Bioassays in Experimental and Preclinical Pharmacology [1 ed.] 9781071612323, 9781071612330

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Karuppusamy Arunachalam Sreeja Puthanpura Sasidharan

Bioassays in Experimental and Preclinical Pharmacology

SPRINGER PROTOCOLS HANDBOOKS

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Springer Protocols Handbooks collects a diverse range of step-by-step laboratory methods and protocols from across the life and biomedical sciences. Each protocol is provided in the Springer Protocol format: readily-reproducible in a step-by-step fashion. Each protocol opens with an introductory overview, a list of the materials and reagents needed to complete the experiment, and is followed by a detailed procedure supported by a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. With a focus on large comprehensive protocol collections and an international authorship, Springer Protocols Handbooks are a valuable addition to the laboratory.

Bioassays in Experimental and Preclinical Pharmacology Karuppusamy Arunachalam Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, People’s Republic of China Department of Basic Sciences in Health, Federal University of Mato Grosso (UFMT), Cuiabá, MT, Brazil

Sreeja Puthanpura Sasidharan C/o Sreeja-P.S. “Sruthilayam”, Kamba, Kinavallur Post Office, Parli, Kerala, India

Karuppusamy Arunachalam Kunming Institute of Botany Chinese Academy of Sciences Kunming, People’s Republic of China Department of Basic Sciences in Health Federal University of Mato Grosso (UFMT) Cuiaba´, MT, Brazil

Sreeja Puthanpura Sasidharan C/o Sreeja-P.S. “Sruthilayam” Kamba, Kinavallur Post Office Parli, Kerala, India

ISSN 1949-2448 ISSN 1949-2456 (electronic) ISBN 978-1-0716-1232-3 ISBN 978-1-0716-1233-0 (eBook) https://doi.org/10.1007/978-1-0716-1233-0 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Pharmacology, the study of drugs, comprises many areas in which experimental pharmacology is among the mainstream focuses in drug discovery. The preclinical and clinical development activities together apply to candidate drug discovery wherein the preclinical stage involves many activities in vitro, involving tissues and cell lines, and in vivo, using animals as experimental systems. The preclinical phase validates the pharmacokinetic and pharmacodynamic properties of the drug in order to support the upcoming clinical phase. So drug discovery involves many steps including active compound identification, pharmaceutical profiling, safety or toxicology and efficacy evaluation, and analysis using in vitro cell lines and in vivo animal models. Even though the in vitro techniques are well established, the drug’s activity or effect in a whole organism needs to be determined through in vivo studies in laboratory animals due to some species’ genetic similarity with humans and also in order to be cost effective and help reduce tragedies in human trials of a novel chemical entity. Thus, these animal models provide fundamental information regarding toxicity and other relevant disease information prior to the initiation of a clinical study of that particular candidate compound. Also, the in vitro studies with relevant cells from the human system play a major role in the analysis of a particular drug. The chapters in this book cover protocols for much of these preclinical pharmacology and toxicology evaluations of any chemical drug and its development through in vitro and in vivo models used to conduct pharmacological research. The protocols mentioned in this book are standardized by the authors during their research. Hence we hope that this 24-chapter book will assist undergraduate and postgraduate students, research scholars, scientists, and other academicians performing research in the field of drug discovery. The authors would like to express their heartfelt thanks to Prof. Michael Heinrich from University College of London (UCL), School of Pharmacy, London, UK, and Prof. Marco Leonti, Universita’ Degli Studi Di Cagliari, Dipartimento Di Scienze Biomediche, Cagliari, Italy, for their support and encouragement throughout our research careers. It would not have been possible to complete this work without the active help of Prof. Domingos Tabajara de Oliveira Martins, Federal University of Mato Grosso, Cuiaba´, Brazil, and our research supervisor Prof. Thangaraj Parimelazhagan, Bharathiar University, Coimbatore, India. We extremely appreciate the efforts by Springer Nature to produce this final edited volume. We gratefully acknowledge the cooperation and support received from David C. Casey from Springer Nature in bringing out this book in outstanding quality. Moreover, we thank all those who have shared their advice, suggestions, and support, including our colleagues and also our families. Kunming, People’s Republic of China and Cuiaba´, MT, Brazil Parli, Kerala, India

Karuppusamy Arunachalam Sreeja Puthanpura Sasidharan

v

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

v ix

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Culture Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phytochemical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preclinical Drug Dose Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicology Studies: In Vitro and In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Standard Procedure for Anaesthesia in Preclinical Experiments . . . . . . . . . . . . . . . General Considerations and Collection of Animal Blood . . . . . . . . . . . . . . . . . . . . . Animal Experiments on the Cardiovascular System . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Experiments on Ulcerative Colitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experiments of Antibacterial Activities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experiments of Antifungal Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Experiment of Wound Healing Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Experiments of Anti-inflammatory Activities . . . . . . . . . . . . . . . . . . . . . . . . Animal Experiment of Anti-nociceptive Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . Experiments of Antioxidant Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Experiments of Hepatoprotective Activities . . . . . . . . . . . . . . . . . . . . . . . . . Animal Experiments of Anti-Diarrhoeal Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Experiments of Central Nervous System (CNS) . . . . . . . . . . . . . . . . . . . . . Experiments of Anti-Cancer Activities (In Vitro and In Vivo) . . . . . . . . . . . . . . . . Animal Experiments of Anti-Diabetic Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Experiments of Gastric Ulcer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein Extraction and Western Blot Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gel Electrophoresis and PCR Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Docking Methods for Drug Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 21 29 33 45 51 57 63 75 91 105 119 137 143 157 167 173 181 191 201 229 241 259

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

263

vii

Abbreviations ΔΨm AED AGS Al(OH)3 ALP ALT AML ANOVA ANXA7 APS AST ATTC BAL BHI BOD BP BSA Caco-2 CAT CCl4 CD CFU CHO-k1 CID CLSI CMC CNS CO2 COX-2 CTGF CVD DAD DAI Dexa DMEM DMSO DNA DOXO DPPH DRI DSS DTNB ECG ED50 EDTA

Mitochondrial membrane potential Animal equivalent dose Adenocarcinoma gastric cell line Aluminum hydroxide Alkaline phosphatase Aminotransferase Acute myeloid leukemia Analysis of variance Annexin A7 Ammonium persulfate Aspartate aminotransferase American type culture collection Bronco-alveolar lavage Brain heart infusion Biochemical oxygen demand Blood pressure Bovine serum albumin Colon adenocarcinoma epithelial cells Catalase Carbon tetrachloride Crohn’s disease Colony-forming units Chinese hamster ovary epithelial cells Collision-induced dissociation Clinical & Laboratory Standards Institute Carboxymethylcellulose Central nervous system Carbon dioxide Cyclooxygenase-2 Connective tissue growth factor Cardiovascular disease Diode array detector Disease Activity Index Dexamethasone Dulbecco eagle modified with nutrient F12 Dimethyl sulfoxide Deoxyribonucleic acid Doxorubicin 2,2-Diphenyl-1-picryl-hydrazyl Dose reduction index Dextran sodium sulfate 5,50 -Dithiobis-(2-nitrobenzoic acid) Electrocardiogram Effective dose 50% Ethylenediaminetetraacetic acid

ix

x

Abbreviations

EGF EGTA ELISA EPM ERKs ESI ESI-MS EtOH FBS FeCl3 FICI FICT GAE GAMA GT GGT GIT GL GPx GSH GSSG H&E H2O H2O2 H3PO4 Hb HED HPLC HRP i.p IBDs IC50 ICH IL-10 IL-13 IL-17 IL-1β IL-4 IL-5 INF- γ i-NOS JNKs KH2PO4 LC-MS LDL L-NAME LOX LPO LPS MAPKs

Epidermal growth factor Ethylene glycol tetraacetic acid Enzyme-linked immunosorbent assay Elevated plus maze Extracellular signal-regulated kinases Electrospray ionization Electrospray ionization mass spectrometry Ethanol Fetal bovine serum Ferric chloride Fractional inhibitory concentration index Fluorescein isothiocyanate Gallic acid equivalent Gamma-glutamyl transferase Gamma-glutamyl transferase Gastrointestinal tract Glucose Glutathione peroxidase Reduced glutathione Glutathione disulfide Hematoxylin and eosin Water Hydrogen peroxide Phosphoric acid Haemoglobin Human equivalent dose High performance liquid chromatography Horseradish peroxidase enzyme Intraperitoneal route Inflammatory bowel diseases Inhibitory concentration 50% International Conference on Harmonization Interleukin 10 Interleukin 13 Interleukin 17 Interleukin 1β Interleukin 4 Interleukin 5 Interferon gamma Induced nitric oxide synthase c-Jun N-terminal kinases Potassium phosphate monobasic Liquid chromatography-mass spectrometry Low density lipoprotein L-NG-Nitro arginine methyl ester Lipoxygenase Lipid peroxidase Lipopolysaccharide Mitogen-activated protein kinases

Abbreviations

MBC MCH MCP-1 MDA MDH MeOH MFC MHB MIC MN MAPK 14 MPO MRM MS MT MTT Na3VO4 NAC NaCl NADPH NaHPO4 NaNO2 NaOH NCCLS NDI NF-κB nNOS NO NOAEL NOEL NOS NSAIDs NTA OD OECD OFT ORT OVA p.o. p38 PAD PAS PBS PCR PDGF PGE2 PI PMN PMSF

