptosis at lower concentrations than the individual drugs. Combination index analysis showed index values < 1 indicating a strong synergistic interaction. Therefore, the combination may enhance the efficacy of gemcitabine at lower doses and minimize toxicity to normal cells. Such findings may enhance the therapeutic index for the prevention and/or treatment of cervical cancer. In addition, analysis of the expression of genes involved in apoptosis and inflammation revealed significant downregulation of Bcl-2, COX-2, and IL-1-β upon treatment with eugenol (Hussain et al. 2011). The combination of sulforaphane (SFN) and eugenol was antagonistic at lower, but synergistic at higher, sublethal doses as reflected in SFN cell cytotoxicity and apoptosis (Hussain et al. 2012). Nevertheless, the combination of 2-methoxyestradiol and eugenol inhibited the growth of prostate cancer cells, significantly reduced the expression of the antiapoptotic protein Bcl-2, and increased the expression of the proapoptotic protein Bax (Ghosh et al. 2009). It was also observed that the combination of eugenol and 5-fluorouracil increased the number of cells in the S and G2/M phases. This indicates that the combination induces apoptosis in human cervical cancer cells (Hemaiswarya and Doble 2013). Taken together, these results reveal synergism between eugenol and several different drugs. β-Elemene recently raised interest in China as a novel antitumor plant drug isolated from the Chinese medicinal herb Zedoary. Studies show that β-elemene increases cisplatin cytotoxicity and cisplatin sensitivity in drug-resistant ovarian tumor cells and in prostate cancer. Similarly, β-elemene increased cisplatin sensitivity and cisplatin cytotoxicity in small-cell lung cancer (SCLC) and carcinomas of the brain, breast, cervix, ovary, and colorectal tract in vitro. β-Elemene induced apoptosis via mitochondrial activation of the caspase-mediated apoptotic pathway, which may account for the increased anticancer potency of cisplatin (Li et al. 2010; Li et al. 2013; Zou et al. 2013a; Zou et al. 2013b). Wang et al. (2012) suggest that β-elemene can improve the effect of chemotherapy in lung cancer as an adjuvant therapy. Bao et al. (2012) also report that β-elemene could inhibit the growth of mouse hepatoma H22 tumor cells in a timeand dose-dependent manner. Liu et al. (2011) report that β-elemene induced protective autophagy and prevented human gastric cancer cells from undergoing apoptosis. Finally, β-elemene could induce apoptosis by inhibiting the Hsp90/Raf-1 complex in glioblastoma cells (Zhang et al. 2012). Thymoquinone (TQ) is a compound extracted from Black Caraway seeds of Nigella sativa. Studies show that TQ inhibited cell proliferation, reduced cell viability, and induced apoptosis (Jafri et al. 2010). TQ downregulated nuclear factor kappaB (NF-kappaB) expression and reduced the secretion of the ENA-78 and Gro-alpha

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cytokines, which are involved in neo-angiogenesis (Jafri et al. 2010). A combination of TQ and cisplatin showed synergism in NSCLC and SCLC cell lines. TQ sensitized pancreatic tumors to gemcitabine and oxaliplatin (Banerjee et al. 2009). Great synergism was observed when TQ and quercetine, a polyphenol from plants, were combined with two platinum drugs, cisplatin and oxaliplatin, in two human epithelial ovarian cancer cell lines, A2780 and its cisplatin-resistant variant A2780 (cisR) (Nessa et al. 2011). TQ improved efficacy and selectivity, and decreased resistance to doxorubicin in several cancer cell lines, such as HL-60 leukemia, 518 A2 melanoma, and MCF-7 breast carcinoma (Effenberger-Neidnicht and Schobert 2011). Several other compounds have been investigated for possible synergy with cancer drugs. Cinnamaldehyde, the organic compound that gives cinnamon its flavor and odor, potentiated the inactivating effect of cis-diamminedichloroplatinum ( cisDDP) in all phases of the cell cycle, suggesting putative synergy (Dornish et al. 1988). Conversely, ibandronate, one of the most potent bisphosphonates, inhibited the growth of prostate cancer cell lines via inhibition of farnesyl-IPP-synthase and exhibited synergy with docetaxel (Epplen et al. 2011). The combination of sorafenib (SF), a multi-kinase inhibitor, and β-ionone (BI), a precursor of carotenoids, decreased focal adhesion kinase (FAK) and Rho protein expression and increased tissue inhibitor matrix metalloproteinase (TIMP)-1 and TIMP-2 protein expression, indicating the selective cytotoxicity of this combination in human hepatoma cells (Huang et al. 2012). Overall, these results show that essential oils can increase the sensitivity of tumor cells to several chemotherapeutic agents. Further investigation is needed to confirm these effects. Acknowledgments This study was supported by CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior).

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Effenberger-Neidnicht K, Schobert R (2011) Combinatorial effects of thymoquinone on the anticancer activity of doxorubicin. Cancer Chemother Pharmacol 67(4):867–874 Eid SY, El-Readi MZ, Wink M (2012) Synergism of three-drug combinations of sanguinarine and other plant secondary metabolites with digitonin and doxorubicin in multi-drug resistant cancer cells. Phytomedicine 19(14):1288–1297 Epplen R, Stöckle M, Engelmann U, Heidenreich A, Ohlmann CH (2011) Differential effects of ibandronate, docetaxel and farnesol treatment alone and in combination on the growth of prostate cancer cell lines. Acta Oncol 50(1):127–133 Ghosh R, Ganapathy M, Alworth WL, Chan DC, Kumar AP (2009) Combination of 2-methoxyestradiol (2-ME2) and eugenol for apoptosis induction synergistically in androgen independent prostate cancer cells. J Steroid Biochem Mol Biol 113(1–2):25–35 Guenther E (1950) In the essential oil. In, vol. IV. D Van Nostrand, New York Hemaiswarya S, Doble M (2013) Combination of phenylpropanoids with 5-fluorouracil as anticancer agents against human cervical cancer (HeLa) cell line. Phytomedicine 20(2):151–158 Huang CS, Lyu SC, Hu ML (2012) Synergistic effects of the combination of b-ionone and sorafenib on metastasis of human hepatoma SK-Hep-1 cells. Invest New Drugs 30(4):1449–1459 Hussain A, Brahmbhatt K, Priyani A, Ahmed M, Rizvi TA, Sharma C (2011) Eugenol enhances the chemotherapeutic potential of gemcitabine and induces anticarcinogenic and anti-inflammatory activity in human cervical cancer cells. Cancer Biother Radiopharm 26(5):519–527 Hussain A, Priyani A, Sadrieh L, Brahmbhatt K, Ahmed M, Sharma C (2012) Concurrent sulforaphane and eugenol induces differential effects on human cervical cancer cells. Integr Cancer Ther 11(2):154–165 Jafri SH, Glass J, Shi R, Zhang S, Prince M, Kleiner-Hancock H (2010) Thymoquinone and cisplatin as a therapeutic combination in lung cancer: in vitro and in vivo. J Exp Clin Cancer Res 29:87 Kim SH, Park EJ, Lee CR, Chun JN, Cho NH, Kim IG, Lee S, Kim TW, Park HH, So I, Jeon JH (2012) Geraniol induces cooperative interaction of apoptosis and autophagy to elicit cell death in PC-3 prostate cancer cells. Int J Oncol 40(5):1683–1690 Kong Y; Zhang JW (2006) Experimental study on rat model of endometriosis treated with tamoxifen and Rhizoma curcumae oil. Sichuan Da Xue Xue Bao Yi Xue Ban 37(4):596–598 Korkina LG, De Luca C, Kostyuk VA, Pastore S (2009) Plant polyphenols and tumors: from mechanisms to therapies, prevention, and protection against toxicity of anti-cancer treatments. Curr Med Chem 16:3943–3965 Lai PK, Roy J (2004) Antimicrobial and chemopreventive properties of herbs and spices. Curr Med Chem 11:1451–1460 Li QQ, Wang G, Reed E, Huang L, Cuff CF (2010) Evaluation of cisplatin in combination with b-elemene as a regimen for prostate cancer chemotherapy. Basic Clin Pharmacol Toxicol 107(5):868–876 Liu RR, Gariboldi MB, Molteni R, Monti E (2008) Linalol, a plant-derived monoterpene alcohol, reverses doxorubicin resistance in human breast adenocarcinoma cells. Oncol Rep 20(3):625– 630 Liu J, Zhang Z, Gao J, Xie J, Yang L, Hu S (2011) Downregulation effects of beta-elemene on the levels of plasma endotoxin, serum TNF-alpha, and hepatic CD14 expression in rats with liver fibrosis. Front Med 5:101–105 M'Barek LA, Mouse HA, Jaâfari A, Aboufatima R, Benharref A, Kamal M, Bénard J, El Abbadi N, Bensalah M, Gamouh A, Chait A, Dalal A Zyad, A (2007) Cytotoxic effect of essential oil of thyme (Thymus broussonettii) on the IGR-OV1 tumor cells resistant to chemotherapy. Braz J Med Biol Res 40(11):1537–1544 Milrot E, Jackman A, Flescher E, Gonen P, Kelson I, Keisari Y, Sherman L (2013) Enhanced killing of cervical cancer cells by combinations of methyl jasmonate with cisplatin, X or alpha radiation. Invest New Drugs 31(2):333–344 Mo H, Elson CE (2004) Studies of the isoprenoid-mediated inhibition of mevalonate synthesis applied to cancer chemotherapy and chemoprevention. Exp Biol Med 229:567–585

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Nessa MU, Beale P, Chan C, Yu JQ, Huq F (2011) Synergism from combinations of cisplatin and oxaliplatin with quercetin and thymoquinone in human ovarian tumour models. Anticancer Res 31(11):3789–3797 Prabuseenivasan S, Jayakumar M, Ignacimuthu S (2006) In vitro antibacterial activity of some plant essential oils. BMC Complementary Altern Med 6:39 Samaila D, Toy BJ, Wang RC, Elegbede JA (2004) Monoterpenes enhanced the sensitivity of head and neck cancer cells to radiation treatment in vitro. Anticancer Res 24(5A):3089–3095 Vieira A, Heidor R, Cardozo MT, Scolastici C, Purgatto E, Shiga TM, L.F. Barbisan LF, Ong TP,Moreno, FS (2011) Efficacy of geraniol but not of b-ionone or their combination for the chemoprevention of rat colon carcinogenesis. Braz J Med Biol Res 44(6):538–545 Wang B, Peng XX, Sun R, Li J, Zhan XR, Wu LJ, Wang SL, Xie T (2012) Systematic review of b-elemene injection as adjunctive treatment for lung cancer. Chin J Integr Med 18:813–823 Yeruva L, Pierre KJ, Elegbede A, Wang RC, Carper SW (2007) Perillyl alcohol and perillic acid induced cell cycle arrest and apoptosis in non small cell lung cancer cells. Cancer Lett 257(2):216–226 Yeruva L, Hall C, Elegbede JA, Carper SW (2010) Perillyl alcohol and methyl jasmonate sensitize cancer cells to cisplatin. Anticancer Drugs 21(1):1–9 Zhang B, Zhang X, Tang B, Zheng P, Zhang Y (2012) Investigation of elemene-induced reversal of tamoxifen resistance in MCF-7 cells through oestrogen receptora (ERa) re-expression. Breast Cancer Res Treat 136:399–406 Zou B, Li QQ, Zhao J, Li JM, Cuff CF, Reed E (2013) b-Elemene and taxanes synergistically induce cytotoxicity and inhibit proliferation in ovarian cancer and other tumor cells. Anticancer Res 33(3):929–940

Chapter 12

Dietary Essential Oils and Cancer Chemopreventive Potential Thomas Prates Ong

Diet, Nutrition, and Cancer Because of the significant health, social, and economic burden posed by cancer, preventive strategies are obviously needed and should be a priority from a public health perspective (Beaglehole et al. 2011). Evidence accumulated in the past few decades reinforces the role of diet as one of the main environmental factors modulating cancer risk (Vineis and Wild 2014). It has been estimated that 35 % of all cancer deaths in the world are due to inadequate dietary habits. As cancer development is usually a long process, there is a large window of opportunity to establish prevention strategies that should focus on several environmental factors including smoking, physical activity and, of course, food consumption (WFCR/AICR 2007; Baena-Ruiz and Salinas-Hernandez 2014). Food patterns that increase the risk of cancer include those characterized by high ingestion of meat products including red and processed meats, fats, refined sugars, alcoholic beverages, low ingestion of fruits, vegetables, and grains. On the other hand, protective patterns include high ingestion of these latter plant-based products, moderate consumption of poultry and fish, and low intake of dietary fats (BaenaRuiz and Salinas-Hernandez 2014). Emphasis has been directed toward the preventive potential of whole fruits and vegetables, as their consumption has been associated with lower incidence and mortality due to cancer in several epidemiological studies (Martin et al. 2013). It is now recommended that the general population should eat at least nine portions of these foods (Liu 2013).

T. P. Ong () Laboratory of Nutrigenomics and Programming, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo and Food Research Center (CEPID/FAPESP), São Paulo, Brazil e-mail: [email protected] © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_12

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The cancer preventive potential of fruits and vegetables has been associated with their chemical composition. In addition to vitamins, minerals, and dietary fiber, fruits and vegetables display complex sets of bioactive components. These bioactive food components (BFCs) belong to several classes, including polyphenolic compounds, sulfur-containing compounds, isothiocyanates, and isoprenic derivatives (Kim and Milner 2011). It is estimated that a regular portion of fruits and vegetables contains more than 100 different bioactive molecules illustrating the complexity of the association between diet and cancer risk (Surh 2003). Although these BFCs are not essential for body function, they have been shown to modulate several cellular and metabolic processes. They presented inhibitory effects during all phases of carcinogenesis (initiation, promotion, and progression), and this could be due to their influences on carcinogen activation and detoxification, DNA repair induction, oxidative stress, inflammation, cell proliferation, death and differentiation, angiogenesis, among others (Milner 2008; González-Vallinas et al. 2013). From a nutrigenomics perspective, BFCs-protective effects are mediated by modulation of different transcriptional programs. These molecular actions involve direct activation of transcription factors and interference with signal transduction pathways (Ong et al. 2011). As BFCs are normally consumed in the diet, present low toxicity and reduced cost, they are considered promising chemopreventive agents (Rabi and Gupta 2008). Cancer chemoprevention was first defined by Dr. Michael Sporn (Sporn et al. 1976) as the use of natural or synthetic compounds to impede, arrest, or reverse carcinogenesis at its earliest premalignant phase. Traditionally, compounds inhibiting the initiation phase of carcinogenesis are termed “blocking” chemopreventive agents. Their mechanisms of action comprise antioxidant effects, scavenging of free radicals, inhibition and induction of phase I and II xenobiotic-metabolizing enzymes, respectively, induction of DNA repair, and blockade of carcinogen uptake. In addition, compounds inhibiting the promotion phase of carcinogenesis are termed “suppressing” chemopreventive agents. They operate through inhibition of cell proliferation, induction of apoptosis in preneoplastic lesions, induction of terminal differentiation, modulation of signal transduction, and alteration of gene expression. Several BFCs present blocking and/or suppressing chemopreventive activities (Steward and Brown 2013). Dietary essential oils from fruits, vegetables, herbs, and spices represent interesting sources of BFCs with cancer chemopreventive potential. Their chemical composition is very complex and about 20–60 different compounds can be found in such oils. One of the main classes is represented by isoprenic derivatives (terpenes and terpenoids) including monoterpenes (geraniol) and sesquiterpenes (farnesol). Monoterpenes contain two isoprenic units (C10) and represent the most prevalent constituents of essential oils (90 %). Sesquiterpenes contain three isoprenic units (C15) and present several chemical structures, similarly to monoterpenes (Bakkali et al. 2008). Isoprenoids are normally consumed by humans in the regular diet in different combinations, and it has been proposed that diet modification and food extract containing these substances could be part of a dietary cancer chemopreventive strategy (Wagner and Elmadfa 2003; Rabi and Gupta 2008). Isoprenoids

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chemopreventive potential has been highlighted for several malignancies, including liver, pancreas, skin, oral, breast, and prostate cancers (Elson and Yu 1994; Crowell 1999; Mo and Elson 2004; Rabi and Bishayee 2009; Yang and Dou 2010; Thoppil and Bishayee 2011; Ong et al. 2012). Isoprenoids represent one of the most diverse class of natural products produced by plants, with more than 40,000 members identified (Rabi and Bishayee 2009). Among them, monoterpene geraniol and sesquiterpene farnesol represent promising essential oil components with cancer chemopreventive potential and will be the focus of this chapter.