Minimum bactericidal concentration Mean corpuscular haemoglobin Monocyte chemoattractant protein-1 Malondialdehyde Malate dehydrogenase Methanol Minimum fungicidal concentration Mu¨eller Hinton broth Minimum inhibitory concentration Mononuclear Mitogen-activated protein kinase 14 Myeloperoxidase Multiple reaction monitoring Mass spectrometry Masson’s trichrome 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide Sodium orthovanadate N-Acetylcysteine Sodium chloride Nicotinamide adenine dinucleotide phosphate Sodium phosphate dibasic Sodium nitrite Sodium hydroxide National Committee for Clinical and Laboratory Standards Nuclear division index Nuclear factor-kappa-B Nitric oxide neuronal synthase Nitric oxide No adverse effects observed No observed effect Nitric oxide synthase Nonsteroidal anti-inflammatory drugs Nitrilotriacetate Optical density Organization for Cooperation and Economic development Open Field Test Object Recognition Test Ovalbumin Per oral P38 protein Photodiode array Periodic acid-Schiff Phosphate-buffered saline Polymerase chain reaction Platelet-derived growth factor Prostaglandin E2 Propidium iodide Polymorphonuclear Proteases inhibitor phenylmethanesulfonyl fluoride

xi

xii

Abbreviations

PNS PPEs RNA RONS RT-PCR s.c. S.E SDB SDS SDS-PAGE SHD SNRI SOD SPF STZ TBARS TBS TBST TC TCA TEMED TER TGF-β1 TGL TIMP-1 TLC TLR TNBS TNF-α TRIS TSA UA UC ULA UV VEGF VEGF-A VLDL VMC

Peripheral nervous systems Personal protective equipment kits Ribonucleic acid Reactive oxygen or nitrogen species Reverse-transcriptase-polymerase chain reaction Subcutaneous Standard error Sabouraud dextrose broth Sodium dodecyl sulfate Sodium dodecyl sulfate polyacrylamide gel electrophoresis Safe human dose Serotonin-norepinephrine reuptake inhibitor Superoxide dismutase Sulfate–phosphate–ferric Streptozotocin Thiobarbituric acid reactive substances Tris-buffered saline Tris-buffered saline with Tween® 20 Total cholesterol Trichloroacetic acid Tetramethylethylenediamine Transepithelial electrical resistance Transforming growth factor beta 1 Triglycerides Tissue inhibitors of metalloproteinases-1 Thin layer chromatography Toll-like receptors 2,4,6 Trinitrobenzenesulfonic acid Tumor necrosis factor-alpha Tris(hydroxymethyl)aminomethane Tryptic Soy Agar Uric acid Ulcerative colitis Ulcerative lesion area Ultra violet spectrum Vascular endothelial growth factor Vascular endothelial growth factor A Very low density lipoprotein Volume mean corpuscular

Chapter 1 Overview Abstract This chapter presents a brief overview of the book. Key words Drug discovery, Toxicity, Introduction

Drug discovery comprises preclinical experimental and clinical pharmacology activities, where the preclinical deals with the identification and characterization of a novel chemical entity leading to its structure framework, as well as testing through in vitro and in vivo for evaluating the metabolic and biochemical processes of the candidate drug, while clinical pharmacology evaluates the pharmacokinetic efficacy and safety of drugs through testing in humans. At present the preclinical pharmacology experiences many developmental activities, including cell, tissue culture, in vitro and in vivo animal tests. The pharmacokinetic and pharmacodynamic properties of the novel compound are analysed in preclinical pharmacology evaluating its safety and efficacy based on its absorption, distribution, metabolism and excretion in the specific organism body. The novel chemical is evaluated in vitro for toxicity in toxicology through using cell lines that are developed nowadays—and of course the evaluation is repeated in vivo using laboratory animals that are genetically most similar to humans—consequently to exploit the action or effect of the chemical on metabolism and on behaviour of animals and thereby in humans. The toxicity investigation helps to fix the dosage of the particular drug for further analyses against various diseases in animals as well as in humans. Free radicals, such as reactive oxygen or nitrogen species (RONS), seem to be most dangerous and the main cause relatively seen to be associated with many diseases like inflammation, ulcer, diabetes, cancer and so on. Even though the organism’s body scavenges these radicals with the help of the endogenous antioxidant system, the diseased conditions or factors reduce its activity Karuppusamy Arunachalam and Sreeja Puthanpura Sasidharan (eds.), Bioassays in Experimental and Preclinical Pharmacology, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-1233-0_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021

1

2

Overview

and develop chances to produce much more reactive species intolerable by the body resulting in the chronic condition of the disease. The antioxidant property of the target drug, via scavenging the radicals directly or by augmenting the antioxidant enzymes, is evaluated following various protocols in vitro and in vivo. Along with the antioxidant activity, the novel chemical is evaluated for its activity against particular diseases or to its symptoms, following the literature, through experimental animal models. Even though the cell line field is well developed, the drug’s activity or effect on the whole organism needs to be determined through in vivo studies due to the genetic similarity of laboratory animals with humans and also to help reduce the cost effects and tragedy in human trials of the target drug. Hence the pharmacological concepts are applied in the evaluation and treatment of cardiovascular and hepatic diseases; various central nervous system disorders; the involvement of different inflammatory mediators in diseases like inflammation, ulcer and colitis; nociception and the drug effect on it; evaluating the management of diabetes; diagnosis of respiratory disease and its treatment; and understanding the cancer treatment. With this, the active drug structure and design is a framework in molecular docking that also helps to analyse the action of the drug on specific binding site of the protein as well as the binding among molecules at an atomic level. The various chapters in this book portray the aims and principles in pharmacology—with special mention to diverse protocols for a variety of diseases and toxicology, including dosage fixation and molecular docking—to the learners. Besides the gel electrophoresis, polymerase chain reaction (PCR) amplification and western blotting are also discussed, which we hope would help the researchers in the drug discovery field as well as the students in better understanding the concepts and performing these experiments.

7

Chapter 2 Cell Culture Assays Abstract Cell culture assays play an important role in drug research along with animal tests with the principle of ‘3Rs’—Reduction, Refinement and Replacement—due to its quick and easy way of drug response analysis, as well as high throughput in assays, simplicity and cost-effectiveness. This chapter explains various procedures, cell viability, cytotoxicity and inflammatory response, for the evaluation of cytotoxicity of novel compounds or extracts. The protocols like propidium iodide used cell cycle assay and apoptosis assay, Alamar Blue method in Caco-2 cells, cell viability test in Caco-2 cells using lipopolysaccharide (LPS) and cytotoxicity against RAW 264.7 for the determination of anti-inflammatory activity—together with the assays for nitric oxide (NO) determination and scratch assay for testing the proliferation of fibroblast cells and inflammatory response in AGS cells model—elucidate the detailed and standardized protocols for the in vitro assays with cell lines. Key words Alamar Blue, NO, LPS, AGS, Scratch assay, Fibroblast cells, Propidium iodide, Annexin V

Aim: To evaluate the cytotoxicity of chemicals and for drug screening through in vitro cell viability and cytotoxicity assays using cultured cells. Introduction: In the drug discovery research, the cell culture-based experiments attain crucial phase due to its efficiency in screening and validating novel drugs in comparison to other in vitro biochemical assays. Cell-based assays gained much importance due to its potentiality to carry out in a high-throughput, cost-effective manner than animal tests, due to the meaningful responses provided by them than the simple lab assays, and because it is quick and easy to identify the responses of compounds in cell-based assays. However, it does not provide information on the interactions of the drug with the target molecule and its effects on the complicated system in an organism, and hence it is generally considered bridging the gap between the experiments using animals and the lab biochemical

Karuppusamy Arunachalam and Sreeja Puthanpura Sasidharan (eds.), Bioassays in Experimental and Preclinical Pharmacology, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-1233-0_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021

3

7

4

Cell Culture Assays

assays—in this case for the cytotoxic test of compounds and the toxicity study of the drug in animal models. In addition, cell culture tests also come under the general ‘3Rs’ principle, that is, Reduction, Refinement and Replacement of animals in experiments, to minimize the animal testing. Therefore, in vitro experiments using cell lines are recommended by the International Conference on Harmonization (ICH) to reduce the usage of animals. Generally, the cell culture assays use all types of general cells, mainly from human, and also specific cells for the experiments, as in the case of cancer studies using particular cancer cell lines [1, 2]. Principle: In the colourimetric assays, the biochemical markers are measured to evaluate the metabolic activity of cells. Reagents used in colourimetric assays develop/change colour in response to the viability of cells, allowing the colourimetric measurement of cell viability via spectrophotometer [3].

1

Cell Cycle Assay: Propidium Iodide Marking [3]

1.1

Adherent Cells

Protocol: 1. Plate the different cell lines in culture medium containing serum and appropriate densities (see Table 1 for an example) in cell culture plates and incubate for 24 h at 37
C in a wet greenhouse containing 5% CO2 (Carbon dioxide) (incubation time required to reach semi-confluency). 2. Remove the culture medium by aspirate and wash the plates two times with serum-free culture medium. 3. Incubate the cells with serum-free culture medium for 24 h (or at least 12 h) at 37
C in a humid oven containing 5% CO2 to synchronize the cells in the G0 stage.

Table 1 Preparation of Adherents for cell cycle assay Plating density 96-Well area: Cell line per cm2 0.31 cm2

6-Well area: 9.3 cm2

100 mm area: 60.1 cm2

Incubation time semiconfluence (h)

Adherents 33.000

1  104/ 100 μL

3  105/ 3 mL

2  106/15 mL 24

MC3T3 30.000

1  104/ 200 μL

2.8  105/ 4 mL

1.8  106/ 15 mL

24

786

6.2  103/ 100 μL

2  105/ 3 mL

1.2  106/ 15 mL

24

3T3

20.000

7

1 Cell Cycle Assay: Propidium Iodide Marking

5

4. Remove the culture medium by aspirate and carry out the treatment with the test compounds (in general, in semiconfluency) for different times (12 and 24 h) and different concentrations, one of them being the 50% inhibitory concentration (IC50). Use serum culture medium in the treatment, but if you are interested in assessing the proliferative effect of the compound, do not use a serum in the culture. Incubate at 37
C in a humid oven containing 5% CO2. 5. Collect the supernatant (containing the non-adherent cells) in a conical tube (Falcon type) and centrifuge at 277  g for 5–10 min. 6. Wash adherent cells two times with phosphate-buffered saline (PBS). Remove the supernatant by suction. 7. Peel the cells with 0.1–0.25% trypsin solution containing 0.02% ethylenediaminetetraacetic acid (EDTA: concentration, volume and time depend on the cell type). 8. Add culture medium containing serum to inactivate trypsin (3–5 mL depending on the volume and concentration of trypsin used). Note the volume of medium added. 9. Transfer the cell suspension to the conical tube containing the non-adherent cells that are centrifuged and homogenized. 10. Count the cells in a Neubauer chamber to determine the concentration. 11. Wash the cells once with PBS (centrifugation: 277  g for 5–10 min). 12. Resuspend the cells in the volume of PBS left in the tube and transfer to Eppendorf tubes. 13. Add 1 mL of PBS and centrifuge at 277  g for 5–10 min. Despise the supernatant. 14. Fix the cells with 2% paraformaldehyde (100 μL/1  106 cells, Electron Microscopy Sciences—20% stock, dilute in PBS) and incubate at 4
C for 30 min. Store at 4
C until the analysis (up to 2 months). 15. Wash the cells by adding 500 μL of PBS/Eppendorf tube (centrifuge at 277  g for 5–10 min). 16. Discard the supernatant with a pipette. 17. Add 90 μL of PBS containing 0.01% saponin to permeabilize the cells. 18. Next, add 10 μL of RNase stock solution 4 mg/mL (Ribonuclease A from bovine pancreas 5 crystallized Type IA—protease-free, essentially salt-free, Sigma, Cat. No. R-4875; prepare in buffer 0.05 M sodium acetate, pH 5.0, containing 0.02 M MgSO4 and freeze at –20
C). 19. Incubate in a bath at 37
C for 1 h.

7

6

Cell Culture Assays

20. Homogenize the cells and transfer to flow cytometry and fluorescence-activated cell sorting (FACS) tubes. 21. Add 300 μL of PBS and mix gently (in the cytometer room). 22. Add 10 μL of propidium iodide (PI) stock solution (25 mg/ mL in PBS) at room temperature (T) in the dark (in the cytometer room) and analyse immediately by flow cytometry. 1.2

1. Plate cell lines K562 and Lucena for synchronization in RPMI culture medium without serum (as shown in Table 2), in T25 bottles for cell culture and incubate for 24 h at 37
C in a humid oven containing 5% CO2.

Cells Suspension

2. Transfer the cells to a conical tube (Falcon type) and centrifuge at 277  g for 10 min. Discard the supernatant. 3. Treat the cells with the test compounds for different times (12 and 24 h) and different concentrations, one of them being the IC50. Use serum culture medium in the treatment (final volume: 10 mL/T25 bottle), but if you are interested in evaluating the proliferative effect of the compound, do not use a serum in the culture. Incubate at 37
C in a humid oven containing 5% CO2. 4. Homogenize the cells and count the cells to determine the concentration in a Neubauer chamber. 5. After determining the volume of cell suspension required (1  106 cells per sample), transfer to a conical tube (Falcon type) and centrifuge at 277  g for 5–10 min. 6. Wash the cells once with PBS (centrifugation: 277  g for 5–10 min). Disperse the supernatant. 7. Resuspend the cells in the volume of PBS left in the tube and transfer to Eppendorf tubes. Table 2 Preparation of human erythroleukaemia cell K562 and treatment of Lucena Volume per bottle, mL

Incubation time synchronization (h)

Incubation time treatment (h)

K562/control 1.5  105

12

24

12 or 24

K562/treated 1.5  10 lucena

10

24

12 or 24

K562/control 1  105

20

24

12 or 24

K562/treated 1  10 lucena

20

24

12 or 24

K562/control 1.5  105

12

 (Annexin V/PI)

424

K562/treated 1.5  10 lucena

10

 (Annexin V/PI)

424

Cell line

Plating density per mL

5

5

5

7

2 Apoptosis Assay: Annexin V/Propidium Iodide Marking

7

8. Add 1 mL of PBS and centrifuge at 277  g for 5–10 min. Disperse the supernatant. 9. Fix the cells with 2% paraformaldehyde (100 μL/1  106 cells, 20% stock, dilute in PBS) and store at 4
C until analysis. 10. Wash the cells by adding 500 μL of PBS/Eppendorf tube (centrifuge at 277  g for 5–10 min). 11. Discard the supernatant with a pipette. 12. Add 90 μL of PBS containing 0.01% saponins to permeabilize the cells. 13. Next, add 10 μL of RNase stock solution 4 mg/mL (Ribonuclease A from bovine pancreas 5 crystallized Type IA—protease-free, essentially salt-free; prepare in buffer 0.05 M sodium acetate, pH 5.0, containing 0.02 M MgSO4 and freeze at 20
C). 14. Incubate in a bath at 37
C for 1 h. 15. Homogenize the cells and transfer to FACS (fluorescence activated cell sorting) tubes. 16. Add 300 μL of PBS and mix gently (in the cytometer room). 17. Add 10 μL of PI stock solution (25 mg/mL in PBS) to room temperature in the dark (in the cytometer room) and analyse immediately by flow cytometry.

2 2.1

Apoptosis Assay: Annexin V/Propidium Iodide Marking [4] Adherent Cells

Protocol. 1. Collect the supernatant (containing the non-adherent cells) in a conical tube (Falcon type) and centrifuge at 277  g for 5–10 min. 2. Wash the adherent cells twice with PBS. Remove the supernatant by suction. 3. Scrap the cells with 0.1–0.25% trypsin solution containing 0.02% EDTA (concentration, volume and time are depend on the cell type). 4. Add culture medium containing serum to inactivate trypsin (3–5 mL depending on the volume and concentration of trypsin used). Note the volume of medium added. 5. Transfer the cell suspension to the conical tube containing the non-adherent cells that are centrifuged and homogenized. 6. Count the cells to determine the concentration in a Neubauer chamber.

7

8

Cell Culture Assays

7. Transfer the necessary volume to new conical tubes and centrifuge at 277  g for 5–10 min. Despise the supernatant. 8. Wash the cells twice with cold PBS (centrifugation: 277  g for 5–10 min). 9. Resuspend cells in Binding Buffer once (100 μL/1  106 cells). 10. Transfer to flow cytometry (FACS) tubes. 11. Add 3 μL of Annexin V conjugated to FITC (Becton Dickinson [BD], Cat. No. 556420), mix gently and incubate for 20 min at room T, in the dark (in the cytometer room). 12. Add 5 μL of PI (25 mg/mL stock solution in PBS) and mix gently. 13. Add 300 μL of Binding Buffer once and mix gently. 14. Analyse by flow cytometry. 2.2

Suspension Cells

1. Homogenize the cells and count to determine the concentration in a Neubauer chamber. 2. After determining the volume of cell suspension required (1  106 cells per sample), transfer to a conical tube (Falcon type) and centrifuge at 1500 rpm for 5–10 min. 3. Discard the supernatant and wash the cells twice with cold PBS (centrifugation: 1500 rpm for 5–10 min). Despise the supernatant. 4. Resuspend cells in Binding Buffer once (50–100 μL/1  106 cells). 5. Transfer to FACS tubes. 6. Add 3 μL of Annexin V conjugated to FITC (BD, Cat. No. 556420), mix gently and incubate for 20 min at room T, in the dark (in the cytometer room). 7. Add 5 μL of PI (25 mg/mL stock solution in PBS) and mix gently. 8. Add 300 μL of Binding Buffer once and mix gently. 9. Analyse by flow cytometry. Note: Prepare the following controls for device calibration (use one sample, e.g. control): l l l

One tube with 1  106 cells without marking One tube with 1  106 cells marked with PI only One tube with 1  106 cells marked only with Annexin V-FITC (Note: mix with a little treated cell in PI and FITC controls)

7

3 Cytotoxicity in Caco-2 Cells by the Alamar Blue Method

2.3

Binding Buffer

9

10 concentrated stock solution 0.1 M Hepes (pH 7.4)

5.9 g

1.4 M NaCl

20.5 g

25 mM CaCl2

0.695 g

H2O Milli Q

250 mL

¼ 0.92 g of CaCl2.2H2O

1. After diluting once (e.g. 10 mL in 100 mL) in Milli Q water (Milli Q H2O), adjust the pH to 7.4. Note: Count 10,000 cells. – When using the same cell suspension for the two assays, first perform the annexin V/PI marking. 2. Then transfer the cells to Eppendorf tubes, once with PBS, fixed with 100 μL of 2% paraformaldehyde for 30 min at 4
C; add 500 μL of cold PBS, centrifuge for 15 min at 2000 rpm, add with 90 μL of saponin 0.01% and 10 μL RNase. Incubate at 37
C for 30 min to 1 h; add 5 μL of PI (25 mg/mL) and perform the analysis.