Cancer Chemoprevention with Isoprenic Derivatives from Dietary Essential Oils In mammalian cells the mevalonate pathway provides precursors for the biosynthesis of cholesterol, dolichols, and ubiquinones that play a role in membrane integrity, steroid and hormone precursors. In addition, it also provides farensyl- and geranylgeranyl pyrophosphate moieties that are needed for the modification of Ras and Rashomologous (Rho) GTPase proteins involved in the regulation of cell proliferation (Fritz 2009). Normally in these cells control of this pathway is exerted by its end product cholesterol or its oxygenated derivatives oxysterols that transcriptionally inhibits the expression of the rate-limiting enzyme for mevalonate and cholesterol synthesis, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (Peffley and Gayen 2003). This negative feedback regulation of this enzyme by cholesterol is mediated by sterol regulatory element-binding protein-1 (SREBP-1), a cholesterol sensor that adjusts cell metabolism according to fluctuations in lipid levels (Bianco et al. 2012). It has also been shown that end products of the mevalonate pathway in plants, namely isoprenic derivatives, posttranscriptionally suppress mammalian HMG-CoA reductase (Elson and Yu 1994; Moreno et al. 1995; Peffley and Gayen 2003). Oral consumption of lemongrass oil (140 mg/day) that contains geraniol and farnesol lowered plasmatic cholesterol in hypercholesterolemic subjects (Elson et al. 1989). In renal cells geraniol was shown to inhibit this enzyme by attenuating its mRNA translational efficiency (Peffley and Gayen 2003). Farnesol was identified as the nonsterol component in the mevalonate pathway inducing HMG-CoA reductase degradation (Meigs et al. 1996). Increased activity of HMG-CoA reductase is a common feature of cancer cells, where its transcriptional feedback inhibition by cholesterol is lost (Elson and Yu 1994; Elson et al. 1999; Mo and Elson 2004). This results in increased production of the mevalonate pathway intermediates—farnesyl and geranylgeranyly pyrophosphates—favoring aberrant signaling of isoprenylated Ras and Rho proteins and increased proliferation of cancer cells (Fritz 2009). In addition, as cholesterol itself has been implied in the growth and development of malignancies including breast cancer, it has been suggested that targeting this molecule or its metabolism should be an important aspect to be considered in breast cancer prevention and treatment (Danilo and Frank 2012). Interestingly, tumor cells retain sensitivity to plant-de-

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rived isoprenoids and mediate inhibitory actions on HMG-CoA reductase. Inhibition of Ras proteins prenylation is a relevant strategy to impede their membrane binding to the inner face of plasma membrane and avoid the associated oncogenic signaling (Blum et al. 2008). Thus, it has been proposed that an effective cancer chemopreventive strategy would comprise inhibition of the mevalonate pathway by targeting HMG-CoA reductase with dietary isoprenoids and thus decreasing the production of farnesyl and geranylgeranyl pyrophosphates, as well as of cholesterol itself that are associated with the cancer process (Goldstein and Brown 1990; Elson and Yu 1994; Mo and Elson 2004; Ong et al. 2012). Accumulating evidence show that in addition to HMG-CoA reductase, dietary isoprenic derivatives present other molecular targets including NFκB, Cox-2, and antioxidant enzymes. This indicates that they represent versatile cancer chemopreventive agents that can influence several aspects of the cancer process.

Monoterpenes (Geraniol) Monoterpenes consumption by humans through the diet is widespread (Cromwell 1999). This particular class of isoprenoids has been shown to present blocking and suppressing cancer chemopreventive effects. Their protective effects during the initiation are mediated by modulation of carcinogen activation (inhibition of phase I enzymes and induction of phase II enzymes). In addition, their effects during the promotion phase involve inhibition of cell growth through apoptosis induction and tumor redifferentiation through different mechanisms, including interference with isoprenylation of cell growth-regulatory proteins (Wagner and Elmadfa 2003; Rabi and Bishayee 2009). The acyclic monoterpene geraniol (3,7-dimethyl-2,6-octadien-1-ol) is present in the essential oils of lemon, lime, orange, and ginger (Khan et al. 2013). Other dietary sources include carrot, nutmeg, blueberry, and blackberry (Wiseman et al. 2007). It is widely used as a common ingredient in consumer products from the flavor and fragrance industries, and its low toxicity coupled with its different biological actions (antioxidant, anti-inflammatory, and anticancer) reinforce geraniol as a promising cancer chemopreventive candidate (Chen and Viljoen 2010). A recent review on geraniol toxicity is available (Lapczynski et al. 2008a). Geraniol metabolism was not extensively studied in mammals. An early study showed that rats orally treated with geraniol presented the following metabolites in urine: geranic acid, 3-hydroxy-citronellic acid, 8-hydroxy-geraniol, 8-carboxy-geraniol, and Hildebrandt acid (Chadha and Madyastha 1984).

In Vitro Studies Among several monoterpenes tested, geraniol was one of the most potent in vitro inhibitor of CYP2B, a phase I enzyme involved in the activation of several

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procarcinogens (Seo et al. 2008). Geraniol elicited cytoprotective and antioxidant actions against t-BHP-induced oxidative stress in murine alveolar macrophages (Tiwari and Kakkar 2009). Geraniol induced cell growth arrest at G0/G1 interphase and induced apoptosis in HEP-G2 cells (Crespo et al. 2013). These effects were accompanied by inhibition of the mevalonate pathway. Interestingly, geraniol was able to both posttranscriptionally and transcriptionally inhibit HMG-CoA reductase expression. In the latter case it was proposed that geraniol would inhibit squalene conversion to lanosterol, and this would deviate carbon through the 24,25-epoxycholesterol shunt, leading to a negative-feedback control of HMG-CoA reductase at the transcriptional level (Crespo et al. 2013). These results show that geraniol effects on HMG-CoA reductase are rather complex and involve modulation of different points in the mevalonate pathway. In a further study in the same cell line, combination of geraniol with simvastatin synergistically inhibited cholesterol biosynthesis and cell proliferation (Polo et al. 2011), showing that targeting the mevalonate pathway through different mechanisms could represent an interesting cancer chemopreventive approach (Mo and Elson 2004). Geraniol treatment caused cell cycle arrest at G0/G1 in MCF-7 breast cancer cells, and this was accompanied by decreased expression of CDK4 and cyclins D1 and D2 (Duncan et al. 2004). Interestingly, although geraniol inhibited HMG-CoA reductase activity, exogenous mevalonate addition was not able to reverse geraniol growth inhibitory effects. The authors of this study suggested that the cell cycle regulatory effects of geraniol do not seem to be related to inhibition of HMG-CoA reductase activity and other mechanism could be relevant, including oncogene prenylation inhibition (Duncan et al. 2004). Geraniol also inhibited the proliferation and induced apoptosis in PC-3 prostate cancer cells in vitro and in vivo (Kim et al. 2011). These effects were associated with modulation of cell cycle regulators and BCL-2 proteins. In addition, combination of this monoterpene with docetaxel elicited synergistic effects on the inhibition of these cells, indicating a possible role of geraniol in the development of new anticancer combinatorial therapeutic strategies (Kim et al. 2011). In addition to apoptosis, inhibitory effects of geraniol on prostate cancer cells were further shown to involve cell death induction through autophagy and to be mediated by AKT and AMPK modulation (Kim et al. 2012). Other HMG-CoA reductase-independent effects of geraniol on cell proliferation include inhibition of polyamine synthesis and membrane and ion channels perturbation (Carnesecchi et al. 2002, 2004).

In Vivo Studies Accumulating in vivo evidence show that geraniol represents a promising chemopreventive dietary agent against different types of malignancies including pancreas, tongue, colon, kidney, and liver cancers. Earlier studies conducted with geraniol showed that treatment of mice (0.5 % in the diet) before injection with P388 leukemia cells increased mice survival time (Shoff et al. 1991). In addition,

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geraniol treatment (23 mmol/kg diet, 350 µmol day) before and after the transplant of Morris hepatoma cells to rats significantly reduced the development of these tumors (Yu et al. 1995). These authors (Yu et al. 1995) also observed inhibitory effects after geraniol administration (6.5 and 65 mmol/kg in the diet) before and after the transplant of B16 melanoma cells to mice. Inhibition of transplanted PC-1 hamster pancreatic adenocarcinomas was further reported after consumption of geraniol (20 g/kg diet), and this effect was not accompanied by alterations in plasma cholesterol levels (Burke et al. 1997). Geraniol chemopreventive effects were reported in a ferric nitrilotriacetateinduced renal carcinogenesis model in rats (Ahmad et al. 2011). Treatment with the monoterpene at the doses of 100 and 200 mg/kg inhibited oxidative stress and renal tumor development. These inhibitory effects were associated with induction of apoptosis, upregulation of xenobiotic metabolizing enzymes, and inhibition of PCNA and NFκB, the latter being an important marker of inflammation (Ahmad et al. 2011). Other authors (Vuch et al. 2012) have reinforced the anti-inflammatory role of geraniol in other contexts including patients carrying a mutation in mevalonate kinase gene. These patients with mevalonate kinase deficiency present recurrent inflammatory episodes that can be inhibited with geraniol treatment (Vuch et al. 2012). A recent study showed that rats orally treated with geraniol (200 mg/kg, thrice a week, 1 week prior to 4-nitroquinoline-1-oxide initiation and throughout the experiment) presented decreased susceptibility to oral tumor development. More specifically, geraniol inhibited the activities of phase I enzymes (cytochrome P450, cytochrome b5, NADPH Cyt P450 reductase, and NADH Cyt b5 reductase) and induced the activities of phase II carcinogen detoxifying enzymes (glutathione-Stransferase, UDP-glucuronosyl transferase, DT-diaphorase) in the liver and tongue tissues (Madankumar et al. 2013). Modulation of NRF2-ARE pathway was shown to be implicated in these chemopreventive activities by geraniol (Madankumar et al. 2013). Inhibition of oral carcinogenesis after consumption of geraniol (250 mg/ kg b.w. starting 1 week before the exposure to DMBA and on days alternate to this carcinogen application) was also reported in golden Syrian hamsters (Vinothkuran and Manoharan 2011). These chemopreventive effects also involved modulation of antioxidant and phase I and II enzymes. In addition, geraniol was further shown to modulate several molecular targets in buccal mucosa that are associated with different processes: cell proliferation (PCNA, cyclin D1, fos), inflammation (NFκB, COX-2), apoptosis (p53, Bax, Bcl-2, caspases-3 and - 9), and angiogenesis (VEGF) (Vinothkumar et al. 2012). Supressing chemopreventive effects by geraniol against skin carcinogenesis were reported in mice (Khan et al. 2013). Topical treatment with the monoterpene (250 µg) 30 min prior to 12-O-tetradecanoyl phorbol-13-acetate application inhibited its promoting effects. These protective effects were associated with geraniol inhibition of oxidative stress and inflammation through modulation of Cox-2 and NFκB p65 subunit expression (Khan et al. 2013). Inhibition of the Ras/Raf/ ERK1/2 signaling pathways in mice skin tumors by geraniol was also reported, and this effect was suggested to involve inhibition of the farnesyl transferase enzyme (Chaudhary et al. 2013).

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Geraniol presented pronounced chemopreventive activities when orally provided (250 mg/kg b.w. daily) to rats during the initiation and selection/promotion phases of the “resistant hepatocyte model of hepatocarcinogenesis” (initiation with diethylnitrosamine (DEN) and selection/promotion with 2-acetylaminofluorene [2AAF]+ partial hepatectomy) (Ong et al. 2006). Monoterpenes inhibited the development of hepatic preneoplastic lesions, and this involved inhibition of cell proliferation and DNA damage and induction of apoptosis. Geraniol-treated animals did not present altered levels of plasmatic cholesterol and HMG-CoA reductase mRNA levels, indicating that its chemopreventive effects were not related to this enzyme (Ong et al. 2006). In a further study suppressing chemopreventive activities in the same model of hepatocarcinogenesis were also reported for geraniol (Cardozo et al. 2011). Treatment of rats with the monoterpene (250 mg/kg b.w. daily) specifically during the promotion phase inhibited the development of hepatic preneoplastic lesions. Geraniol did not inhibit cell proliferation in these lesions and alter HMG-CoA reductase, suggesting that its protective effects were independent of this enzyme. In addition, the monoterpene induced apoptosis in hepatic preneoplastic lesions, and this seems to be due to inhibitory actions of RhoA activation. Interestingly, these authors also verified that geraniol-treated animals presented increased hepatic levels of the monoterpene, showing that it is feasible to achieve tissue levels sufficient for chemoprevention after daily ingestion of this compound (Cardozo et al. 2011). Geraniol also presented inhibitory actions against nimesulide-induced hepatotoxicity in rats, and this involved prevention of mitochondrial dysfunction and oxidative stress (Singh et al. 2012). Geraniol was also tested for its chemopreventive potential in rats subjected to a dimethyl-hydrazine-based colon carcinogenesis model and orally treated with the monoterpene (250 mg/kg b.w.) alone or combined with another isoprenic derivative beta-ionone (160 mg/kg b.w.) (Vieira et al. 2011). Geraniol, but not beta-ionone, presented inhibitory effects on colonic preneoplastic lesions development, which were associated with apoptosis induction and Bcl-2 protein reduced expression. Geraniol-treated animals presented increased levels of this monoterpene in colonic tissue indicating good bioavailability. Contrary to the expectations by authors, combination of the acyclic monoterpene geraniol with the cyclic one beta-ionone does not seem to be an efficient chemopreventive strategy since it did not alter the development of preneoplastic lesions (Vieira et al. 2011). The combination of geraniol with 5-fluorouracil resulted in synergistic inhibitory effects on the growth of transplated human colon tumors in mice (Carnesecchi et al. 2004).

Sesquiterpenes (Farnesol) Farnesol (3,7,11-trimethyl-2,6,10-dodecatrien-1-ol) is endogenously produced through dephosphorylation of farnesyl-pyrophosphate, a metabolite of the mevalonate pathway (Joo and Jetten 2010). It is also naturally found in the essential oils of chamomile and lemongrass (Joo and Jetten 2010). Farnesol metabolism was not extensively studied in mammals. An early study showed that rats orally treated

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with farnesol presented in urine different dicarboxylic acids probably derived from farnesol oxidation to farnesoic acid (Bostedor et al. 1997). In addition, it was demonstrated that farnesol can be oxidized to a prenyl aldehyde, possibly by an alcohol dehydrogenase in rat mitochondrial and peroxisomal fractions (Westfall et al. 1997).

In Vitro Studies Farnesol has been shown to effectively suppress cellular growth and induce apoptosis in several cancer cell lines. Although initial studies showed HMG-CoA reductase inhibition to be an important step of these anticancer actions, increasing evidence show that farnesol also presents HMG-CoA reductase-independent actions and other molecular targets, including CTP:phosphocholine cytidylyltransferase, involved in phosphatidylcholine biosynthesis and NFκB (Joo and Jetten 2010). Through a global gene expression analysis, it was observed that induction of endoplasmic reticulum stress is a relevant feature of farnesol-induced apoptosis in lung cancer cells (Joo et al. 2007). Anticancer effects of farnesol involve transcriptional regulation of genes associated with cellular growth and metabolism (Duncan an Archer 2006). Farnesol was initially identified as the activator of the nuclear receptor FXR that was termed “farnesoid X activated receptor” (Forman et al. 1995). This sesquiterpene has also been shown to activate other transcriptional factors, including PPAR-alpha and -gamma. Growth inhibition of MCF-7 breast cancer cells by farnesol was shown to involve thyroid hormone receptor-mediated signaling (Duncan and Archer 2006).

In Vivo Studies Farnesol has been shown to present chemopreventive effects during all stages of carcinogenesis through different mechanisms (Joo and Jetten 2010). Farnesol administered subchronically (doses up to 1000 mg/kg/day for 28 days) to rats was minimally toxic (Horn et al. 2005). No mortality, effects on body weight, hematology and coagulation parameters, gross- or microscopic-organ alterations were observed in farnesol-treated rats. In addition, no evidence of toxicity was observed under clinical examination. In this study, farnesol induced the activity of several phase I and II enzymes in the liver (Horn et al. 2005). While phase II enzymes induction could represent a relevant cancer chemopreventive mechanism, care should be taken when considering combinatorial chemopreventive interventions with farnesol because of potential CBA−drug interactions as this isoprenic derivative also induces phase I enzymes. A recent review on farnesol toxicity is available (Lapczynski et al. 2008b). Initial studies conducted with farnesol showed inhibition of transplanted PC-1 hamster pancreatic adenocarcinomas after consumption of sesquiterpene (20 g/kg diet), and this effect was not accompanied by alterations in plasma cholesterol levels (Burke et al. 1997). In addition, farnesol presented chemopreventive activities in

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an azoxymethane-based model of carcinogenesis (Rao et al. 2002). Consumption of farnesol in the diet (1.5 %) before initiation and throughout the experiment resulted in inhibition of aberrant crypt foci in rats, and this effect was not accompanied by alterations in serum total cholesterol levels (Rao et al. 2002). Farnesol chemopreventive effects against colon carcinogenesis could be mediated by antioxidant and anti-inflammatory actions (Khan and Sultana 2011). These authors observed that treatment of rats with farnesol (100 mg/kg b.w., daily for 7 days) prior to initiation with DMH inhibited oxidative stress through induction of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, catalase, glutathione-S-transferase, and quinone reductase. In addition, farnesol supplementation ameliorated DMHinduced inflammation in the colonic tissue (Khan and Sultana 2011). Similarly, farnesol treatment (100 and 200 mg/kg b.w., daily for 2 weeks) inhibited rat lung inflammation and oxidative stress induced by benzopyrene (Qamar et al. 2012). The finding that farnesol also modulated this carcinogen metabolism suggests that it could also play a role in antilung cancer initiation (Qamar et al. 2012). Farnesol chemopreventive effects were also reported in a mice model of skin carcinogenesis (initiation with DMBA and promotion with TPA) (Chaudhary et al. 2009). Farnesol was topically administered (25, 50, and 100 mg/kg b.w.) 30 min prior to each TPA administration and a dose-dependent inhibitory effect on skin tumor development was observed. Interestingly, at the lower doses, farnesol inhibited the Ras/Raf/ERK1/2 signaling pathway in skin tumors, whereas at the highest dose it presented the opposite effects (Chaudhary et al. 2009). Apoptosis induction and cell proliferation inhibition were observed in rats treated with farnesol (250 mg/kg b.w. daily) before a 70 % partial hepatectomy (Chagas et al. 2009). More specifically it was suggested that farnesol induced a G0/G1 arrest leading to early apoptosis induction that was accompanied by a reduced number of proliferating hepatocytes at S phase (Chagas et al. 2009). Farnesol presented pronounced chemopreventive activities when orally provided (250 mg/kg b.w. daily) to rats during the initiation and selection/promotion phases of the “resistant hepatocyte model of hepatocarcinogenesis” (initiation with DEN and selection/promotion with 2-AAF + partial hepatectomy) (Ong et al. 2006). The sesquiterpene inihibited hepatic preneoplastic lesions development, and this involved inhibition of cell proliferation and DNA damage but not induction of apoptosis. Farnesol-treated animals presented reduced levels of plasmatic cholesterol and increased levels of HMG-CoA reductase mRNA levels. The latter effect could be due to a compensatory upregulation of HMG-CoA reductase gene expression following an eventual degradation of the enzyme by farnesol, as also shown for lovastatin a competitive inhibitor of this enzyme. Based on these results it was suggested that HMG-CoA reductase represents a relevant target for farnesol chemopreventive activities during hepatocarcinogenesis. On the other hand it was further verified that farnesol treatment did not alter protein FXR expression (Ong et al. 2006). In a previous report, oral consumption of a single dose of farnesol (500 mg/kg b.w.) increased 1000-fold farnesol hepatic levels (Keller et al. 1996). This result shows that farnesol is highly bioavailable and can accumulate in the liver and reinforce its chemopreventive potential for hepatocarcinogenesis chemoprevention.

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Conclusions and Perspectives Geraniol and farnesol represent isoprenoids with promising blocking and suppressing chemopreventive potential against several cancers. They have elicited protective effects in both in vitro and in vivo model systems. In addition to HMG-CoA reductase, they present several other molecular targets. The low toxicity and regular consumption through diet are further characteristics that reinforce their role as cancer chemopreventive agents. Importantly, bioavailability, metabolism, safety, and mechanism of action are aspects that should be deeply investigated, especially in clinical trials (Rabi and Gupta 2008). Two recent studies conducted by Miller et al. (2010, 2013) for d-limonene, one of the first monoterpenes to be systematically investigated for its cancer chemopreventive activities, are good examples in this regard. In an open-label pilot clinical study conducted among women with newly diagnosed operable breast cancer, daily consumption of 2 g of limonene 2–6 weeks before surgery significantly reduced cyclin D1 expression. Importantly, limonene, but not its major active circulating metabolite perillic acid, was shown to preferentially accumulate in the breast tissue (Miller et al. 2013). In addition, consumption of a d-limonene-rich lemonade daily for weeks (500–600 mg limonene/day) resulted in increased levels in the plasma and adipose tissue, indicating that the monoterpene presents oral bioavailability (Miller et al. 2010). These studies reinforce the potential of d-limonene for chemoprevention of cancer in lipid-rich tissues including the breast. Thus, adoption of a similar approach would be key to advance the knowledge on the feasibility of using geraniol and farnesol as dietary cancer chemopreventive agents in humans. Acknowledgment T.P. Ong laboratory is supported by grants and fellowships from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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Moreno FS, Rossiello MR, Manjeshwar S, Nath R, Rao PM, Rajalakshmi S, Sarma DS (1995) Effect of beta-carotene on the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat liver. Cancer Lett 96:201–208 Ong TP, Heidor R, de Conti A, Dagli ML, Moreno FS (2006) Farnesol and geraniol chemopreventive activities during the initial phases of hepatocarcinogenesis involve similar actions on cell proliferation and DNA damage, but distinct actions on apoptosis, plasma cholesterol and HMGCoA reductase. Carcinogenesis 27:1194–1203 Ong TP, Moreno FS, Ross SA (2011) Targeting the epigenome with bioactive food components for cancer prevention. J Nutrigenet Nutrigenomics 4:275–292 Ong TP, Cardozo MT, de Conti A, Moreno FS (2012) Chemoprevention of hepatocarcinogenesis with dietary isoprenic derivatives: cellular and molecular aspects. Curr Cancer Drug Targets 12:1173–1190 Peffley DM, Gayen AK (2003) Plant-derived monoterpenes suppress hamster kidney cell 3-hydroxy-3-methylglutaryl coenzyme a reductase synthesis at the post-transcriptional level. J Nutr 133:38–44 Polo MP, Crespo R, de Bravo MG (2011) Geraniol and simvastatin show a synergistic effect on a human hepatocarcinoma cell line. Cell Biochem Funct 29:452–458 Qamar W, Khan AQ, Khan R, Lateef A, Tahir M, Rehman MU, Ali F, Sultana S (2012) Benzo(a) pyrene-induced pulmonary inflammation, edema, surfactant dysfunction, and injuries in rats: alleviation by farnesol. Exp Lung Res 38:19–27 Rabi T, Bishayee A (2009) Terpenoids and breast cancer chemoprevention. Breast Cancer Res Treat 115:223–239 Rabi T, Gupta S (2008) Dietary terpenoids and prostate cancer chemoprevention. Front Biosci 13:3457–3469 Rao CV, Newmark HL, Reddy BS (2002) Chemopreventive effect of farnesol and lanosterol on colon carcinogenesis. Cancer Detect Prev 26:419–425 Seo KA, Kim H, Ku HY, Ahn HJ, Park SJ, Bae SK, Shin JG, Liu KH (2008) The monoterpenoids citral and geraniol are moderate inhibitors of CYP2B6 hydroxylase activity. Chem Biol Interact 174:141–146 Shoff SM, Grummer M, Yatvin MB, Elson CE (1991) Concentration-dependent increase of murine P388 and B16 population doubling time by the acyclic monoterpene geraniol. Cancer Res 51:37–42 Singh BK, Tripathi M, Chaudhari BP, Pandey PK, Kakkar P (2012) Natural terpenes prevent mitochondrial dysfunction, oxidative stress and release of apoptotic proteins during nimesulidehepatotoxicity in rats. PloS One 7:e34200 Sporn MB, Dunlop NM, Newton DL, Smith JM (1976) Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids). Fed Proc 35:1332–1338 Steward WP, Brown K (2013) Cancer chemoprevention: a rapidly evolving field. Brit J Cancer 109:1–7 Surh YJ (2003) Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 3:768–780 Thoppil RJ, Bishayee A (2011) Terpenoids as potential chemopreventive and therapeutic agents in liver cancer. World J Hepatol 3:228–249 Tiwari M, Kakkar P (2009) Plant derived antioxidants—Geraniol and camphene protect rat alveolar macrophages against t-BHP induced oxidative stress. Toxicol In Vitro 23:295–301 Vieira A, Heidor R, Cardozo MT, Scolastici C, Purgatto E, Shiga TM, Barbisan LF, Ong TP, Moreno FS (2011) Efficacy of geraniol but not of beta-ionone or their combination for the chemoprevention of rat colon carcinogenesis. Braz J Med Biol Res 44:538–545 Vineis P, Wild CP (2014) Global cancer patterns: causes and prevention. Lancet 383:549–557 Vinothkumar V, Manoharan S (2011) Chemopreventive efficacy of geraniol against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. Redox Rep 16:91–100 Vinothkumar V, Manoharan S, Sindhu G, Nirmal MR, Vetrichelvi V (2012) Geraniol modulates cell proliferation, apoptosis, inflammation, and angiogenesis during 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. Mol Cell Biochem 369:17–25

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Chapter 13

Cancer and Aromatherapy: A View of How the Use of Essential Oils Applies to Palliative Care Rita de Cássia da Silveira e Sá

Introduction Aromatherapy or aromatic medicine is defined as the use of essential oils extracted from herbs, flowers, and other plant parts to produce physiological and pharmacological responses when absorbed into the body through the olfactory system, through the skin, or through diffusion across mucous membranes (Diego et al. 1998; Muzzarelli et al. 2006). It is regarded as a therapeutic procedure widely employed around the world for the treatment of physical and psychological disorders in addition to being used for health improvement and well-being. For this reason, it is seen as a valuable tool for the management of depression and other stress-related diseases as they can unleash neurochemical responses that can lead to relaxation (d’Angelo 2002; Moss et al. 2003; Perry and Perry 2006). Essentially, aromatherapy is concerned with quality of life and tries to deal with the adversities. It relieves tension, pain and insomnia, exerts calming effects, enhances mood, and improves cognition (Alexander 2001; de Valois and Clarke 2001). Different methods are used to deliver aromatherapy, including diffusers, baths, compresses, and massage. As a complementary therapy, massage brings about a range of psychological and physiological changes, including improvements in blood and lymph flow, reduction in muscle tension, increase in pain threshold, improvement of mood, reduction of blood pressure, and relaxation of the mind. It is thus used frequently for a range of symptoms like anxiety and stress, back pain, and other musculoskeletal conditions (Bowers 2006; Lee et al. 2012). Because of these advantages, massage is becoming increasingly popular in cancer palliation and in combination with essential oils has given aromatherapy an important means for its application in hospital settings.

R. de Cássia da Silveira e Sá () Department of Physiology and Pathology, Health Science Center, Federal University of Paraíba, João Pessoa, PB, Brazil e-mail: [email protected] © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_13

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Essential oils are a mixture of a variable number of volatile substances produced by plants. They comprise a large variety of components, which display a multitude of effects on the body and mind (d’Angelo 2002; Bakkali et al. 2008; De Sousa 2012). Essential oils exhibit a wide range of pharmacological applications, being particularly known for their antiseptic, anti-inflammatory, sedative, and antimicrobial properties (Bakkali et al. 2008). In the plant, they exert many actions, such as insect attraction, nourishment, neutralization of infectious agents, and cellular repair (d’Angelo 2002). Psychological and physiological effects of fragrances have been empirically known from ancient times. Some oils employed in modern aromatherapy can be traced back historically making aromatherapy the oldest medicinal practice still in use. As early as the prehistoric man who made use of aromatic plants to cover the bodies of the deceased to help the souls in their departure, the principles behind contemporary aromatherapy were set thousands of years ago by different civilizations, beginning with the ancient Egyptians followed by the Chinese, the Greeks, and the Romans (Cooke and Ernst 2000; d’Angelo 2002; Herz 2009). The ancient Egyptians used aromatherapy oils for various purposes, but its primary function was embalming the dead with oils such as cinnamon and cedar wood. Later, they started using oils as perfumes and during healing ceremonies as a calming agent. The Chinese used them in the form of incense when performing massage therapy and also during religious rituals. The ancient Greeks gave a significant contribution to the development of medicinal aromatherapy as evidenced by the work of Hippocrates, around 450 BC, who recommended the use of aromatic oils to aid the treatment of patients with rheumatism and arthritis, while the Romans became famous in history for their aromatic baths and for the use of aromatic oils as aphrodisiacs. At a later time, aromatherapy became a subject of interest in Europe during the Renaissance with the rediscovery of ancient Greek and Roman texts about old herbal medicines and aromatherapy. In modern times, aromatherapy was employed to treat soldiers in the Second World War (Herz 2009). The term aromatherapy was devised by René-Maurice Gattefosse (1881–1950), a French chemist who worked in the perfume business but had a special interest in studying the therapeutic properties of essential oils. Owing to an accident that took place in his laboratory, he was able to investigate the healing properties of essential oils after severely burning his hands in an explosion. He plunged his hands into a vessel filled with pure lavender oil, which reduced the swelling and accelerated the healing process without scarring (Cooke and Ernst 2000; Herz 2009). Following Gattefosse′s footsteps, a French doctor, named Jean Valnet, used chamomile, clove, lemon, and thyme essential oils to treat gangrene and battle wounds during the Second World War. Over the years, aromatherapy has evolved from a ritualistic and empirical experience to a more scientific context in the complementary and alternative medicine. Earlier, the choice of oil selection for medical and psychiatric use was basically dependent on tradition rather than science; however, a better understanding of the mechanism of action of the aromatic constituents is now providing a more scientific point of view for the oil selection and the appropriate therapeutic dosing.