3

Cytotoxicity in Caco-2 Cells by the Alamar Blue Method [5]

3.1

Principle

In this assay, the Alamar Blue (blue dye resazurin) is used as an agent to analyse the viability of cells, where the reductase or diaphorase-type enzymes in the mitochondria of living cells convert blue to pink resorufin and the absorbance of this colour change is measured spectrophotometrically [6].

3.2

Protocol

1. Human colon adenocarcinoma epithelial cells (Caco-2) are from the American-Type Culture Collection (ATCC, HTB-37 code) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% foetal bovine serum (FBS, Gibco) and 1% non-essential amino acid and 1% glutamine. 2. The culture medium is supplemented with 50 U/mL of penicillin and 50 U/mL of streptomycin. The cells are maintained at 37
C in a 5% CO2 atmosphere. 3. The cytotoxicity of extract/drug is assessed in Caco-2 cells by the AlamarBlue® test. 4. For evaluation, Caco-2 cells are seeded in 24-well plates at a density of 1  105 cells/well. 5. The culture medium is replaced 3 times a week for 21 days to ensure complete differentiation of the cells.

7

10

Cell Culture Assays

6. After a period, the cells are with the culture medium removed and is iced with PBS; subsequently, they are exposed to various concentrations of extract (3.125–400 μg/mL, in dimethylsulfoxide [DMSO] solution) and incubated for 24 h. 7. Doxorubicin (DOXO) (0.0058–58 μg/mL) is used as positive control. 8. The cell medium is removed and replaced with 1 mL of Alamar Blue solution (10% v/v in culture medium) in each well, and then the plates are incubated for 6 h in an oven at 37
C, 5% CO2. 9. After incubation, absorbance is measured in a plate reader at 570 and 600 nm. 10. The percentage of Alamar Blue reduction is calculated and compared to the doxorubicin (positive) and negative (medium + Alamar Blue) controls.

4 4.1

Viability in Caco-2 Cells Stimulated with Lipopolysaccharide [7] Protocol

1. To determine cell viability, Caco-2 cells are plated in 24-well plates at a density of 1  105 cells/well. 2. The culture medium is replaced three times a week for 21 days to ensure complete differentiation of the cells. 3. Afterwards, the cells are treated with the extract (1, 10 and 100 μg/mL) for 1 h and co-treated with lipopolysaccharide (LPS, 0.5 ng/mL) for 24 h. 4. The cell medium is removed and replaced with 1 mL of Alamar Blue solution (10% v/v in culture medium) in each well, and then the plates are incubated for 6 h in an oven at 37
C and 5% CO2. 5. After incubation, absorbance is measured in a plate reader at 570 and 600 nm. 6. The percentage of Alamar Blue reduction is calculated and compared to the controls as mentioned earlier.

4.2 Caco-2 Cells Stimulated by LPS

1. Caco-2 cells stimulated with LPS (1 μg/mL) are treated with extract or vehicle. 2. Transepithelial electrical resistance (TER) is measured daily to assess the function of the epithelial barrier with the use of an epithelial resistance meter for Caco-2 cell monolayers. 3. TER is measured for approximately 21 days (540  12 Ω cm2), until a steady-state occurs indicating that the barrier function is stabilized.

7

5 Determination of Nitric Oxide (NO) by In Vitro Experiment

5

11

Determination of Nitric Oxide (NO) by In Vitro Experiment [8]

5.1

Principle

The catalytic reaction on amino acid L-arginine by nitric oxide synthase (NOS) synthesises nitric oxide (NO) in living organisms. The NOS isoform, NOS induced form to express in macrophages and leukocytes to synthesize NO in response to inflammatory stimuli or bacterial products like LPS through activation of the toll-like receptor 4 present in the bacterial cell surface or inflammatory cytokines like interferon gamma (IFN-γ) by the autocrine production mechanisms of IFN-γ. The synthesis of NO in macrophage cell culture in vitro is determined through measuring the nitrite (NO2) level [8].

5.2

Materials

l

Murine macrophages of the RAW 264.7 strain.

l

LPS at a concentration of 1 μg/mL.

l

Interferon-gamma (INF-γ) at a concentration of 1 ng/mL.

l

5.3

Protocol

LPS/INF-γ plus at a concentration of 1 μg/mL/1 ng/mL, respectively.

l

RPMI-10% culture medium (without phenol red).

l

24- and 96-well plates. 1. Murine macrophages of the RAW 264.7 strain is grown in 75 cm2 cell culture flasks in RPMI-1640 (Sigma) medium, supplemented with streptomycin antibiotic (10 mg/mL), penicillin (6 mg/mL) and 10% foetal bovine serum (FBS), maintained at 37
C and 5% CO2. 2. The cell culture, after acquiring semi-confluence, is iced once with the Hanks buffer solution, trypsinized and counted in a Neubauer chamber, adjust the number of cells to 1  106 cells/ well in complete RPMI (without phenol red) medium. 3. From this suspension, the cells are seeded in 24-well plates and incubated at 37
C and 5% CO2 for 3 h to adhere the cells to the plate. 4. Next, different concentrations of the test samples are prepared (between 100 and 6.25 μg/mL, in the ratio 1:2), which is added to the cells after removing the medium, incubated at 37
C and 5% CO2 for 30 min. 5. As a negative control, the cells are cultured with the diluents of the test samples. 6. After the incubation period, the cells are stimulated synergistically with LPS 1 μg/mL (Lipopolysaccharide from Escherichia coli Serotype 0111: B8—Sigma, St. Louis, MO, USA) and IFN-γ 1 ng/mL (Recombinant Mouse IFN-γ/BD Pharmingen, San Diego, CA, USA) at 37
C, in the presence of 5% CO2.

7

12

Cell Culture Assays

7. After 24 h of incubation, the nitrite dosage is done by the colourimetric method, based on the Griess reaction [9]. The supernatants (0.1 mL) are incubated with an equal volume of the Griess reagent (1% sulphanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride, 2.5% H3PO4) at room temperature for 10 min. The absorbance is read on an ELISA reader on a 540 nm filter (Titertek Multiskan Reader). 8. The nitrite concentrations are calculated by extrapolation to a standard sodium nitrite (NaNO2) curve (100–0.78 μM) and the data expressed in μM nitrite. 5.4 Calculation of LPS (B8 Sigma) Nitrite Test

1. Weigh approximately 1.50 mg of LPS and dilute in 500 μL of Hanks (1); it has 3000 μg (take to the vortex).

5.4.1 Stock Solution

3. Aliquot and store at –20
C.

2. Sonic the Eppendorf for approximately 3 min. l

Dilution LPS 1:100.

l

C1∙V1 ¼ C2∙V2.

l

3000 μg  V1 ¼ 100  3 mL.

l

5.4.2 Usage Solution

V1 ¼ 100/3000 ¼ 33 μL þ 1 mL of RPMI medium ¼ 10% (without phenol).

l

2 μg/mL LPS ¼ (1 μg/mL LPS).

l

C1∙V1 ¼ C2∙V2.

l

100 μg  V1 ¼ 2  4 mL.

l

V1 ¼ 4/100 ¼ 80 μL þ 3 mL of RPMI medium ¼ 10% (without phenol).

Note: Add 1 mL of the prepared LPS concentrations (2 μg/mL) to the RAW cell culture per well and add 1 mL of RPMI-10 ¼ 1 μg/ mL. 5.5 Calculation of INF-γ Nitrite Test

l

20,000 ng/mL (aliquots stored at –20 refrigerator).

5.5.1 Stock Solution

l

Dilution LPS 1:100.

l

C1∙V1 ¼ C2∙V2.

l

20,000 ng  V1 ¼ 100  2 mL.

l

5.5.2 Usage Solution

C in brown-LI

V1 ¼ 100/20,000 ¼ 10 μL + 1.990 μL of RPMI medium ¼ 10% (without phenol).

l

2 ng/mL INF-γ ¼ (1 μg/mL INF-γ).

l

C1∙V1 ¼ C2∙V2.

l

100 μg  V1 ¼ 2  3 mL.

l




V1 ¼ 4/100 ¼ 80 μL + 3 mL of RPMI medium ¼ 10% (without phenol).