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The Interrelation Between the Olfactory System and the Immune System The human brain is capable of identifying a very large number of different odors as odor molecules enter the nose and trigger a series of events that begin with the sensitization of olfactory receptor cells located in the upper posterior region of the nasal cavity, followed by the transmission of the scent stimulus via the olfactory nerves to the hypothalamus and the limbic system (Fig. 13.1). Interconnecting pathways provide further neural activation that may produce stimulation or inhibition of the immune system and the endocrine system (d’Angelo 2002; Broughan 2005). In aromatherapy, after essential oils and their constituents enter the body through the nose, skin, or mucous membranes, they reach the bloodstream and eventually the brain, leading to a subsequent chain of chemical reactions that have a strong influence on mood and behavior. Studies conducted in rodents have provided evidence to suggest that the effects of aromatherapy on mood, physiology, and behavior result from the interaction of odors or their ability to act on the autonomic nervous system/central nervous system and/or the endocrine system (Elisabetsky et al. 1995; Herz 2009). Indeed, this is possible because of the direct connection of

Fig. 13.1  The basic flow of olfactory information in the brain begins with the contact of odor molecules with the cilia of the olfactory receptors embedded in the membrane of nasal cavity that is then transduced into neural messages that reach the limbic system, which is the area of the brain that regulates emotion and memory, and influences visceral responses to those emotions. It also influences motivation, mood, and sensation of pain and pleasure. It comprises the hypothalamus, amygdala, hippocampus, and other nearby areas such as the cingulated gyrus

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olfactory stimuli to the brain, particularly the hypothalamus and the limbic region, which play important modulatory roles on motivational and emotional, learning, and memory behaviors (Alexander 2000). Experiments carried out in rats and mice showed that the removal of the olfactory bulb induced aggressive behavior in the animals as well as abnormal levels of neurotransmitters and hormones (Leonard and Tuite 1981; Fujiwara et al. 2002). In the hypothalamus, active compounds of the oils can activate receptors, increasing the release of neurotransmitters that enhance the hypothalamic–pituitary axis function and the activation of neuroreceptors on cells of the immune system (d’Angelo 2002). In the limbic system, they also activate receptors, culminating in the release of neurotransmitters, including encephalin, serotonin, endorphin, and noradrenalin. Serotonin exhibits calming and relaxing effects while noradrenalin works as a stimulant. Encephalin and endorphins give a feeling of well-being and reduces pain (Lawless 1994; Kyle 2006). Olfactory responses are directly linked to previous experiences or emotions and related behaviors. Moreover, the perceptive sensitivity can be potentiated by the expectation created by a deliberate contact with smelling odors, contributing to behavioral, mental, and emotional displays produced in response to a given scent (Alexander 2000). Positive environmental reactions to odors are capable of affecting health and the state of mind, generating a sense of harmony and well-being, thereby enabling the patient to improve his general condition. In addition to diseases, humans are continually exposed to stressful situations which can have harmful influence on the individual′s organism. For instance, it is well known that in a stressed state the lymphoid organs may become atrophied and, as a consequence, reduce the immune responses (Fujiwara et al. 2002). In this context, various studies have addressed the effects of odorants on stress-induced immunosuppression in mice, showing that immunosuppression was back to normal after inhalation or olfactory stimulation with different fragrances, such as lemon, rose, lavender, and citrus lemon, but not jasmine and cardamom, suggesting that some odorants have antistress effects (Shibata et al. 1990, 1991; Fujiwara et al. 1998). The immune system can be stimulated by the nervous system to produce chemical messengers (e.g., corticotrophin and endorphins), which influence the functioning of the central nervous and endocrine systems. The interaction between those two systems facilitates the flow of information about the state of the immune system to the brain, which will then influence the psychological state (Alexander 2001). In humans, several trials have reported the clinical effects of fragrances. In particular, a study developed to examine the effects of citrus fragrance on neuroendocrine hormone levels and immune function in depressed patients ( n = 20) showed no significant therapeutic effects between the citrus fragrance and antidepressant-treated groups (Komori et al. 1995a, 1995b). Although the mean levels of urinary cortisol and dopamine were higher in those groups when compared with health subjects, they were reduced and returned to normal levels after treatment with citrus fragrance, exhibiting mean levels significantly lower than those of the individuals treated with the antidepressants (Fujiwara et al. 2002). Data available in the literature have given enough evidence to suggest that essential oils have beneficial effects on the immune system by reducing stress, improving

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mood and brain activity, which are important factors for sustaining good health and the healing process (Alexander 2001). Corroborating this assumption, a well-founded theory supports the idea that essential oil odorants act as physiological motivators, strengthening the immune system and positively influencing the overall health of the individual by enhancing recovery from illness or changing our susceptibility to disease. In general, essential oils are capable of modulating the immune system by interfering with brain chemicals in command of affective states, or mood, which affect immunity; conditioning agents to control the immune response, and exerting pharmacological effects directly on cellular immune functions following tissue application (Alexander 2001). In the course of cancer treatment, the series of psychological stressors, the physical and emotional experiences endured by patients can activate stress-response mechanisms, including the hypothalamic–pituitary–adrenal axis (HPA) (Sephton et al. 2000). The prolonged activation of the endocrine response can produce HPA axis dysfunction, being the alteration in circadian cortisol rhythms a relevant sign of such event (Yehuda et al. 1996; Chrousos and Gold 1998; McEwen 1998; Rosmond et al. 1998; Sephton et al. 2000). As an example, normally cortisol levels are highest during the early morning hours and decrease during the day; however, in advanced breast cancer up to 70 % of patients exhibit flattened circadian profiles, consistently high levels, or irregular fluctuations (van der Pomper et al. 1996; Touitou et al. 1996). Furthermore, dysregulation of the cortisol response may interfere with tumor resistance as evidenced by the ability of cortisol to accelerate tumor growth via immunosuppressive actions (McEwen et al. 1997) or effects on metabolic processes (Romero et al. 1992), which can result in dysregulation of immune activity and trafficking of immune cells (Gatti et al. 1993; Kronfol et al. 1997). A follow-up with breast and ovarian cancer patients with altered cortisol rhythms showed altered patterns of circulating leukocytes, neutrophils, platelets, and serum proteins (Touitou et al. 1995). Maladaptive immune responses constitute a serious dysfunctional stress response that can be improved by palliative care. To this end, massage therapy with or without oil is believed to positively affect the interrelation among stress, stress hormones, and immune function by possibly suppressing the HPA axis activity and subsequently causing decreases in cortisol levels (Diego et al. 2001; Hughes et al. 2008). Hence, immune function may improve as a result of the increase in number and activity of natural killer cells (Hughes et al. 2008).

Essential Oils and Aromatherapy Many studies have investigated the effect of essential oils and their constituents on the organism. One example involves the evaluation of the sedative properties of lavender oil ( Lavendula angustifolia) in mice injected with caffeine, which showed that the hyperactive animals became less agitated after inhaling lavender oil (Buchbauer et al. 1991; Cawthorn 1995). To a certain extent, those findings corroborate the medicinal folk use of herbal pillows utilized to improve sleep or to reduce stressful situations (Cawthorn 1995).

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Experiments carried out in humans quite often displays disputed results owing to the subjectiveness of the tests and individual perceptions and responses to them. Expected psychological and physiological effects may not always be significantly expressed in a given experimental protocol leaving enough argument for questionable interpretation. In two experimental studies conducted to verify the occurrence of physiological changes in groups of people who received a 20-min foot massage with or without lavender oil or neroli oil, contradictory findings were noted in the aromatherapy group. Traditionally, both oils are considered to have physiological and psychological effects, such as soothing effects on the nervous system and reduction of tension and anxiety. Contrary to what was expected, neroli oil failed to produce significant changes in the physiological parameters, whereas the application of lavender oil did produce alterations including reduced heart rate, blood pressure, and respiratory rate (Woolfson and Hewitt 1992; Cawthorn 1995). Most essential oils are safe and toxicity rarely occurs, but when it does, it is primarily attributed to misuse and accidental ingestion. The use of the right dosage and method of application are two important issues that should be carefully considered, since studies carried out in animal models showed that some essential oils used in the wrong doses or too high a concentration have contributed to tumor development and unwanted side effects to the skin, liver, and other organs (Tisserand and Balacs 1995, 2000). Additionally, an essential oil may be safe when applied in one way but may not be safe when used in another way. For instance, the application of some oils can be considered safe when inhaled, and yet may be irritating if applied to the skin even in low concentrations. The safety of essential oil application to the skin is of primary importance in aromatherapy, as some oils, such as bergamot, lemon, and orange, can cause severe burns or skin cancer (phototoxicity) in case there is exposure to natural sunlight or sun-bed radiation after application to the skin, whereas this would not result from inhalation (Tisserand and Balacs 1995). Not all essential oils are suitable for use in aromatherapy (e.g., onion and camphor oil), and specifically in oncology, for safety measures, the use of fennel, aniseed, clary sage, sage, and geranium should be avoided in estrogen-sensitive cancers for being potentially carcinogenic essential oils (Tisserand and Balacs 2000; Bowers 2006). As aromatherapy relies on the premise that aromatic plants have the ability to influence mood, behavior, and well-being, it provides an interesting display of therapeutic and physiological properties (van der Watt et al. 2008; Yim et al. 2009).

Anxiety, Depression, and Palliative Care Anxiety is a physiological and psychological condition that involves emotional, somatic, behavioral, and cognitive components (Seligman et al. 2001; Lee et al. 2011). For decades, the treatment of anxiety disorders has been based on the use of pharmacological and psychological medications which are well-known for their undesirable side effects (Lippa et al. 2005). Therefore, in an attempt to overcome the hindrance of traditional medicine, the health-care system began to develop palliative care programs to provide symptom

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relief and pain management, as in palliative care a number of measures are undertaken to improve the quality of life of patients in all disease stages, including those facing life-threatening illnesses and no longer showing response to curative treatments. In this context, anxiety is an aggravating sequela of such conditions, which can bring devastating outcomes to the treatment itself and further increase to the patient’s low mood, sleep, and appetite disorders (Wilkinson et al.1999; Kyle 2006). For that matter palliative care is thus seen as a useful tool in the battle against disease treatments and their side effects that have contributed to turn aromatherapy into the most commonly used complementary alternative medicine for treating anxiety symptoms in the world (Hadfield 2001). In addition to anxiety, depression is another dysfunction that aromatherapy is being currently adopted worldwide as an alternative treatment (Brown and Gerbarg 2001). Depression affects behavior, cognition, and the neurovegetative system, producing severe impediment in the individual′s life. It can appear as a secondary symptom in patients with life-threatening diseases, such as cancer. Indeed, several studies have evidenced the benefits of aromatherapy massage on cancer patients. In one trial, for instance, cancer patients receiving aromatherapy massage showed significant improvement in anxiety and/or depression at 6 weeks when compared with those receiving usual care (Wilkinson et al. 2007). In another study, significant reduction of physical parameters, including systolic and diastolic pressure, heart rate, and respiratory rate, was observed on patients diagnosed with primary malignant brain tumor and who had completed a course of radiotherapy. It was hypothesized that the state of being relaxed often obtained after aromatherapy massage is involuntary and caused by reduced activity of the sympathetic nervous system, which, in turn, could account for the lowering of blood pressure, heart, and respiratory rates (Hadfield 2001). Besides human trials, the antidepressant effects of aromatherapy have also been investigated in animal models. It is believed that the pathophysiology of depression may involve monoamine deficiency or imbalance and reduced activity of serotonin (5-HT) pathways (Cowen 2008; Yim et al. 2009). A study conducted in mice showed that the use of lemon oil increased the metabolic turnover of 5-HT in the prefrontal cortex and striatum, an effect similar to that displayed by selective serotonin reuptake inhibitors (SSRIs)—a class of antidepressants that includes fluoxetine (Prozac, Sarafem, Ladox, and Fontex) and escitalopram (Lexapro and Cipralex)—to improve depressive symptoms by preventing the reuptake of 5-HT by the presynaptic neuron, thus maintaining higher levels of this substance in the synapse and increasing its function (Castro et al. 2003; Cowen 2008).

Cancer and Aromatherapy Cancer is a common disease and many people experience postoperative stress, depression, and anxiety, facing a pessimistic perspective in quality of life, which becomes increasingly compromised (Burgess et al. 2005; Billhult et al. 2008). Palliative cancer care, as opposed to terminal care, is a way of giving patients who are

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not necessarily going to recover from their illness but often live for many years, means to better deal with the disease (Buckle 1997; Bowers 2006). It is aimed to help patients to cope with their pain and to offer relieve by controlling symptoms at all stages to ease suffering by giving the patient psychological, social, and spiritual support (Bowers 2006). Aromatherapy massage is considered the most commonly employed complementary therapy and the most recognized benefits for cancer patients are relaxation, relief from muscle tension, improve sleep, reduce stress and stress-related symptoms, and reduce side effects of cancer therapy (Fellowes 2004; Bowers 2006). In a particular study, a chemotherapy patient exhibiting severe nausea and finding that everything smelt like chemotherapy chemicals reported that the scent of lavender oil, placed under his pillow, made him feel relaxed and helped his sleep (de Valois and Clarke 2001). It is well accepted that emotional distress such as anxiety can interfere or even stop physical recovery following cancer treatments. Anxiety is indeed a significant side effect for oncology patients that in extreme cases can cause the delay or even suspension of treatment. If not treated, heightened anxiety can also induce other symptoms worsening the patient′s well-being (Quattrin et al. 2006; Benney and Gibbs 2013). The use of aromatherapy massage for relieve of symptoms of anxiety is a recognized practice in cancer palliative care; however, the inhalation of aromatherapy oils has been reported to be ineffective in reducing anxiety (Graham et al. 2003; Wilkinson et al. 2007). Furthermore, aromatherapy benefits seem to be more significant at short term and intermediately with no apparent long-term effects (Benney and Gibbs 2013). A follow-up of cancer patients receiving palliative care showed that four weekly sessions of aromatherapy massage enhanced clinical anxiety and/or depression up to 2 and 6 weeks after the end of treatment, but did not at 10 weeks post-intervention (Wilkinson et al. 2007). Apart from the conventional methods of application, an intervention conducted in acute cancer patients in the UK used a personalized aromatherapy device, known as “Aromastick,” intended to provide patients with an independent handling tool for the management of their nausea, anxiety, and sleep problems. The advantage of this device lies in the fact that it meets the need for a discrete aromatherapy intervention as the aroma stays close to the patient when holding it near the nose while breathing in. Since in a hospital setting there may be patients suffering chemotherapy-induced nausea and vomiting, aromatherapy can become intrusive due to the hypersensitivity to smells developed by these patients. In a trial that followed up 160 patients 1 week after they began using aromastick, 77 % reported enjoying at least one benefit from its use. In particular, 65 % of nauseous patients felt more relaxed and 51 % felt less stress; 47 % of nauseous patients felt that it helped setting their nausea, and 55 % of patients with sleep problems expressed improvement in their sleep. It was also observed that apparently aromastick effects were directly related to the frequency of use (Stringer and Donald 2011). Table 13.1 shows results of some trials which demonstrate a trend of improvement of symptoms of cancer patients using aromatherapy.