7

5 Determination of Nitric Oxide (NO) by In Vitro Experiment

13

Note: Add 1 mL of the prepared concentrations of INF-γ (2 μg/ mL) to the RAW cell culture per well and add 1 mL of RPMI10% ¼ 1 ng/mL. 5.6 Calculation of LPS/INF-γ Nitrite Test

l

Usage solution: Prepare 4 μg/4 ng of LPS/INF-γ (¼ 2 μg or 2 ng/mL).

l

Remove 600 μL of the 1:100 dilutions of LPS.

l

μg/mL-Solution A.

l

C1∙V1 ¼ C2∙V2.

l

100 μg  V1 ¼ 4  15 mL.

l

V1 ¼ 4/100 ¼ 600 μL + 15 mL of RPMI medium ¼ 10% (without phenol).

l

Remove 600 μL of the 1:100 dilution of INF-γ.

l

ng/mL-Solution B.

l

C1∙V1 ¼ C2∙V2.

l

100 ng  V1 ¼ 4  15 mL.

l

V1 ¼ 4/100 ¼ 600 μL + 15 mL of RPMI medium ¼ 10% (without phenol). – Pour one solution into the other (A + B) of the prepared concentrations of LPS/INF-γ (4 μg to ng/mL); homogenize (vortex) ¼ 2 μg or 2 ng/mL. – Add 1 mL/well to the RAW cell culture and add 1 mL of RPMI-10% ¼ 1 μg or 1 ng/mL. – Collect 100 μL (24 h) of the supernatant and incubate with an equal volume of Griess (remove the reagent 30 min before the test).

5.7 Calculation of L-NAME (N5751) Nitrite Test

l

Prepare 10 mM.

l

Molecular weight: 269.7.

l

269 g ¼ 1 mol ¼ 1 L.

l

1 mol ¼ 103 mM.

l

269 g ¼ 103 mM.

l

X ¼ 10 mM.

l

X ¼ 2.69 g.

l

2.69 g ¼ 1000 mL (1 L).

l

X ¼ 10 mL.

l

X ¼ 0.0269 g or 26.9 mg/10 mL.

l

Weight 13.45 mg and dilute in 5 mL of RPMI-10% medium (without phenol).

7

14

Cell Culture Assays

5.8 Preparation of the Standard Nitrite (NaNO2) Curve

l

Weight 6.9 mg of sodium nitrite and dilute in 1 mL of medium10% and mix (vortex).

l

Dilution 1: 100.

5.8.1 Stock Solution

l

Remove 10 μL (stock solution) + 990 μL (half-10%).

l

1:10 dilution (corresponds to 100 μM).

l

l

Remove 100 μL (stock solution) + 900 μL (half-10%) and proceed with serial dilution in the ratio of 1:2 with a preferred final volume of 500 μL. For accurate values, make eight points for the standard curve. – Add 100 μL of the sample and/or concentrations of the sodium nitrite standard per well. – Add 100 μL of the Griess Reagent per well. – Incubate for 10 min at room temperature and measure absorbance at 540 nm. – Subtract the white absorbance from the absorbance of each well (samples). – Determine the nitrite concentration of the samples from the standard curve.

6 Analysis of Specific Cytotoxicity Against RAW 264.7 for Determination of Antiinflammatory Activity In Vitro [10] 6.1

Principle

MTT assay is a colourimetric experiment to estimate cell metabolic process, which depends on the capacity of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to its unsolvable formazan with purple colour [11].

6.2

Protocol

1. Cell viability in the presence of extract is assessed by the MTT test. 2. Briefly, the cells are cultured in microplates of 96 wells in DMEM medium for cell culture without segmented filamentous bacteria (SFB) and incubated in an oven at 37
C in humidified 5% CO2 atmosphere. 3. After 24 h, the medium is gently removed and replaced with DMEM supplemented with extract in concentrations ranging from 0.5 to 40 μg/mL. The cells are then treated with Escherichia coli lipopolysaccharides (LPS) 100 ng/mL; the microplates are incubated in a 5% CO2 oven for 44 h when 20 μL of MTT solution and incubation is continued for another 4 h.

7

6 Analysis of Specific Cytotoxicity Against RAW 264.7 for Determination of. . .

15

4. The medium is then removed, and formazan crystals are solubilized in 100% DMSO. 5. The amount of MTT-formazan obtained is directly proportional to the number of viable cells that are determined by measuring the absorbance at 590 nm in the microplate reader. 6. The positive control used is dexamethasone in concentrations ranging from 0.001 μM at 10 μM. 6.3 Cell Viability Test: General Cytotoxicity Analysis for Eukaryotic Cells

1. The evaluation of cell viability is performed by the MTT. 2. MTT is an insoluble salt metabolized by mitochondrial enzymes from viable cells, giving rise to crystals of formazan with blue colouring. 3. The test is performed on tumour and non-tumour cell lines. 4. RAW 264.7, 3T3, HepG2 cells, MDA-MB 231 and HCT-116 are inoculated in microplates at 1  104 cells/well. 5. At other cells, suspensions are seeded in 96-well microplates containing 1  105 cells/well. 6. The plates are pre-incubated for 24 h at 37
C to allow cells to adapt before adding samples. 7. Subsequently, the extract is added to the cells at a concentration of 50 μg/mL. 8. The plates are incubated for 48 h at 37
C in an atmosphere of 5% CO2 and 5% relative humidity. 9. After the incubation period with the samples, 20 μL of MTT solution (5 mg/mL in phosphate buffer) is added to each well and incubated for 4 h. 10. At the end of this period, the supernatant is removed and 200 μL of DMSO is added to dissolve formazan crystals. 11. The groups included are control, treated with 0.1% DMSO (interference control of the solvent-diluent system), etoposide (positive control for tumour cells), silymarin (positive control for HepG2) and a cell control (CC) in which there is just medium and cells. 12. All samples are solubilized in DMSO, before dilution. 13. For comparison, the cytotoxicity of the extract is evaluated under the experimental conditions. 14. All experiments are carried out in triplicate and expressed as the average of three independent experiments. 15. Optical density (OD) is evaluated in a spectrophotometer at 590 nm. 16. The results are expressed as a percentage of cell proliferation, compared to 0.1% DMSO.

7

16

7

Cell Culture Assays

Scratching of Fibroblast Cells (Scratch Assay [12])

7.1

Principle

In this method, fibroblast cells are migrated into the gap or cell-free area prepared early by removing cells from that area by thermal, chemical or mechanical damage and which are estimated.

7.2

Protocol

1. Fibroblasts of the N3T3 strain, grown in medium containing 10% simulated body fluid (SBF), is placed in a cell culture plate with 24 wells, at a concentration of 5  105 cells/well and made a risk in the cell monolayer using a 10-micropipette tip. 2. The remnants of the cell are debrided by washing with phosphate-buffered saline (PBS). 3. The DMEM medium with 10% SBF is used as a negative control and the platelet-derived growth factor (PDGF, 5 ng/ mL) as a positive control. 4. The extract is tested at concentrations of 0.8, 4 and 20 μg/mL through incubation for 12 h at 37
C and 5% CO2 atmosphere. 5. The contraction rate is calculated using three images per well, captured in an inverted optical microscope coupled to a digital photographic camera and the images evaluated using ImageJ® software.

8

Evaluation of the In Vitro Inflammatory Response in AGS Cells 1. Adenocarcinoma gastric cell line (AGS) cells grown in Dulbecco’s modified Eagle’s medium (DMEM) are seeded in 96-well acrylic microplates (2  104 cells/well), and incubated for 24 h at 37
C with an atmosphere containing 5% CO2 to allow these cells to adhere to the plate. 2. After this period, the culture medium is removed by pouring the microplates, and in its place are added treatments prepared with culture medium (growth control), extract (6.25–800 μg/ mL) or doxorubicin (0.1–100 μM), and the plates incubated again for 24 or 72 h. 3. After the incubation time, the treatments are removed by pouring the plates to add 200 μL of the Alamar Blue developer (10% in DMEM medium supplemented with foetal bovine serum) in each well and incubated again for 6 h. 4. The reading is performed on a spectrophotometer at 540 nm (for the oxidized state of the reagent) and at 620 nm (for the reduced state of the reagent [5]). 5. The results are expressed in terms of 50% inhibitory concentration (IC50), which refers to the concentration of the drug that inhibits 50% of cell proliferation. IC50 values 13) for 20 min and subject to 25 V, 30 mA electrophoresis run for 30 min. 8. At the end of electrophoresis, the slides are incubated in neutralization solution (0.4 M Tris, pH 7.5) for 15 min, fixed in 96% ethanol for 5 min, stained with GelRed™ and read under a fluorescence microscope at 400 magnification. 9. Genetic damage is identified by the presence of a comet-like tail formed by fragments of DNA, quantified by the ratio between the intensity of the comet’s tail and the total intensity of the comet, multiplied by 100 and expressed as % of DNA in the tail. 10. Precisely, 100 nucleoids per slide are counted, properly photographed and evaluated with the aid of the TriTek Comet ScoreTM Freeware software.

4

Effect on Cell Cycle Principle: The extract/drug complex induces double-stranded DNA breaks that prevent its entry into the mitotic phase, promoting cell-cycle

38

Toxicology Studies: In Vitro and In Vivo

arrest. Excessive accumulation of DNA damage by continuous or higher exposure to drugs results in p53 accumulation, activates p53/bax signalling pathway and leads to cell apoptosis [10]. Protocol: 1. CHO-K1 cells (105–206 cells/well) are seeded in 12-well plates and then treated with the extract (100 μM) or etoposide (10 and 100 μM) as a positive control. 2. Some wells received only complete grown medium to assess the normal cell cycle. 3. After 24 h of culture, cells are harvested, iced twice with ice-cold phosphate-buffered saline (PBS), fixed with cold 70% ethanol and kept at 20
C until use. 4. Then, cells are washed three times with PBS, and stained with a PBS solution of ribonuclease-A (50 μg/mL) and propidium iodide (20 μg/mL) for 90 min. 5. Cell-cycle distribution is determined by flow cytometry (AccuriTM C6, BD, California, USA), and analysed using ModFit LT Software v5.0.

5

In Vivo Acute Toxicity Study [11] Principle: The in vivo toxicity study evaluates the physiological or behavioural and neurological changes (tremors, urination, hyperactivity etc.) in animals on administering the sample.

5.1

Materials

l

Extract/drug.

l

Distilled water.

l

Beaker.

l

Glass rod.

l

12 mice (male).

Protocol: 1. The control group must consist of five animals. 2. The remaining mice should be separated into groups with five animals in each group according to distribution. Group 1 receives 500 mg/kg dose of the extract; Group 2 receives 1500 mg/kg dose of extract. 3. Administer equivalent dose to the animals, and the animals in the control group administer the vehicle (distilled water).

5 In Vivo Acute Toxicity Study

39

4. Evaluate the parameters in animals of each group that received a dose of extracts on placing an animal in the control group to compare. 5. Evaluate the reactions obtained on the first day, at the times determined in the Malone Table (Table 1), as well as daily until a cycle of 14 days of evaluation is closed. 6. After this period, sacrifice 30% of the animals treated with the highest dose (Group 2) and control animals of its group to perform a histological slide, as well as weighing and measuring their vital organs. Table 1 The Hippocratic Screen, according to Malone and Robichaud [11] Date: Animal tested:

Sex:

Weight:

Drug used:

Vehicle:

Concentration:

Volume injected:

Administration route:

Dosage:

Identification mark:

No:

Colour:

Activity

Central nervous system

Parameters

Time of injection Minutes Hours Days 0 15 30 1 2 4 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Stimulating Motility Respiratory frequency Tail erection Exophthalmos Stereotype movements Paw licking Mouth scratching Tail biting Clonic convulsions Tonic convulsions Fine tremors Coarse tremors Depressing Motility Respiratory frequency Catatonia Palpebral ptosis Analgesia Anaesthesia Loss of corneal reflex Ataxia Dyspnoea Alienation of environment Back tonus Loss of sense (paw) Exophthalmos Paralysis Sedation

(continued)

40

Toxicology Studies: In Vitro and In Vivo

Table 1 (continued) Other effects

Ear

Urination

Pallor Cyanosis Hyperaemia Increased Decreased Colour Diarrhoea Contortion Reaction of flight Passivity Aggressiveness Grunts Drooling Fasciculations Mydriasis Tail erection Tail tremor Pupil diameter

Heart:

Arrhythmia:

Stop systole:

Diastole:

Intestines:

Motility:

No motility:

Hypermotility:

Blood:

Pre-coagulated:

Coagulated:

Not coagulated:

Gallbladder:

Extended:

Normal:

Full:

Liver: Lungs:

Other observations:

Note: These parameters are written on observing the peculiar features or differences in animal organs

7. For other groups, sacrifice a treated animal and its control, weighing and measuring its vital organs.

6

Subchronic Toxicity (Repeated Doses) Principle: The toxicity of a sample is evaluated for 30 days of administration to animals on analysing several behavioural, haematological and biochemical parameters. Protocol: 1. Subchronic toxicity is assessed by single daily exposure of rats to the vehicle and 3 doses of extract/drug (50, 250 and 1000 mg/kg) per oral (p.o.), for 30 days, according to the method described by Chan et al. [12]. 2. The rats (n ¼ 6/group) are kept in individual metabolic cages to measure feed and water consumption, excretion of faeces

6 6 Subchronic Toxicity (Repeated Doses)

41

and urine and variation in body weight. The volume of treatments administered is adjusted according to the variation in animal weights, in order to keep the doses constant. 3. Data are collected every 2 days; however, for statistical analysis, these are grouped into sets of 6 days and expressed as D0, D1, D2, D3, D4 and D5 (Table 2). 4. Any symptoms or clinical signs of toxicity, such as changes in the skin, hair, eyes and mucous membranes in the gastrointestinal, respiratory, central (CNS) and peripheral nervous systems (PNS), as well as behavioural manifestations in general, are noted. 5. On the 31st day of the trial, the animals are anaesthetized with ketamine and xylazine (100 and 10 mg/kg, respectively) to collect blood via the inferior vena cava for haematological examinations (red blood cells, haemoglobin [Hb], haematocrit [Ht], volume mean corpuscular [VMC], mean corpuscular haemoglobin [MCH], platelets, T leukocytes, neutrophils, lymphocytes, eosinophilic monocytes, basophils) and serum intended for biochemical analyses (uric acid [UA], alkaline Table 2 Effect of subchronic oral treatment of extract/drug for 30 days on body weight, cumulative weight gain, water and feed intake, and excretion of faeces and urine in rats Period of treatment (days) Parameters

D0

D6

D12

D18

D24

D30

Extract (100 mg/kg) Body weight (g) Cumulative weight gain (g) Water intake (mL) Feed intake (g) Faeces output (g) Urine output (mL) Extract (400 mg/kg) Body weight (g) Cumulative weight gain (g) Water intake (mL) Feed intake (g) (continued)

42

Toxicology Studies: In Vitro and In Vivo

Table 2 (continued) Period of treatment (days) Parameters

D0

D6

D12

D18

D24

D30

Faeces output (g) Urine output (mL) Extract (1000 mg/kg) Body weight (g) Cumulative weight gain (g) Water intake (mL) Feed intake (g) Faeces output (g) Urine output (mL) Vehicle (10 mL/kg) Body weight (g) Cumulative weight gain (g) Water intake (mL) Feed intake (g) Faeces output (g) Urine output (mL)

phosphatase [ALP], gamma-glutamyl transferase [GAMA GT], glucose [GL], total cholesterol [TC], triglycerides, albumin, total proteins, aspartate aminotransferase [AST], alanine aminotransferase [ALT], urea and creatinine; Table 3). 6. Animals are sacrificed by deepening the anaesthesia to remove organs (lung, heart, liver, kidneys, spleen, stomach and brain) for determining their relative weights [(organ weight/body weight)  100] and kept in 4% paraformaldehyde solution for 24 h for histopathological analysis. 7. After a period of fixation in paraformaldehyde, the organs are dehydrated and impregnated with paraffin in an automatic histological processor. 8. Approximately, 3 μm sections of each organ are obtained to make the slides in a microtome and later stained, and morphological changes, necrosis/degeneration, and mononuclear (MN), polymorphonuclear and neovascularization cells are analysed.

6 Subchronic Toxicity (Repeated Doses)

43

Table 3 Effect of subchronic oral administration of extract/drug on haematological and biochemical parameters in rats after 30 days of treatment

Biochemical parameters

Vehicle

Extract 100 mg/kg

Extract 400 mg/kg

Extract 1000 mg/kg

Haematological parameters Red blood cells (106/mm3) Haemoglobin (g/dL) Haematocrit (%) MCV (fl) MCH (pg) MCHC (%) Platelets (103/mm3) Total leukocytes (103/mm3) Neutrophiles Lymphocytes Eosinophil Monocytes Biochemical parameters Glucose (mg/dL) Urea (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) Alanine aminotransferase (UI/L) Aspartate amino transferase (UI/L) Alkaline phosphate (UI/L) Total cholesterol (mg/dL) Triglycerides (mg/dL) Total proteins (mg/dL) Gama GT MCV mean corpuscular volume, MCH mean corpuscular haemoglobin, MCHC mean corpuscular haemoglobin concentration, Gama GT gamma-glutamyl transferase

44

Toxicology Studies: In Vitro and In Vivo

References 1. McGaw LJ, Elgorashi EE, Eloff JN (2014) Cytotoxicity of African medicinal plants against normal animal and human cells. Toxicol Surv Afr Med Plants 2014:181–233 2. Sharwan G, Jain P, Pandey R, Shukla SS (2015) Toxicity profile of traditional herbal medicine. J Ayurvedic Herb Med 1(3):81–90 3. Robyn K (1996) Toxicology and herbs: an introduction. Austr J Herb Med 8:100–111 4. OECD (Organization for Economic Cooperation and Development) (2001) Guideline for testing of chemicals: acute oral toxicity–acute toxic class method. Guideline, 423. OECD, Paris 5. OECD (2008) Test no. 425: acute oral toxicity: up-and-down procedure: OECD Guidelines for the Testing of Chemicals. OECD, Paris 6. Nakayama K, Nomoto M, Nishijima M, Maruyama T (1997) Morphological and functional characterization of haemocytes in the giant clam Tridacna crocea. Journal of Invertebrate Pathology 69:105–111 7. Arunachalam K, Asceˆncio SD, Soares IM, Aguiar RWS, da Silva LI, de Oliveira RG, Balogun SO, de Oliveira Martins DT (2016)