Table 13.1  Summary of experimental studies using aromatherapy in health subjects, in cancer palliative care, and other conditions Essential oil Experimental treatment Sample Results Randomized No 69 80 % of patients Aromatherapy massage Marjoram, lavender, rose, Improved well-being Weekly for 6 months (4 h eucalyptus, chamomile, Relaxing per session) geranium, neroli Cancer patient Roman chamomile Aromatherapy massage 51 Improved quality of life and physi- Yes Cancer patients cal symptoms Reduction in anxiety 52 Reduction in anxiety Yes Lavender, rosewood, lemon, Aromatherapy massage rose valerian eight sessions Cancer patients No 58 50 % of patients Varied oil blends Aromatherapy massage Reduction in anxiety and six sessions depression Cancer patients 103 Reduction in anxiety and physical Yes Roman chamomile Aromatherapy massage symptoms 3-week course Advanced cancer patients No 8 Relaxed feeling 1 week after Lavender, Roman chamomile Aromatherapy massage session 30-min session Reduction in systolic and diastolic Primary malignant brain blood pressure, heart rate, and tumor respiratory rate No 769 (treatment) Reduction in pain, tension, difLavender, rosewood, sandal- Aromatherapy massage ficulty sleeping, and emotional wood, rose, geranium, sweet 3-year patient audit distress orange, cypress, eucalyptus, Cancer patients Less constipation and nausea de Valois and Clarke (2001)

Hadfield (2001)

Wilkinson et al. (1999)

Kite et al. (1998)

Corner et al. (1995)

Wilkinson (1995)

Reference Evans (1995)

13 Cancer and Aromatherapy: A View of How the Use of Essential … 259

Lavender

Varied oil blends

Lavender

Varied oil blends

Lavender, rosemary, citronella

Table 13.1 (continued) Essential oil Lavender tea tree

Aromatherapy massage— six sessions Cancer patients Humidified aromatherapy treatment Cancer patients Aromatherapy massage six sessions (1 h) Conditions: schizophrenia, psychotic depression, anxiety with depression Aromatherapy massage 30-min session weekly for 4 weeks Breast cancer

Experimental treatment Essential oil infused aqueous cream Breast cancer patients with skin rashes due to chemotherapy Inhalation aromatherapy 10-min session Healthy females

42

8

17

Small alteration in blood pressure, pulse, pain, depression, and well-being Reduction in depression in six patients Improved mood—30 % patients after each session and 10 % after six weeks Reduction in anxiety and depression after second and fourth treatment

Increased blood flow and decreased systolic blood pressure (lavender) Decreased blood flow and increased systolic blood pressure (rosemary) Decreased blood flow (citronella) Reduction in stress

9

11

Results No significant difference between aromatherapy and conventional treatment

Sample 32

Dunwoody et al. (2002) Louis and Kowalski (2002) Edge (2003)

Soden et al. ( 2004)

No

No

Yes

Saeki and Shiohara (2001)

Yes

No

Reference Gravett (2001)

Randomized Yes

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Aromastick

Varied form 40 oil blends

Varied from 20 oil blends

Sandalwood

Sweet orange, geranium, basil

Lavender, bergamot cedarwood

Table 13.1 (continued) Essential oil Blend of lavender and chamomile

Experimental treatment Aromatherapy massage Weekly for 4 weeks Cancer patients Inhalation aromatherapy during radiotherapy One session (duration not mentioned) Anxious cancer patients Aromatherapy massage 30-min session twice for 4 weeks Patients with depressive disorders Aromatherapy massage Weekly for 4 weeks Palliative care patients Aromatherapy massage Weekly for 4 weeks Breast cancer Aromatherapy massage 20-min session once Cancer patients (hematology transplant unit) Inhalation aromatherapy Cancer patients Reduction in depression

Reduction in anxiety

Reduction in anxiety and depression Reduction in anxiety Reduction in cortisol serum level

5

250

288

39

Reduction in stress, sleep disturbance, and nausea Relaxing effect

No improvement in the state of anxiety

398

160

Results Improved mood, quality of life, and physical symptoms

Sample 46

Stringer et al. (2008)

Evans (1995)

No

Wilkinson et al. (2007)

Kyle (2006)

Okamoto et al. (2005)

Graham et al. (2003)

Reference Wilcock et al. (2004)

Yes

Yes

Yes

No

Yes

Randomized Yes

13 Cancer and Aromatherapy: A View of How the Use of Essential … 261

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Although the focus of this chapter is the use of aromatherapy in cancer palliative care, it is important to comment on the role essential oil constituents play in cancer prevention and treatment. An increasing number of studies are providing relevant evidence about the anticancer mechanisms of essential oils and their action as chemopreventive agents (Bhalla et al. 2013), since chemotherapy has been considerably ineffective in certain types of cancer, including pancreatic cancer, demanding the search for new chemotherapeutic approaches (Stark et al. 1995; Matos et al. 2008). For example, in vitro studies showed the potential anticancer effect of Chios mastic gum ( Pistacia lentiscus L.), whose aromatic constituents have been found to be related to suppression of lung, prostate, and colon cancers (Giaginis and Theocharis 2011). Preclinical cancer models have shown the chemopreventive and chemotherapeutic property of the monoterpene perillyl alcohol ( p-mentha-1,8-diene7-ol) in rat mammary tumors and associated liver metastases (Haag et al. 1992; Haag and Gould 1994), and pancreatic tumors originated from hamster and human cell lines (Stark et al. 1995; Stayrook et al. 1997). Perillyl alcohol was reported to be selectively effective on tumor cells but not on normal ones, and to be able to change tumor cells to a differentiated state (Belanger 1998; Fonseca et al. 2008). Various mechanisms seem to be involved in the anticancer activity of essential oils and they include the following properties (Bhalla et al. 2013): antioxidant (PazElizur et al. 2008), antimutagenic (Bakkali et al. 2006), antiproliferative (Lampronti et al. 2006), improvement of immune function (Yoon et al. 2009), enzyme induction and improved detoxification (Hayes et al. 1987; Ip et al. 1992), modulation of multidrug resistance, and synergistic mechanism of volatile constituents (Ravizza et al. 2008).

Conclusion The use of aromatherapy to reduce levels of psychological distress and improve the quality of life for cancer patients is becoming increasingly incorporated into clinical practice, particularly in palliative care settings. The therapeutic use of essential oils combined with massage appears to be an excellent tool to aid relaxation and relief of some symptoms by influencing body, mind, and spirit. Moreover, it is not dangerous to health, providing it is administered by trained professionals. Despite the information available on essential oil safety and cancer, more research is needed since most of the reports are concerned with the use of rodents treated orally and submitted to long-term treatments at high dose levels. Unfortunately, this is not representative of essential oil use in aromatherapy practice. Nevertheless, the benefit of aromatherapy as a palliative care should not be overlooked in the sense that it brings hope and stimulates an optimistic attitude in the management of cancer patients by creating a more cooperative environment and by giving patients an opportunity to talk about their expectations, worries, and fears in a safe and supportive setting. Therefore, if even subjectively aromatherapy is providing aid to

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emotionally, psychologically, and physically vulnerable cancer patients, this should counterbalance the lack of research evidence important to show its clinical efficacy. Finally, despite the controversy over the benefits and risks aromatherapy and other complementary therapies offer to patients, it cannot be ignored the fact that these practices are being widely employed in cancer care settings. A significant body of evidence in the effectiveness of complementary therapy is available in the literature and should provide a helpful guide for health-care professionals who are increasingly recognizing the need for safe and noninvasive supportive care interventions to improve the overall quality of life of patients.

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Saeki Y, Shiohara M (2001) Physiological effects of inhaling fragrances. Int J Aromather 11(3):118–125 Seligman MEP, Walker EF, Rosenhan DL (2001) Abnormal psychology. WW Norton & Company, New York Sephton SE, Sapolsky RM, Kraemer HC, Spiegel D (2000) Diurnal cortisol rhythm as a predictor of breast cancer survival. J Nat Cancer Inst 92(12):994–1000 Shibata H, Fujiwara R, Iwamoto M et al (1990) Recovery of PFC in mice exposed to high pressure stress by olfactory stimulation with fragrance. Int J Neurosci 51:245–247 Shibata H, Fujiwara R, Iwamoto M et al (1991) Immunological and behavioural effects of fragrance in mice. Int J Neurosci 57:151–159 Soden K, Vincent K, Craske S et al (2004) A randomized controlled trial of aromatherapy massage in a hospice setting. Palliat Med 18:87–92 Stark MJ, Burke YD, McKinzie JH et al (1995) Chemotherapy of pancreatic cancer with the monoterpene perillyl alcohol. Cancer Lett 96: 15–21 Stayrook KR, McKinzie JH, Burke YD et al (1997) Induction of the apoptosis-promoting protein Bak by perillyl alcohol in pancreatic ductal adenocarcinoma relative to untransformed ductal epithelial cells. Carcinogenesis 18:1655–1658 Stringer J, Donald G (2011) Aromasticks in cancer care: an innovation not to be sniffed at. Comp Ther Clin Pract 17:166–121 Stringer J, Swindell R, Dennis M (2008) Massage in patients undergoing intensive chemotherapy reduces serum cortisol and prolactin. Psycho-Oncol 17:1024–1031 Tisserand R, Balacs T (1995) Essential oil safety. Churchill Livingstone, New York Tisserand R, Balacs T (2000) Essential oil safety: a guide for health care professionals. Churchill Livingston, London Touitou Y, Levi F, Bogdan A et al (1995) Rhythm alteration in patients with metastatic breast cancer and poor prognostic factors. J Cancer Res Clin Oncol 121:181–188 Touitou Y, Bogdan A, Levi F et al (1996) Disruption of the circadian patterns of serum cortisol in breast and ovarian cancer patients: relationships with tumour marker antigens. Br J Cancer 74:1248–1252 van der Pompe G, Antoni MH, Heijnen CJ (1996) Elevated basal cortisol levels and attenuated ACTH and cortisol responses to a behavioral challenge in women with metastatic breast cancer. Psychoneuroendocrinology 21:361–374 van der Watt G, Laugharne J, Janca A (2008) Complementary and alternative medicine in the treatment of anxiety and depression. Curr Opin Psychiatry 21:37–42 Wilcock A, Manderson C, Weller R et al (2004) Does aromatherapy massage benefit patients with cancer attending a specialist palliative care day centre? Palliat Med 18:287–290 Wilkinson S (1995) Aromatherapy and massage in palliative care. Int J Palliat Nurs 1(1):21–30 Wilkinson S, Aldridge J, Salmon I et al (1999) An evaluation of aromatherapy massage in palliative care. Palliat Med 13:409–417 Wilkinson SM, Love SB, Westcombe AM et al (2007) Effectiveness of aromatherapy massage in the management of anxiety and depression in patients with cancer: a multicenter randomized controlled trial. J Clin Oncol 25:532–539 Woolfson A, Hewitt D (1992) Intensive aromacare. Int J Aromather 4(2):2–14 Yehuda R, Teicher MH, Trestman RL et al (1996) Cortisol regulation in posttraumatic stress disorder and major depression: a chronobiological analysis. Biol Psychiatry 40:79–88 Yim VWC, Adelina KYN, Hector WH et al (2009) A review on the effects of aromatherapy for patients with depressive symptoms. J Alt Comp Med 15(2):187–195 Yoon WJ, Kim SS, Oh TH et al (2009) Cryptomeria japonica essential oil inhibits the growth of drug-resistant skin pathogens and LPS-induced nitric oxide and pro-inflammatory cytokine production. Pol J Microbiol 58:61–68

Chapter 14

Perillyl Alcohol: A Pharmacotherapeutic Report Molecular Genetics of Malignant Gliomas Disclose Targets for Perillyl Alcohol Clovis O Da Fonseca and Thereza Quirico-Santos

Historical Overview of Gliomas The incidence of all of primary brain and central nervous system (CNS) tumors is estimated at 18.71 per 100,000 individuals per year according to data from the USA Central Brain Tumor Registry. Primary brain tumors are one of the top ten causes of cancer related deaths in Europe and North America (Canadian Cancer Society’s Steering Committee 2010). Gliomas are tumors that arise from glial cells, and include astrocytoma, glioblastoma (GBM), oligodendroglioma, ependymoma, mixed glioma, malignant glioma, and a few more rare histologies. They are classified according to their presumed cell of origin and extent of brain infiltration—circumscribed or diffusely infiltrating—and include astrocytomas, oligodendrogliomas, and ependymomas (Fig. 14.1). GBM are more common among men than women (male:female ratio is 1.58:1) and twice as common in Caucasian versus African Americans (Ostrom et al., 2014). The median age of patients at time of diagnosis is approximately 64 years. Approximately 5 % of patients with malignant gliomas have a family history of gliomas. Some of these familial cases are associated with rare genetic syndromes, including neurofibromatosis types 1 and 2 and Li–Fraumeni syndrome (Farrell and Plotkin 2007).

C. O. Da Fonseca () Faculty of Medicine, Fluminense Federal University, Niterói, RJ, Brazil e-mail: [email protected] T. Quirico-Santos Department of Cell and Molecular Biology, Institute of Biology, Fluminense Federal University, Niterói, RJ, Brazil © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_14

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C. O. Da Fonseca and T. Quirico-Santos

Fig. 14.1  Primary brain tumor distributions. (From CBTRUS Central Brain Tumor Registry of the United States CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2007–2011. Reprinted with permission from Oxford University Press)

Pathological Features of Astrocytomas Astrocytomas are heterogeneous in nature and may show diffuse infiltration of adjacent and distant brain structures (Kleihues et al. 2002)(Kleihues and Cavanee, 2002). These neoplasms are histologically graded based on criteria set by the World Health Organization (WHO) into four prognostic grades (Table 14.1). Pylocytic astrocytomas, which are benign and relatively circumscribed, are classified as grade I tumors. Low-grade diffuse astrocytomas, classified as grade II astrocytoma, are histologycally characterized by moderate cellularity, mild nuclear atypia, and rare or absent mitotic figures and are moderately proliferative and invasive. Anaplastic astrocytoma (AA) or grade III astrocytomas are characterized histologically by increased cellularity, nuclear atypia, and mitotic activity. These tumors are more proliferative and infiltrative compared with grade II. GBMs, classified as grade IV astrocytomas, are histologically similar to AA but in addition are characterized by the presence of necrosis, glomeruloid microvascular proliferation, and pseudopallisading. GBMs are significantly more proliferative, invasive, and angiogenic compared with grade II and grade III astrocytomas. Malignant gliomas typically contain both neoplastic and stromal tissues which contribute to their histologic heterogeneity. The prognosis for patients with malignant gliomas remains dismal: the median survival of AA patients is 2–3 years, and that for GBM patients is 10–12 months.

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Table 14.1  The incidence rate of all primary malignant and non-malignant brain and CNS tumors.

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Molecular Genetics of Malignant Gliomas Traditionally, GBMs have been classified into primary or secondary subtypes on the basis of clinical presentation and features, although both subtypes are indistinguishable at a morphological level. Approximately, 95 % of GBMs present as de novo primary tumors and typically occur in older patients (England 2013). Secondary GBMs arise and progress from lower grade astrocytomas, are quite rare, and tend

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Fig. 14.2  Frequency of genetic alterations during malignant glioma progression. (Modified from Ohgaki et al. (2007))

to occur in patients below the age of 45. It is now known that both subtypes constitute distinct disease with specific genetic differences documented between primary and secondary GBMs (Fig. 14.2). Primary GBMs are characterized by epidermal growth factor receptor ( EGFR) gene amplification and mutation; loss of heterozygosity (LOH) of chromosome 10q containing phosphatase and tensin homolog ( PTEN); overexpression of mouse double minute 2 (MDM2); and deletion of p16. The hallmarks of secondary GBMs include mutations of p53 and retinoblastoma ( RB); overexpression of platelet-derived growth factor (PDGFA; PDGF-α); and loss of 19q (Fig. 14.2).