Gallesia integrifolia (Spreng.) Harms: in vitro and in vivo antibacterial activities and mode of action. J Ethnopharmacol 184:128–137 8. Manshian BB, Singh N, Doak SH (2013) The in vitro micronucleus assay and kinetochore staining: methodology and criteria for the accurate assessment of genotoxicity and cytotoxicity. In: Genotoxicity Assessment. Humana, Totowa, NJ, pp 269–289 9. Collins AR (2004) The comet assay for DNA damage and repair. Mol Biotechnol 26(3):249 10. Zhou Y, Li S, Li J, Wang D, Li Q (2017) Effect of microRNA-135a on Cell Proliferation, Migration, Invasion, Apoptosis and Tumor Angiogenesis Through the IGF-1/PI3K/Akt Signaling Pathway in Non-Small Cell Lung Cancer. Cell Physiol Biochem 42:1431–1446. https://doi.org/10.1159/ 000479207 11. Malone MH, Robichaud RC (1962) A Hippocratic screen for pure or crude drug materials. Lloydia 25(4):320 12. Chan PK, O’hara GP, Hayes AW (1982) Principles and methods for acute and subchronic toxicity. Principles Meth Toxicol 12:17–19

Chapter 6 Standard Procedure for Anaesthesia in Preclinical Experiments Abstract Anaesthesia has been an inevitable part of animal studies since it provides a condition of unconsciousness, analgesia, muscle relaxation and a-reflexia with constant immobility that are most required to perform the in vivo experiments. Hence, standard procedures are significant with skilled techniques, which are conferred in this chapter with pre- and post-procedures, calculation examples for anaesthetic agent preparation, standard volumes of agents administered in animals and the animal handling. Key words Ketamine, Xylazine, Anaesthesia, Preclinical, Animals

Aim: The aim of this experiment is a standardization of the main methods of induction of anaesthesia in laboratory animals (mice/rat). Introduction: In biomedical research, the animal models play a vital role as they provide clear picture to understand the physiology of other living organisms as well as about the pathophysiology of diseases that aid fundamental information for the drug discovery. Anaesthesia is a condition of unconsciousness, analgesia, muscle relaxation and a-reflexia, providing constant immobility and thus is an unavoidable part in the experiments using laboratory animals [1]. Based on the nature of the anaesthetic agents, anaesthetic regimen can be inhaled or are injectable, and the skilled techniques in this lessen the animals’ suffering and stress during the course since the animals do not willingly endure human exploitation [1]. Also, it is observed that the anaesthetic agents like ketamine, halothane/isoflurane and propofol and the anaesthesia protocols have great influence in the survival of animals and also in the results since it can affect many factors in animal’s body including physiological parameters, cerebral metabolism and also neurotransmission. Therefore a safe/ standard procedure in anaesthesia is to be followed with skilled techniques that consider these parameters and animal welfare [2]. Karuppusamy Arunachalam and Sreeja Puthanpura Sasidharan (eds.), Bioassays in Experimental and Preclinical Pharmacology, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-1233-0_6, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021

45

46

Standard Procedure for Anaesthesia in Preclinical Experiments

Principle: In common, anaesthesia could affect a few physiological parameters (blood oxygen saturation, pressure, the cerebral blood flow etc.) that would be affecting the postoperative record. Most of the anaesthetic drugs reduce the cerebral metabolism and they often affect the neurotransmission of nerve impulses; for that, the body temperature and other physiological parameters have to be monitored during anaesthesia [3].

1

Planning and Anaesthetic Care [4] Protocol: 1. In all situations in which it is necessary to anaesthetize an animal, it is very important that the researchers plan and effectively put in place the appropriate care before, during and after each procedure. 2. The use of anaesthetic agents significantly changes the animal’s physiology, and without the necessary care and due planning the result can be disastrous. 3. The degree of the changes caused varies, but every anaesthetic agent generates hypothermia and decreased cardiovascular (bradycardia) and respiratory (Bradypus) activities. 4. After the procedure, these changes persist until the animal’s recovery, and therefore it is necessary to care for the animal immediately after the procedure and in some cases, for a few more days afterwards. 5. The animal’s recovery time varies depending on the agent used.

2

Pre-procedure 1. Before starting a procedure that requires the use of anaesthetic or sedative agents, researchers should assess some factors. 2. Factors related to the animal include age, sex, species, temperament, lineage and health status. 3. Factors related to the procedure include the technique selected, the duration of the procedure, the degree of pain/ discomfort the procedure may cause the animal and the training of the people involved. 4. Factors related to the laboratory consider whether the environment and materials available are suitable for the procedure. 5. Factors related to the post-procedure period include the choice of the anaesthetic as it can cause undesirable effects on the animal. In cases where the procedure is not terminal, that is,

3 Experimental Procedure

47

the animal must recover from anaesthesia, it is important to know what the expected effects of administering the chosen anaesthetic are. 6. Depending on the case, administration of medications such as antibiotics (when there is a need to minimize the risk of infection in the postoperative period) and analgesics are required before the start of the procedure. 7. It should be remembered that animal transport promotes increased heart rate, weight loss, increased plasma adrenaline concentration, noradrenaline, glycemia, cortisol and free fatty acids, and causes alteration of carbohydrates, plasma proteins, osmolarity and lipid metabolism. In addition, it promotes neutrophilia and lymphopenia. It is further observed that these changes remain for approximately 7 days and, depending on the animal’s genotype, they can last for several weeks. 8. Therefore, before starting any procedure with the animals, reserve at least 7 days (preferably 14 days) for acclimatization to the new environment even if the animal has only moved from one section to another within the same container. 9. The clinical inspection of the animal before the anaesthetic procedure provides important information about its health status and observes the hydration status and the presence of clinical respiratory signs that are very common in rodents (e.g. Mycoplasma Pulmonis infection). 10. Another important care is with the body mass when calculating the doses of the drugs. This must be precise to avoid overdose and weight loss that inevitably occurs in the post-surgical period. In the case of loss of 10–15% of the animal’s body weight in a few days, euthanasia is recommended and also indicated when the total loss reaches 20% of the body weight. 11. It is not necessary to fast in rodents, as they do not vomit; in addition, they become hypoglycaemic very quickly when fasted. 12. The restriction of food should only occur if it is really necessary and is specified in the research protocol approved by Institute Ethical Committee. 13. In the case of water supply, the restriction must occur at least 60 minutes before anaesthetic induction.

3

Experimental Procedure During the experimental procedure, the following precautions must be taken into account: 1. Maintain the sterility of the environment.

48

Standard Procedure for Anaesthesia in Preclinical Experiments

2. Handle the tissues carefully, as in this way the pain after the surgical procedure is reduced, as well as the risk of developing infections and contamination on collected parts. 3. Reset fluid loss (administer the heated fluid, to prevent a sudden drop in the animal’s temperature; see Subheading 3.1). 4. Keep the animal warm (using hot plates, thermal bag, lamp, bubble wrap etc.), as it has a small body and loses heat more easily. Attention should also be paid to ensure that the animal does not develop hyperthermia. 5. Take into account the anatomical particularities and the indications and contraindications of anaesthetics according to the species. 6. Avoid drying the eyes by excessive contact with air; apply a sterile protective gel/ointment or simply cotton soaked in saline during the period the animal is anaesthetized. 7. The animal can have accelerated metabolism and be susceptible to hypothermia due to low weight/area ratio corporeal. 8. Check cannibalism behaviour (it is important to always leave water and food at will). 9. Rats are fast-growing, have a life span of 2.5–3 years, are intelligent and females are lighter. 10. Mice are preferred for genetic studies as they are very prolific, small, and easy for domestication and maintenance. 11. Blood collection can be performed on the tail, orbital sinus and cardiac puncture. 12. The ideal temperature of 20–25
C and ventilation with 100% air renewal should be maintained. 3.1 Physiological Parameters

3.2

Contention

Rat

Mice

Heart rates (HR, BPM)

250–350

570

Respiratory (FR, MPM)

70–90

180

Temperature

37.5

37.5

Weight

250–700 g

30–40 g

1. In physical restraint by the base of the tail, do not hold for too long because they do spinning and hence there are chances of breaking the tail, and they try to climb the hand to bite. 2. Another way is by holding the skin on the back of the neck. 3. In mechanical containment, containment rollers are used, where the animals are only with the tail out of the roll.

References

3.3 Via and Volumes mL/kg

3.4 Calculations for Animal Anaesthesia Agent

49

Animals Oral Subcutaneous (SC) Intraperitoneal (IP) Intramuscular (IM) Intravenous (IV)

l

Rat

10

5–10

5–10

0.5

0.5

Mice

10

2–3

2–3

0.3

0.2

Ketamine 10,000 mg/100 mL ¼ 100 mg/mL (stock). We have to do C1  V1 ¼ C2  V2. C1 ¼ 100 mg=mL, V1 ¼ ?mL: If we prepare for 100 mL (V2) of ketamine at 7 mg/mL (C2), then: C2 ¼ 7 mg=mL, V2 ¼ 100 mL: V1 ¼ 7  100 ¼ 7 mL: It is the volume of ketamine (stock) to be taken.

l

Xylazine 2000 mg/100 mL ¼ 20 mg/mL (stock). C1 ¼ 20 mg=mL, V1 ¼ ?mL: If we prepare for100 mL (V2) of ketamine at 7 mg/mL (C2), then: C2 ¼ 0:8 mg=mL, V2 ¼ 100 mL V1 ¼ 0:8  100 ¼ 4 mL: It is the volume of xylazine (stock) to be taken to make 100 mL of the solution with a concentration of 0.8 mg/mL.

l

Mix the two (11 mL) and make up with distilled water.

l

Saline solution 0.9%. 0.9 g ¼ 100 mL X ¼ 1000 mL. X ¼ 9 g of sodium chloride and add distilled water up to 1 L.