Neural Stem Cell-Related Pathways in Glioblastoma Multiforme Recent studies suggest that a small subpopulation of cancer stem cells (CSCs) has the capacity to repopulate solid tumors such as GBM, drive malignant progression, and mediate radio- and chemoresistance. GBM-derived CSCs share the fundamental stem cell properties of self-renewal and multipotency with neural stem cells and may be regulated by miRNAs (González-Gómez 2011).

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In fact, when we try to understand the laws of physico-chemistry, as the principle of Le Chatelier, we can find answers that can explain the phenomena that occur in the process of tumor recurrence after radiation therapy and chemotherapy. According to the principle of Le Chatelier, “Any system in equilibrium, when actuated by an external agent evolves in such a way as to minimize this action” and considering the morphology of spheroidal tumors fed from their surfaces, we can conjecture that (1) in all spheroidal tumors, the number of neoplastic cells grow proportionally to the tumor volume, that is to say, proportionally to the third power of the radius, and (2) the number of veins and arteries that penetrate the surface of the tumor increases linearly with the area of the spheroid or proportionally to the square of the spheroid radius. Thus, as the tumor grows the supply of nutrients and oxygen becomes severely limited relative to cell requirements (loss of stoichiometry) and consequently, there is an increase in production and storage of lactic acid and other abnormal metabolites within the cell, compromising their normal metabolism. If there is rapid production and storage of lactic acid and other abnormal metabolites within the cell, self-poisoning might occur, followed by apoptosis or necrosis. If the supply of nutrients and oxygen is slightly below the basal cell metabolism requirements, a slow progressive storage of lactic acid and anomalous metabolites will take place within the cell. This slow storage of metabolites allows the cell a degree of self-control. Initially, the internal pressure is approximately preserved. When the levels of lactic acid and anomalous metabolites reach critical values, the following facts occur in succession: (1) the cells stop to divide; (2) to minimize the toxicity of intracellular environment, the cells take up water through an osmotic process and increase their volume and internal pressure (dilution of metabolites). This mechanism is an immediate consequence of Le Chatelier’s principle, and aims at self-preservation of the cell. Thus, the penetration of oxygen and nutrients in these cells by a driving process is inhibited, and only occurs by a diffusion process which is slow and inefficient. Under such conditions, the cell assumes a state of latency (suspended animation) with high pressure and very low metabolism. Eventually, through mechanisms such as thermal agitation they are capable of toxins excretion and return to their original exacerbated metabolism. These cells under a state of latency (high pressure, low metabolism, and acid environment) are resistant to radiation and chemotherapy and may account for tumor recurrence following treatment. Notwithstanding, a third group of cells, generally located near arteries, with unrestricted metabolism (cell requirements are completely supplied by the local vascular system) accounts for the fast growth of tumor. Under such conditions these cells have low internal pressure and low acid environment being very susceptible to radiation and chemotherapy treatments. We studied the populations of cells in deep position and peripheral position of gliomas through in vitro experiments. We found larger number of cells in deep location than in peripheral location (Fig. 14.3). Aiming at the development of therapeutic strategy, our group studied the effect of POH as an agent that has mechanism of action on the cell membrane. In fact, the

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Fig. 14.3  In vitro culture of tumor tissues. Higher concentration of cells identified in the central part of the tumor (R. Holanda—Laboratory of Cellular Morphogenesis—Rio de Janeiro Federal University)

POH is a molecule with a polar tail (hydrophilic) termination and a covalent (hydrophobic) head. When in contact with the plasma membrane, which is amphipatic, the polar end of the POH is connected electrically to the membrane, mechanically altering its stability. The hydrophobic end prevents the POH from fully penetrating the cell membrane (Joy Bowles 2004, 3rd Edition). In this condition, the POH remains attached to the cell for a certain period of time which depends on tumor temperature, pH, and cell environment, eventually being uncoupled by thermal agitation mechanisms. If the tumor cell undergoes a process of division with the POH attached to it, the mechanical disturbance produced by the POH will cause the membrane to burst and kill the cell. In normal cells, not in the fast reproduction process, POH binds to the plasma membrane and eventually is disengaged by the energy supplied from the thermal agitation before the cells undergo reproduction (Duelund 2012).

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Novel and Investigational Therapies for Malignant Gliomas The standard therapy for newly diagnosed malignant gliomas involves maximal surgical resection, radiotherapy, and chemotherapy, yet this does little to improve life expectancy. The dismal prognosis of patients diagnosed with GBMs is attributed to the highly infiltrative nature of these tumors, as well as the heterogeneous molecular profiles of seemingly histological similar GBMs. The objective of many investigational therapies is to (1) target cellular pathways or specific molecules implicated in pathogenesis and (2) identify molecular features of the tumor that predict a response, so that patients who are most likely to benefit can be selected for a particular treatment. In fact, most gene alterations induce cell-cycle dysfunction on a complex molecular level. Further insight into tumor genesis by means of genomic assays may aid in predicting the clinical behavior of GBM and in providing individualized potential targets for therapeutic agents.

The Ras Pathway as a Target for GBM Therapy Gliomas are suitably targeted by molecular therapy because they display a set of defined molecular lesions and signaling pathway alterations that may be used as targets for therapy. Primary GBMs, those arising as de novo lesions, commonly overexpress EGFR and its ligand-independent mutant EGFRvIII (Mischel and Cloughesy 2003). This results in signaling through Ras/MAPK and PI3K/Akt pathways. Target molecules extensively studied include EGFR, PDGFR, PTEN, telomerase, and signal pathway modulators for PI3K/Akt and Ras/MAPK pathways. Therapies targeting these specific molecules may result in killing tumor cells effectively while keeping normal cells intact. Ras is an integral signaling element and has been characterized as the primary switch that transmits external signals through numerous intracellular signaling pathways. Ras belongs to a superfamily of small molecular weight guanine nucleotide-binding proteins with the intrinsic ability to hydrolyze guanosine 5V-triphosphate known as GTPases. At least 20 members of this superfamily have been identified. Three genes encode Ras proteins: N-RAS, H-RAS, and K-RAS. The three Ras proteins coded by these genes are closely related to one another and are similar in their ability to interact with regulators and effectors. Ras proteins are a class of nucleotide-binding proteins that play pivotal roles in the control of normal and transformed cell growth. Experimental studies on the structure, function, and regulation of Ras proteins indicate that they are key intermediates in signal transduction pathways that mediate proliferative and other types of signals largely from upstream of receptor kinases, which control a wide variety of cellular processes, including growth, differentiation, apoptosis, cytoskeletal organization, and membrane trafficking (Karp 2001; Clark 2003). For Ras transduce the extracellular signals provided by growth factors and cytokines, it must be associated with

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the inner surface of the plasma membrane. Ras proteins are a family of membraneassociated small GTPases that transmit signals from cell surface receptors such as EGFR, EGFRvIII, and PDGFR, promoting diverse cellular effects such as proliferation survival and angiogenesis (Heldin and Lennartsson 2013). Membrane anchorage of Ras, required for functional activity in signal transduction, is facilitated by posttranslational modifications resulting in covalent attachment of a farnesyl group to the cysteine in the C-terminal CAAX motif. This attachment is mediated by farnesyltransferase (Karp 2001). These lipophilic modifications facilitate the association of isoprenylated proteins with an intracellular membrane which is a functional requirement. Farnesylation of Ras enhances its ability to stimulate downstream signaling enzymes, including mitogen-activated protein kinase in mammalian cells. Unfarnesylated Ras proteins do not associate the plasma membrane and are incapable of cellular transformation. Farnesyl transferase inhibitors represent a new class of agents that target signal transduction pathways responsible for the proliferation and survival of diverse malignant cell types. Although these agents were developed to prevent a processing step necessary for membrane attachment and maturation of Ras proteins, recent studies suggest that farnesyltransferase inhibitors block the farnesylation of additional cellular polypeptides, thereby exerting antitumor effects independent of the presence of activating ras gene mutations (Karp 2001). Although GBM does not display Ras mutations, it may have enhanced expression of Ras. Also, GBM expresses high levels of ligand-dependent and ligand-independent growth factor (EGF and PDGF) receptors. Activation of these receptors leads to tyrosine kinases activation and functional upregulation of the Ras signaling pathway or expresses the activated form of this protein (Guha 1997). Overexpression and activation of receptor tyrosine kinases, such as PDGFR and EGFR, lead to proliferation of human malignant astrocytoma cells. Although oncogenic mutations affecting Ras are not prevalent in human malignant astrocytomas, they might be elevated in these tumors secondary to the mitogenic signals originating from activated receptor tyrosine kinases. In support of this hypothesis, high levels of Ras. GTP, similar to those found in oncogenic Ras-transformed fibroblasts, were present in 4 established human malignant astrocytoma cell lines that express PDGFR and EGFR in 20 operative malignant astrocytoma specimens (Ding 2001). Stimulation of PDGFRs and EGFRs induced tyrosine phosphorylation of the Shc adaptor protein and its association with Grb2, suggesting a mechanism by which Ras may be activated in human malignant astrocytoma cells. Furthermore, blocking Ras activation by expression of the H-Ras-Asn dominant-negative mutant, or by farnesyl transferase inhibitors, decreased in vitro proliferation of the human astrocytoma cell lines (Feldkamp 2001). These results support the hypothesis that proliferative signals from receptor tyrosine kinases expressed by human malignant astrocytoma cells use the Ras mitogenic pathway. Pharmacological inhibitors of the Ras pathway may therefore be of therapeutic value in these presently terminal tumors. Previous studies have demonstrated that astrocytomas express elevated levels of activated Ras.GTP despite the absence of activating Ras mutations. The importance of increased Ras activity in GBM is

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supported by studies demonstrating reduced growth of preclinical GBM if treated with dominant-negative Ras mutants or Ras pathway inhibitor drugs. Furthermore, overexpresssion of H-Ras in a transgenic mouse model resulted in early death from growth of multifocal malignant astrocytomas (Levin 1999). Overall, the current data suggest that activation of the Ras signaling pathway in malignant gliomas is due to aberrant expression and overactivity of membrane tyrosine kinase receptors, including EGFR, PDGFR, FGFR, and IGF-IR.

Perillyl Alcohol Inhibition of the Ras/MAPK Pathway Over the past three decades, we have made great strides in the treatment of most, but not all, brain tumors. Dramatic advances have occurred in diagnostic imaging, neurosurgery, neuroanesthesia, radiotherapy, and chemotherapy for CNS tumors. Unfortunately, our progress has not yet met our expectations. Due to the infiltrative nature of most primary brain tumors, neurosurgery can never be expected to be curative for the majority of gliomas. As infiltrative tumors interdigitate with normal brain cells and are not highly sensitive to irradiation, one cannot expect radiotherapy to be curative without serious damage to normal brain cells. The hope for a cure, then, rests with chemotherapy. The ability to treat most advanced malignancies with classic cytotoxic DNA-damaging agents is limited, with little curative potential and rare durable remissions. This has led to emphasis on the development of new therapeutic agents with novel mechanism of action. In fact, the increasing discovery of new drugs and the introduction of new medications in the pharmaceutical market have led to a need to develop techniques to better control the occurrence of toxic and collateral effects of these products. In this context, cancer researchers now have a unified concept to guide their search for specific genetic abnormalities. The genes and proteins that participate in the conversion of normal into malignant cells are also involved in the key processes that convert extracellular events that culminate in division and growth. The search for new chemotherapeutic drugs has increased, especially for those that have a natural origin. Diverse mevalonate-derived products of secondary metabolic pathways present in plants have both chemotherapeutic and chemopreventive properties. The mevalonate pathway produces isoprenoids that are vital for diverse cellular functions, ranging from cholesterol synthesis to growth control. Several mechanisms for feedback regulation of low-density-lipoprotein receptors and of two enzymes involved in mevalonate biosynthesis ensure the production of sufficient mevalonate for several end products. Manipulation of this regulatory system could be useful in treating certain forms of cancer (Goldstein 1990). Perilyll alcohol, also known as p-mentha-1,8-diene-7-ol and sometimes as 4-isopropenyl-cyclohexenecarbinol, is composed of two isoprene units produced by the mevalonate pathway. It has been found to be active in inducing apoptosis in tumor cells with no impact on normal cells and can, in fact, turn back tumor cells to a differentiated state (Belanger 1998). Although the mechanism by which POH exerts

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its anticancer activity is not clear, a number of potentially important drug-related activities have been observed in preclinical studies, including cellular effects such as an early G1 arrest and the induction of apoptosis, biochemical effects such as the inhibition of posttranslational modification of proteins involved in signal transduction and differential gene regulation with overexpression of M6P/IGF-II and TGF-b type II receptor genes. Indeed, it has been postulated that the anticarcinoma activity of POH involves a decrease in the levels of isoprenylated Ras and Ras-related proteins, thereby reducing the physiological functioning of these proteins (Ariazi 1999). Protein isoprenylation involves the posttranslational modification of a protein by the covalent attachment of a lipophilic farnesyl isoprenoid group to a Cys residue at or near the carboxyl terminus. Isoprenoid substrates for prenyl protein transferase enzymes include farnesyl pyrophosphate and geranylgeranyl pyrophosphate, two intermediates in the mevalonate pathway (Goldstein 1990). This action was widely attributed to the inhibition of farnesyl protein transferase activity. Farnesylation of Ras also greatly enhances its ability to stimulate downstream signal transduction enzymes, including mitogen-activated protein kinase in mammalian cells (Itoh 1993). Farnesylation is the most critical part of the process that leads to the activation of Ras (Feldkamp 1999), and farnesyl transferase inhibitors exert their antitumor effect, in part, by inhibiting Ras-mediated signaling. Nevertheless, farnesyl transferase inhibition may also block signaling from other pathways that also require farnesylation, including the Rho B and PI3K/Akt pathways (Jiang 2000). Also, follow-up studies revealed that POH suppresses the synthesis of small G proteins and HMG CoA reductase (Mo 2004). In addition, POH has been shown to induce apoptosis and cause a G0/G1 arrest in liver tumors (Mills 1995), colon cancer cell (Reddy 1997), mammary carcinomas cells (Shy 2002), leukemic cell lines (Sahin 1999), and a transitory G2 arrest and Fas-mediated apoptosis in prostate cells (Jeffers 1995) and GBM cells (Rajesh 2003). In pancreatic ductal adenocarcinoma cells, the apoptotic effect of POH appears to involve the increase of the proapoptotic protein Bak in a wild-type p53-dependent way (Stayrook 1997). Our experiments (Fernandes 2005) showed that in vitro treatment of POH consistently inhibited proliferation, produced marked changes in cell morphology, inhibited protein synthesis, caused marked alteration in membrane permeability, and drastic changes in the cytoarchitecture of C6, U87MG, and A172 cells. We have previously shown that in in vivo treatment of GBM cells, POH showed inhibition of cell migration and antimetastatic activity in the model of the chick embryo with C6 cell line (Teruszkin 2002). In this work we have indicated that POH inhibits proliferation of the C6 glial cell line. POH was logarithmically diluted in concentrations of 30 % through 0.0003 % and showed inhibition cell proliferation of 78.36 % in concentration of 30 %; 69.87 % in concentration of 3 %; and 67.04 % in concentration of 0.03 % (Fig. 14.4). Also POH 1.6 µM was cytotoxic to GBM cells (Fig. 14.5).

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Fig. 14.4  Effect of perillyl alcohol on C6 cells proliferation. The dilutions of POH are displayed in the horizontal axis, ranging from 30 to 0.0003 %. The vertical axis represents percentage of growth comparing with untreated control cells. Treatment with POH 30–0.03 % caused significant inhibition of cell growth (80–60 % approximately), while concentrations below 0.3 % showed little effect

Fig. 14.5  U87MG cell line viability and morphology in the presence of perillyl alcohol. a POH was cyotoxic to GBM cells after 24 h drug addition in a dose-dependent manner. Each point on the graph represents a concentration in ascending order (100, 250, 500, 750, 1000, 1500, 2000 µM). Results were expressed as mean ±SD of at least three experiments performed in triplicate. GBM cells morphology incubated with b medium, c 1.5 mM POH, and d 2 mM POH was observed by optical microscopy with × 20 of magnification. ( Master Degree Thesis—Maximino Alencar Bezerra Júnior—under guidance Prfa Lidia Amorim and Clovis O. Da Fonseca)

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Perillyl Alcohol for the Treatment of Recurrent Malignant Gliomas GBMs are the most aggressive primary brain tumors and their heterogeneity and complexity often render them nonresponsive to various conventional treatments. The development of novel therapies based on the understanding of the basic biology of brain tumors is needed to improve the treatment of malignant gliomas providing the data for the development of substances that have activity in the cell membrane unlike the alkylating agent. In this respect, aiming at the development of therapeutic strategy, our group studied the effect of POH as an agent that has mechanisms of action on the cell membrane. For example, we have used mass spectrometrybased proteomics to study the modifications these tumoral cells undergo during the process of apoptosis when exposed to POH (Fischer 2010); by identifying and quantifying more than 4000 proteins, we were able to observe key changes in the differential expression of proteins linked to several pathways including that of RAS, homeostasis, induction of apoptosis, metallopeptidase activity, and ubiquitin-protein ligases activity to name a few. The results also proposed alterative mechanisms of the POH-activated process through the phosphorylation of glycogen synthase kinase 3 (GSK-3) and the inhibition of extracellular signal-regulated kinases (ERK). In face of these results, we further investigated the effects of POH on the tumor by comparing the proteome of resistant and non-POH-resistant cells. Needless to say, such is fundamental so that advances in the treatment with POH can be done as tumoral cells are likely to gain resistance at some stage. That said, in a previous and preliminary report, we disclosed fundamental data on alterations that A172 cells undergo to become resistant to POH (Fischer 2010); this study addressed a membrane-enriched fraction of the A172 cells. Such was performed as we strongly believe that proteins anchored to the membrane compose the key components for understanding the chemotherapeutic effects of POH. We hypothesize this because POH is a molecule with a polar tail (hydrophilic) termination and a covalent (hydrophobic) head. When in contact with the plasma membrane, which is amphipatic, the polar end of the POH is connected electrically to the membrane, mechanically altering its stability. The hydrophobic end prevents the POH from fully penetrating the cell membrane (Duelund 2012). In this condition, the POH remains attached to the cell for a certain period of time which depends on tumor temperature, pH, and cell environment, eventually being uncoupled by thermal agitation mechanisms. If the tumor cell undergoes a process of division with the POH attached to it, the mechanical disturbance produced by the POH causes the membrane to burst and kill the cell. Although the microscopic events of this POH attachment are not well understood, it may act on the sequence of reactions within the cell nucleus further enhancing its division process. In normal cells, not in the fast reproduction process, POH binds to the plasma membrane and eventually is disengaged by the energy supplied from the thermal agitation before the cells undergo reproduction (Duelund 2012). These compounds are known to be biologically active and exhibit antimicrobial, anticancer, and anti-inflammatory effects that could all be membrane mediated. This fact may account for the low side effects associated with POH therapy.

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Intranasal Perillyl Alcohol—Clinical Trials in Recurrent Malignant Gliomas As Ras activation plays an important role in the pathogenesis of GBM (Dontula 2013), it makes sense that inhibition of elevated Ras activity represents a pharmacological-based approach for a potential treatment of these tumors. Perillyl alcohol inhibits Ras/MAPK signaling pathway, making it feasible to begin considering its use for patients with relapsed GBM. Phase I and phase II trial protocols in humans indicate that oral administration of POH affects mainly the gastrointestinal tract, causing nausea, vomiting, and diarrhea. No evidence of hepatic, renal, or neurobiological toxicity has been observed and the maximum tolerated dose determined was 8.4 g/m2 per day delivered orally in four doses with and modest clinical responses were attained in trials (Bailey 2008). In accordance with these directions, we are developing a new method for POH delivery, that is, POH inhalation. Intranasal delivery allows POH to cross the blood–brain barrier and reach the CNS, eliminating the need for systemic delivery and reducing the side effects. Based on the favorable therapeutic ratio observed in vitro and in vivo treatment, commercial availability, low cost, and low toxicity, we are developing phase I and phase II clinical trials that delivers POH by inhalation in patients with relapsed GBM. This trial was approved by the Brazilian Committee of Ethics and Research (CONEP 9681 no. 25000.009267/2004-25, July 12, 2004). After our in vitro and in vivo studies we determined the administration of POH 23 drops diluted in 3 ml of mineral water with pH above 7 to alkalinize the acidity of peritumoral edema (PTBE), in a common nebulizer. The POH was administered by inhalation four times daily in an interrupted administration schedule in patients with recurrent malignant gliomas after standard therapy. In this study, all patients received the starting dose of 23 drops (66.7 mg) four times totaling 266, 8 mg/ daily, with dose escalation up to 46 drops (133, 4 mg) four times daily totaling 533, 6 mg/daily. The cohort included 198 patients (117 men and 81 women) with measurable contrast-enhancing tumor image, Karnofsky index ≥ 70 %, mean age 53.4 years (range 19–83) with primary GBM n = 154), grade III astrocytoma (AA; n = 26) and anaplastic oligodendroglioma (AO; n = 15). POH inhalation schedule four times daily started with 66.7 mg/dose totaling 266 mg/day with escalation to 133.4 mg/ dose totaling 533.6 mg/day. Clinical toxicity and overall survival following treatment were compared with tumor size, topography, extent of peritumoral edema, and histological classification.

Patients and Methods This clinical trial approved by the Hospital Medical Research Ethics Committee and the Brazilian Ministry of Health (CONEP 9681 no. 25000.009267/2004-25, July 12, 2004) complies with the principles laid down in the Declaration of Helsinki.

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Informed consent was obtained from each patient and the next-of-kin before beginning the study.

Patient Selection This prospective study was carried out from July 2006 to July 2012 with patients attending the outpatient neurosurgical unit in the Antonio Pedro University Hospital and included in the phase I/II clinical trial to assess the efficacy of intranasal administration of the monoterpene POH. The cohort was included after appropriate informed consent of 198 adult patients (random selection) with recurrent malignant glioma and under symptomatic treatment after failing response to current standard treatment that included surgery, and/or radiation, and multimodal chemotherapy specific for GBM. Diagnosis and histological classification of malignant glioma was based upon WHO criteria. Eligibility criteria included patients older than 18 years with recurrent GBM with at least two relapses, measurable contrast-enhancing tumor image on magnetic resonance, Karnofsky index ≥ 70 %, adequate hematological clinic laboratory-based measures, stable heart rhythm, and no clinical evidence of congestive heart failure or unstable angina. Exclusion criteria included pregnancy, hematologic malignancy, occurrence of seizures, recurrence within the last 96 h before inclusion; concomitant infectious or inflammatory processes; acute cerebrovascular or hemorragic event. According to WHO classification patients’ distribution were as follows: 155 (78 %) with GBM IV, 27 (14 %) with AA and 16 (8 %) AO. All patients were followed by magnetic resonance imaging (MRI) and clinical evaluation every 3 months. Adverse events were graded according to Common Terminology Criteria for Adverse Events—Version 4.0 (CTCAE). The cohort included patients older than 18 years old with recurrent malignant glioma, with measurable contrast enhancing tumor on CT scan and/or MRI, Karnofsky index 70 % or higher. The majority of patients had adequate hematologic functions (hemoglobin; blood cell counts) and clinic laboratory-based measures (bilirubin, serum glutamic oxaloacetic transaminase, alkaline phosphatase, creatinine kinase) within the normal range; stable heart rhythm, no clinical evidence of congestive heart failure or unstable angina. At the moment of the study, none were under radiation therapy or chemotherapy for more than 4 weeks and were receiving only symptomatic palliative treatment. All patients included in the present study were under palliative symptomatic treatment because they had failed current standard treatment for malignant glioma recurrence. A correlation of initial symptoms and clinical presentation, with topographic localization (lobar or deep) and size of the tumor in the brain tissue was done based on brain MRI image. Lobar tumors were defined as any tumoral lesion away from the basal ganglia. All tumors limited to or involving the basal ganglia were classified as deep gray matter lesions. Tumor size was measured on axial contrast enhanced scans using the scale of the largest perpendicular diameters of the enhancing lesion.

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Extent of PTBE, mass effect, and midline shift (cm) were evaluated at initial and during the course of treatment with monoterpene POH. The area occupied by peritumoral edema was determined on the same axial slices used for tumor size measurements by subtracting tumor from edema diameters. The comparison was kept constant with same type of axial imaging (CT or MRI). It was also considered the histological classification as AO, AA, or GBM, previous treatment (biopsy, surgical excision, radiotherapy, chemotherapy), and overall survival after recurrence.

Drug Administration and Dose Escalation Perillyl alcohol was formulated for intranasal delivery and the preparation supplied by the University Pharmacy according to the following patent: BR application number 0107262-5 December 17, 2001. Perillyl alcohol 0.3 % v/v POH (55 mg) was administered by inhalation four times daily. All patients received POH four times daily by intranasal (inhalation) delivery from initial dose (66.7/dose; totaling 266.8 mg/daily), and escalation 133.4 mg/dose, totaling 533.6 mg/daily.

Statistical Analysis Statistical analysis was carried out using Kaplan–Meier curves, log rank tests, and univariate and multivariate Cox regression models. The association of categorical variables with survival was assessed using Kaplan–Meier curves and log rank tests, and the significance of continuous variables was assessed using univariate Cox regression models.

Results Demographic characteristic of patients are specified in Table 14.1. Patients were stratified into two groups: recurrent primary GBM and recurrent secondary GBM derived from low-grade glioma. Gross total resection was performed in 103 patients, whereas 95 patients underwent subtotal resection or stereotactic biopsy. According to CTCAE, five patients had nasal aching, and two patients had epistaxis after prolonged use with 133.4 mg/dose, totaling 533.6 mg/daily POH. After specific local treatment, patients improved clinical condition and POH treatment was reduced to lower doses. Any evidence of local or systemic infection, gastrointestinal toxicity, and hematological side effects, even in those patients with prolonged treatment with intranasal POH for more than 4 years was not observed. The most frequent complaint at onset was intense headache (53 %), but patients also reported focal neurological signal—hemiparesis (29 %); convulsive seizures

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Table 14.2  Patient characteristics ( n = 198) GBM 155 (78 %) Histology Tumor type Recurrent primary GBM 144 (92 %) Recurrent secondary GBM 11 (8 %) 51 (38–64) Age, years; mean (range) Gender Male 92 (59 %) Female 63 (41 %) Initial predominant symptom (GBM, AA, AO) Headache Seizures Focal neurological deficit Short-term memory loss Temporary confusion Behavioral changes Site of tumor Lobar 122 (78 %) Deep 33 (21 %) Treatment of the initial tumor Gross total resection 78 % Subtotal resection or biopsy 22 % Radiotherapy 100 % Chemotherapy 100 % PFS with POH intranasally (month 6th) 48 % Edema on initial tumor (MRI) None 5 (9.6 %) 24 (46.2 %) < 5 cm 28 (53.8 %) > 5 cm Edema on recurrent tumor (MRI) 19 (36.5 %) < 5 cm 33 (63.4 %) > 5 cm Midline shift on initial tumor (MRI) None 8 (15.3 %) 19 (36.5 %) < 1 cm 25 (48.7%) > 1 cm Midline shift on recurrent tumor (MRI) 18 (35.5 %) < 1 cm 34 (64.5 %) > 1 cm

AA 27 (27 %)

AO 16 (8 %)

52 (41–64)

49 (28–69)

16 (59 %) 11 (41 %)

9 (56 %) 7 (44 %)

53 % 25 % 52 % 11 % 14 % 7% 22 (80 %) 5 (20 %)

16 (100 %) None

77 % 23 % 100 % 100 % 60 %

100% 0% 100% 100% 66%

1 (10 %) 7 (70 %) 2 (20 %)

3 (60 %) 2 (40 %) none

6 (60 %) 4 (40 %)

5 (100 %) 1

2 (20 %) 6 (60%) 2 (20%)

3 (60 %) 2 (40 %) none

4 (40 %) 6 (60 %)

3 (60 %) 2 (40 %)

(25 %); nausea and mental confusion (14 %); visual dizziness and lack of memory (11 %); behavior alteration, deficit of language and cognition (7 %), sleepiness (4 %) before diagnostic confirmation of malignant brain tumor. Prevalent complaints at tumor recurrence were neurological deficits (51 %); intense headache (43 %); and seizures in 24 % (Table 14.2).

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Fig. 14.6  Magnetic resonance imaging (MRI) images showing intranasal POH administration effect in two grade II astrocytoma patients. One patient had a decrease in the tumor size between initial MRI (a) and after 1 year (b), 2 years (c), or 3 years (d) of POH treatment. Conversely, we can observe persistent tumor size in initial MRI (e), and after 4 years (f) of POH treatment

Long-term treatment (up to 4 years) with daily intranasal POH administration as single chemotherapy drug stabilized ( n = 3) and improved clinical condition ( n = 2) of patients with grade II astrocytoma (Fig. 14.6). However due to small number of AA ( n = 27) patients included, further studies are required to determine the effect of treatment on the natural history of the disease mainly due to evident reduction of tumor size in comparison with corresponding MRI taken before patient inclusion in POH treatment. However, it is important to establish a relation between the effectiveness of treatment with topography and histo-molecular characteristics of the tumor, because even patients with dull response to POH treatment showed albeit reduction of tumor size after intranasal POH treatment. A 6-month progression-free survival rate including partial responses and stable disease of glioma patients according to the histological diagnosis was GBM: 48 %; AA: 60 %; and AO: 67 %. Survival rate after 24 months of POH treatment was 6.2 % for primary GBM, 63 % for secondary GBM comprising 15 % for AA, and 56 % for AO. Kaplan–Meier curves showed that patients with secondary GBM had high survival rate in comparison with primary GBM ( p < 0.0001) (Graph 1-A) evidenced by a sharp decline of tendency line. Patients with AA showed a significant ( p = 0.0199) decline of tendency line compared with AO (Graph 1-B), which conversely had prolonged survival rate further evidenced by a mild and steady decline of tendency line.

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Fig. 14.7  Effect of intranasal POH treatment in recurrent oligoastrocytoma patient with recurrent oligoastrocytoma and extensive peritumoral edema failed to respond to POH treatment. MRI obtained before (a) and 4 months after POH treatment (b). Good responder patient showed in comparison with MRI (c) obtained before entrance in POH protocol a marked reduction (d) in the tumor size after 4 years of POH treatment. Before treatment HE staining (e) shows increased glomeruloid microvascular and glial cell proliferation with atipia, hypercromatism, and nuclear pleomorphism. Brain biopsy fragment obtained from intracranial drainage of hematoma 4 years after intranasal POH treatment shows (f) apparently normal histological characteristics of glial and neuronal cellularity

Lobar location was present in 80.8 % of glioma patients (Table 14.2), and as previously reported it was also observed a correlation of tumor topography with therapeutic response to intranasal administration of POH (Da Fonseca CO 2009). Patients with tumoral lesion in the basal ganglia survived longer than those with tumors at lobar localization. Likewise, presence of peritumoral edema, midline shift, and requirement for continued use of steroids also influenced morbidity and tumor invasive recurrence. Furthermore, maintenance of extensive brain edema (> 5 cm) and midline shift (> 1 cm) since the initial MRI scan despite regression of tumor size, determined a poor response to POH treatment and shorter survival than those patients with moderate edema (Fig. 14.7). Nonetheless, patients with either

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Fig. 14.8  Effect of intranasal POH treatment in recurrent GBM. Representative brain image (MRI) of a patient with recurrent primary GBM. Note a decrease in the tumor size between the initial MRI (a and b) and 6 months after treatment (c and d)

recurrent AA or GBM showed good response to intranasal POH administration with no PTBE (Fig. 14.8). In the present study, we used steroidal therapy withdrawn as parameter indicative of morbidity and quality of life. In this context, good responders to treatment included patients with prolonged survival who were not using steroids for a long period of time. On the contrary, patients still under treatment with high dosage of steroidal drugs besides persistence of large peritumoral edema always showed negative response to POH treatment, and morbidity was often associated with recurrent infections, and deep venous and carotid artery thrombosis. Prolonged POH inhalation chemotherapy did not cause cumulative toxicity, and neither altered clinical chemistry (hepatic, renal, lung) and hematological parameters. Glioma patients (19 %) with good response to POH treatment and improvement of clinical–neurological status without evidence of MRI tumor recurrence had POH dosage reduced to the lower concentration. However, full adhesion to POH treatment was of utmost importance because discontinuation (Fig. 14.9) caused tumor recurrence even after early MRI image showing marked reduction of the tumoral lesion.

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Fig. 14.9  Tumor recurrence after treatment discontinuation. Note a decrease in the tumor image size of a patient with secondary GBM at the time of inclusion in POH trial (a); image obtained 3 years after continuous treatment with intranasal POH (b); and brain MRI obtained 3 months after patient have quit treatment (c)

Future Directions Patients with worse prognosis and poor response to intranasal POH treatment had tumoral lesion in the supratentorial region which presents a dense microvascular network toward the brainstem. The tumoral microenvironment is reach in growth factors and tissue proteases that diffuse through the peritumoral stroma and activate intracellular pathways, thereby promoting extensive neovascularization and generation of epigenetic events that promote new oncogenic mutations, and lost-in-function of different tumor suppressor genes (De Palma and Hanahan 2012; Esteller 2006). Indeed, abnormal methylation of CpG promoter regions of several genes has been recognized as an important mechanism of epigenetic gene silencing, contributing to changes in gene expression and resistance to alkylating drugs (Di Vinci 2012). For development of new therapeutic approaches, it is important to determine major molecular pathways responsible for proliferation, invasion, angiogenesis, and anaplastic transformation of glioma cells. We demonstrate that intranasal administration of a p21-Ras lipophilic inhibitor that easily crosses the blood–brain barrier is a safe and noninvasive strategy capable to prolong overall survival of patients with recurrent malignant glioma considered at terminal stage. As a cytotoxic agent, POH inhibits cell cycle, upregulates the pro-apoptotic protein Bax and also TMZ-resistant and TMZ-sensitive glioma cells, independently of O6-methylguanine-DNA methyltransferase (MGMT) expression. POH cytotoxicity upon glioma cell lines resulted from the effect on the endoplasmic reticulum (ER) stress pathway, as shown by increased expression of glucose-regulated protein-78 (GRP78), transcription factor-3 (ATF3) activation, and C/EBP-homologous protein (CHOP), and also by arresting survival pathways, such as mTOR and Ras (Cho 2012). Thus, we can envisage in a near future the synthesis of biological active hybrid molecules containing POH as a carrier conjugated to drugs specifically targeting critical regulators of cell proliferation, as a promising antitumor

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Fig. 14.10  Effect of intranasal POH associated with oral temozolomide plus radiation therapy treatment in initial GBM. Representative brain image (MRI) of a patient with primary GBM. Note a decrease in the tumor size between the initial MRI (a), after 3 months (b), and 6 months after treatment (c)

therapeutic strategy successfully employed to treat brain tumors. In this context, POH intranasal has demonstrated greater efficacy when administered with other chemotherapeutic agents (Fig. 14.10).

References Ariazi EA, Satomi Y, Elis MJ, Haag JD, Shi W, Satler CA, Gould MN (1999) Activation of the transforming growth factor beta signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol. Cancer Res 59:1917–1928 Bailey HH, Attia S, Love RR, Fass T, Chappell R, Tutsch K, et al (2008) Phase II trial of daily oral perillyl alcohol (NSC 641066) in treatment-refractory metastatic breast cancer. Cancer Chemoth Pharm 62:149–157 Belanger JT (1998) Perillyl alcohol: applications in oncology. Altern Med Rev 3:445–448 Cho HY, Wang W, Jhaveri N et al (2012) Perillyl alcohol for the treatment of temozolomideresistant gliomas. Mol Cancer Ther 11: 2462–2472 Da Fonseca CO, Silva JT, Lins IR, Simão M, Arnobio A, Futuro D, Quirico-Santos T (2009) Correlation of tumor topography and peritumoral edema of recurrent malignant gliomas with therapeutic response to intranasal administration of perillyl alcohol. Invest New Drugs 27(6):557–564 Ding H, Roncari L, Shannon P, Wu X, Lau N, Karaskova J, Gutmann DH, Squire JA, Nagy A, Guha A (2001) Astrocyte-specific expression of activated p21-ras results in malignant astrocytoma formation in a transgenic mouse model of human gliomas. Cancer Res 61: 3826–3836 Dontula R, Dinasarapu A, Chetty C, Pannuru P, Herbert E, Ozer H, Lakka SS (2013) MicroRNA 203 Modulates glioma cell migration via robo1/ERK/MMP-9 signaling. Genes Cancer 4(7–8):285–296. Duelund L, Amiot A, Fillon A, Mouritsen OG (2012) Influence of the active compounds of Perilla frutescens leaves on lipid membranes. J Nat Prod 75(2):160–166 England B, Huang T, Karsy M (2013) Current understanding of the role and targeting of tumor suppressor p53 in glioblastoma multiforme. Tumour Biol 34(4):2063–2074.

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Farrell CJ, Plotkin SR (2007) Genetic causes of brain tumors: neurofibromatosis, tuberous sclerosis, von Hippel-Lindau, and other syndromes. Neurol Clin 25(4):925–946, viii Feldkamp MM, Lau N, Guha A (1999) Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of anti-proliferative, proapoptoic and antiangiogenic effects. Oncogene 18:7514–7526 Feldkamp MM, Lau N, Roncari L, Guha A (2001) Isotype-specific Ras. GTP-levels predict the efficacy of farnesyl transferase inhibitors against human astrocytomas regardless of Ras mutational status. Cancer Res 61:4425–4431 Fernandes J, Fonseca CO, Teixeira A, Gatass CR (2005) Perillyl alcohol induces apoptosis in human glioblastoma multiforme cells. Oncol Rep 13(5):943–947 Fischer Jde S, Liao L, Carvalho PC, Barbosa VC, Domont GB, Carvalho Mda G, Yates JR 3rd (2010) Dynamic proteomic overview of glioblastoma cells (A172) exposed to perillyl alcohol. J Proteomics 73(5):1018–27. Goldstein JL, Brown MS (1990) Regulation of the mevalonate pathway. Nature 343:425–430 González-Gómez P, Sánchez P, Mira H (2011) MicroRNAs as regulators of neural stem cellrelated pathways in glioblastoma multiforme. Mol Neurobiol 44(3):235–249 Guha A, Feldkamp MM, Lau N, Boss G, Pawson A (1997) Proliferation of human malignant astrocytomas are dependent on Ras activation. Oncogene 15:2755–2765 Heldin CH, Lennartsson J (2013) Structural and functional properties of platelet-derived growth factor and stem cell factorreceptors. Cold Spring Harb Perspect Biol 5(8):a009100 Itoh T, Kaibuchi K, Masuda T, Yamamoto T, Matsuura Y, Maeda A, Shimizu K, Takai Y (1993) The post-translational processing of ras p21 is critical for its stimulation of mitogen-activated protein kinase. J Biol Chem 15:3025–3028 Jeffers L (1995) The effect of perillyl alcohol on the proliferation of human prostatic cell lines. Proc Am Assoc Cancer Res 36:303 Jiang K, Coppola D, Crespo NC, Nicosia SV, Hamilton AD, Sebti SM, Cheng JQ (2000) The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis. Mol Cell Biol 20:139–148 Karp JE, Kaufmann SH, Adjei AA, Lancet JE, End DW (2001) Current status of clinical trials of farnesyltransferase inhibitors. Curr Opin Oncol 3:470–476 Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G, Burger PC, Cavenee WK (2002) The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 61(3):215–225 Levin VA (1999) Chemotherapy for brain tumors of astrocytic and oligodenglial lineages: the past decades and when are heading. Neuro-oncology 1:69–80 Mills JJ, Chari SS, Boyer IJ, Gould MN, Jirtle RL (1995) Induction apoptosis in liver tumors by the monoterpene perillyl alcohol. Cancer Res 55:979–983 Mischel PS, Cloughesy TF (2003) Targeted molecular therapy of GBM. Brain Pathol 13(1):52–61 Mo H, Elson CE (2004) Studies of the isoprenoid-mediated inhibition of mevalonate synthesis applied to cancer chemotherapy and chemoprevention. Exp Biol Med (Maywood) 229:567–585 Ohgaki H et al (2007) Genetic pathways to glioblastoma: a population-based study. Am J Pathol 170(5):1445–1453 Ostrom QT, Gittleman H, Liao P, Rouse C, Chen Y, Dowling J, Wolinsky Y, Kruchko C, BarnholtzSloan J (2014) CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2007–2011. Neuro Oncol 16:15 Rajesh D, Stenzel RA, Howard SP (2003) Perillyl alcohol as a radio-/chemosensitizer in malignant glioma. J Biol Chem 19:35968–35978 Reddy BS, Wang CX, Samaha H, et al (1997) Chemoprevention of colon carcinogenesis by dietary perillyl alcohol. Cancer Res 57:420–425 Sahin MB, Perman SM, Jenkins G, Clark SS (1999) Perillyl alcohol selectively induces G0/G1 arrest and apoptosis in Bcr/Abl-transformed myeloid cell lines. Leukemia 13:1581–1589 Teruszkin I, Alves S, Silva HN, Curie CM, Bozza M, Fonseca CO, Da Costa CMG (2002) Effects of perillyl alcohol in glial cell line in vitro and anti-metastatic activity in chorioallantoic membrane model. Int J Mol Med 10:785–788

Chapter 15

Conclusion and Future Perspectives Damião Pergentino de Sousa

In this book, several aspects of essential oils related to the treatment or prevention of cancer were discussed. This disease is currently one of the most severe health problems in the world, and still stands out as a main cause of death. Despite many efforts by the scientific community and real advances in the study of cancer, there are significant increases in new cases every year. Furthermore, the therapeutic arsenal used to restore patient’s health is still quite limited and of only moderate efficiency with respect to toxicology and therapeutic aspects, especially in the treatment of solid tumors. The history of cancer research and development of new drugs has shown the importance of natural products for obtaining effective therapeutic agents. Natural anticancer substances such as taxol and vinblastine have complex chemical structures. Their chemical synthesis is a challenge for the pharmaceutical industry as well as academic researchers. Essential oils are natural products widely found in the plant kingdom, especially in fruits such as orange, with high production in some countries, and which also yields an essential oil containing high percentages of limonene, a substance with antitumor properties. Limonene and other constituents of essential oils such as perillyl alcohol are structurally simple molecules. Easy isolation from nature or from chemical synthesis makes them interesting compounds with therapeutic potential in cancer, especially due to their low production costs. The therapeutic use of essential oils in combination with conventional cancer treatments may well represent a new strategy for clinical therapy against certain tumor types. Because of volatility and high lipid solubility of essential oils, the inhalation route is a convenient way of administration with the purpose of clinical application. The analgesic, anti-inflammatory, and relaxing activities commonly reported for essential oils also make them potential adjuvants for clinical protocols of treatment and represent a new therapeutic option to improve patient’s quality of life, reducing the physical and emotional disorders caused by cancer.

D. P. de Sousa () Department of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa, PB, Brazil e-mail: [email protected] © Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7_15

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Index

A Anticancer, 13, 14, 81, 88, 95, 98, 103, 203, 278 development of, 241 drugs with, 135, 189, 224 Anticarcinogenesis, 217 Antitumor, 111, 112, 120, 128, 226 Antitumor activity, 121, 128, 131, 157, 202 essential oil, 141–144, 150, 153, 154, 156, 158, 160, 161 croton regelianus, 141 cymbopogon flexuosus, 142–144 guatteria friesiana, 150, 153 lindera umbellata, 153, 154 lippia gracilis, 156 pyrolae herba, 158, 160, 161 Aromatherapy, 162, 251–253, 256, 257 Aromatic plants, 20, 67, 202, 232, 256 C Cancer biology, 2, 209 Cancer therapy, 10, 11, 148, 182, 210 effects of, 258 Chemoprevention, 114, 125, 135, 238, 243 Chemopreventive, 217 Chemotherapy, 10–13, 130, 188 Chemotypes, 20, 47 Clinical study, 191, 246 Cytotoxicity, 99, 101, 102, 121, 140, 143, 222 essential oil, 146, 148–150, 152 eucalyptus benthamii, 146, 148–150 guatteria pogonopus, 152 D Diagnostic structures, 74 Diet, 237, 238 Docking, 113, 116, 118

E Epidemiology, 7 Essential oils, 19, 20, 41, 42, 79, 84, 85, 144, 202, 232, 252, 289 assessments of, 20 biological role, 204 contribution of, 161, 162 important trade, 47, 48 locations of, 74, 78 naturally-occurring, 131 sesquiterpenes from, 207–210 with ISO, 48 F Food, 20, 112, 231, 237 H Histochemical method, 78 I Inhalation, 254, 256, 258, 279, 281 M Malignant gliomas, 128, 185, 267, 268 investigational therapies for, 273 molecular genetics of, 269, 270 treatment of, 278 with recurrent, 279 Massage, 251, 255–258 Molecular modeling, 111, 112 Monoterpenes, 20, 22, 25, 43, 85, 94, 98, 112, 188, 190, 191, 240 antineoplastic activity of drugs by, enhancement of, 189 monoterpenes of, 178 physico-chemical properties of, 185, 187

© Springer International Publishing Switzerland 2015 D. P. de Sousa (ed.), Bioactive Essential Oils and Cancer, DOI 10.1007/978-3-319-19144-7

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292 N Natural products, 13, 20, 81, 82, 95, 135, 210, 289 Nutrition, 237–239 O Organic synthesis, 84, 97, 106 P Palliative care, 255–257, 262 Perillyl alcohol, 114, 126–129, 178, 182, 183 inhibition of, 275, 276 treatment of, 278, 279 Phenylpropanoids, 20, 22, 24, 25, 43, 48, 87, 112, 217 Q Quality control, of botanical drugs, 73

Index R Risk factors, 10 environmental, 7 S secondary metabolites, 252 Sesquiterpene, 20, 22, 25, 37, 88, 89, 120 role of, 202–205, 243 Structure activity relationships (SAR), 111, 114 T Terpenes, 22, 24, 188 Toxicity, 13, 20, 34, 128, 140, 223, 285 V volatile, 252