References 1. Gargiulo S, Greco A, Gramanzini M, Esposito S, Affuso A, Brunetti A, Vesce G (2012) Mice anesthesia, analgesia, and care, part I: anesthetic considerations in preclinical research. ILAR J 53 (1):E55–E69 2. Luca C, Salvatore F, Vincenzo DP, Giovanni C, Attilio ILM (2018) Anesthesia protocols in laboratory animals used for scientific purposes. Acta Bio Med 89(3):337

3. Alstrup AKO, Smith DF (2013) Anaesthesia for positron emission tomography scanning of animal brains. Lab Anim 47(1):12–18 4. Hedenqvist P, Hellebrekers LJ (2003) Laboratory animal analgesia, anesthesia, and euthanasia. Handb Lab Anim Sci 1:413–455

Chapter 7 General Considerations and Collection of Animal Blood Abstract The collection, processing and storage of biological samples from the animals in in vivo methods for the evaluation of various parameters of the study follow systematic approach since it may affect the results. Therefore, the procedure of collection, area of collection (blood) and storage of samples need significant assurance of perfect quality. In this chapter, the protocols for various ways of blood collection and pre-procedure steps are described. The stepwise standardized procedures of blood collection, including vein blood collection, collection from tail vein, through cardiac puncture, from posterior vena cava and orbital sinus, are discussed with safety measures for the storage. Key words Vein blood, Vena cava, Cardiac puncture, Orbital sinus, Blood collection

Aim: To standardize the blood sample collection in laboratory animals (mice/rat). Introduction: Humans have depended on animals for various purposes from time immemorial, and even now mankind is depending specifically on laboratory animals like rodents, rabbits and monkeys for research work. Even though in vitro assays and cell-based assays have developed in drug research, these results fail at occasions to compete with the complicated physiological environment in animals and other higher organisms [1]. Hence, research depending on animals is growing in trend, and the collection, processing and storage of organs, tissues and blood as biological samples in the animal model are inevitable part of scientific research for examining different parameters and different methods existing for it. The systemic approach on sample collection is important, which include the collection of samples, its preparation, storage and handling since it assures high-quality sample for examining and perfect parameter results [2]. Therefore, the procedure for sample collection should be stressless and handled carefully to avoid contamination from secretions or debris or any unwanted tissue of animal since it affects

Karuppusamy Arunachalam and Sreeja Puthanpura Sasidharan (eds.), Bioassays in Experimental and Preclinical Pharmacology, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-1233-0_7, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021

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the parameters and results in the study. Also, the parameters show variations on differences in the sample collecting area or procedure, handling, and the housing environment [3, 4]. Therefore, since the animal models are an unavoidable part of drug research, the ‘3Rs’ principle—Reduction, in the number of animals, Refinement, involving the alternate methods to avoid the suffering of animals, and Replacement, of laboratory animals— proposed by William Russell plays a vital part in laboratory techniques for the use of animals with ethical aspects [5]. Principle: Blood collection of experimental animals is one of the vital protocols in pharmacological/biochemical studies. During the blood sample collection from laboratory animals, the animals should be acclimatized well and with minimum stress since stress could affect the outcome of the experiment. Protocol: 1. The approach of blood series has to be defined inside the protocol permitted by means of the Institute Animal Ethics Committee. 2. Blood is withdrawn from venous, arterial blood vessels or heart chambers. 3. Once in two weeks is ideal for non-rodents. 4. All non-terminal blood series without replacement of fluids is limited up to 10% of general circulating blood volume in healthy, every day, grown-up animals on an unmated occasion, and the series can be repeated after three to four weeks. 5. In case repeated blood samples are required at quick intervals, a maximum of 0.6 mL/kg/day of an animal’s total blood extent may be removed in each 24 hours. 6. If it includes repeated blood sample collection, the samples may be withdrawn through a brief cannula. 7. This can also reduce pain and pressure within the experimental animals. 8. The approximate blood quantity per animal to collect is 55–70 mL/kg body weight. 9. All the manner of blood series should follow the approach described by Hoff [6].

1

Vein Blood Collection 1. The lateral saphenous vein is used for sampling whilst taking aseptic precautions.

2 Tail Vein Blood Collection

53

2. The back of the hind leg is shaved with electric-powered trimmer till saphenous vein is seen (hair removal cream also can be used). 3. The animal is constrained manually or using an appropriate animal restrainer. 4. The hind leg is immobilized and slight strain can be implemented lightly above the knee joint. 5. The vein is punctured using a 20 G needle and sufficient quantity of blood is gathered with a capillary tube or a syringe with a needle. 6. The punctured site is compressed to prevent the bleeding. 7. Whilst amassing blood, the nearby anaesthetic cream may be applied on the collection site. Not more than three attempts are made, but non-stop sampling must be averted and accumulation greater than four samples in a day (24-h length) is not really useful.

2

Tail Vein Blood Collection 1. This technique is used for accumulating a large volume of blood pattern (as much as 2 mL/withdrawal). 2. The animal is made comfortable in a restrainer at the same time as maintaining the temperature around 24–27  C. 3. The tail is rubbed from the base to the top as it result in leucocytosis. 4. If the vein is not seen, the tail is dipped into warm water (40  C). 5. The local anaesthetic agent needs to be implemented on the surface of the tail 30 min earlier than the experiment. 6. A 23 G needle is inserted into the blood vessel and blood is accumulated using a capillary tube or a syringe with a needle. 7. In case of no difficulties, 5 cm or at least 1 cm of the bottom of the tail and skin is cut open and the vein is pricked with bleeding lancet or needle and blood is collected with a capillary tube or a syringe with a needle. 8. Having completed blood collection, pressure/silver nitrate ointment/solution is implemented to prevent the bleeding. 9. If a couple of samples are washed, the temporary surgical cannula can be used. 10. Restrainer is washed frequently to keep away/save you from pheromonally triggered stress or remove contamination.

54

3

General Considerations and Collection of Animal Blood

Cardiac Puncture Blood Collection 1. Requirements consist of animal, anaesthetic agent, towel and cotton, 19–25 G needle with 1–5 mL syringe, surgical blade, tube (internal diameter of 0.1–0.3 mm) for thoracotomy, plastic disposable bag and blood sample series tubes. 2. In general, the cardiac puncture is usually recommended at the end of the examination in unmated and perfect experimental animals for collecting a large quantity of blood. 3. During this blood sample collection, an animal is in overdose anaesthesia for sacrifice. 4. An appropriate needle is used for blood sample collection with or without thoracotomy. 5. In this, blood sample is taken from the heart, preferably from the ventricle, slowly to keep away from collapsing of coronary heart.

4

Posterior Vena Cava 1. Posterior vena cava blood collection is usually recommended at the end of the study. 2. An animal should be anaesthetized, ‘Y’- or ‘V’-shaped cut inside the abdomen is made, and the intestines are lightly removed. 3. The liver is pushed forward and the posterior vena cava (among the kidneys) is identified. 4. Also, 21–25 G needle is inserted to gather blood from the posterior vena cava. 5. This procedure is repeated three to four times to acquire more volume of the blood sample.

5

Orbital Sinus 1. This approach is utilized with recuperation in test conditions and this technique is likewise called periorbital, back orbital and orbital venous plexus dying. 2. Blood collection is gathered under mild anaesthesia. 3. The effective ophthalmic sedative drug is applied to the eye before sleeping. 4. The animal is scuffed with thumb and index finger of the unused hand, and the skin around the eye is pulled rigid.

References

55

5. A capillary tube is embedded into the average canthus of the eye (30-degree point to the nose). 6. Slight thumb pressure is sufficient to cut the tissue and enter the plexus/sinus. 7. When the plexus/sinus is punctured, blood get through the capillary tube. 8. When the necessary volume of blood is collected from plexus, the capillary tube is delicately expelled and cleaned with sterile cotton. 9. Draining can be stopped by applying delicate finger pressure. 10. Thirty minutes after blood assortment, an animal is checked for postoperative and periorbital injury. References 1. Bajpai VK, Rather IA, Kim K (2016) Isolation of mouse internal organs for molecular and histopathological studies. Bangladesh J Pharmacol 11 (2):485–488 2. Golubeva Y, Rogers K (2009) Collection and preparation of rodent tissue samples for histopathological and molecular studies in carcinogenesis. Methods Mol Biol 511:3–60 3. Kumar M, Dandapat S, Sinha MP, Kumar A, Raipat BS (2017) Different blood collection methods from rats: a review. Balneo Res J 8 (2):46–50

4. Parasuraman S, Raveendran R, Kesavan R (2010) Blood sample collection in small laboratory animals. J Pharmacol Pharmacother 1 (2):87 5. Andersen ML, Winter LM (2019) Animal models in biological and biomedical researchexperimental and ethical concerns Anais da Academia Brasileira de Cieˆncias (Proceedings of the Brazilian Academy of Sciences), 91 6. Hoff J (2000) Methods of blood collection in the mouse. Lab Anim 29(10):47–53

Chapter 8 Animal Experiments on the Cardiovascular System Abstract Cardiovascular disease (CVD) is caused by the imbalance in the working of heart, blood and blood vessels and the changes in life style increase its risk factors, while its treatment relies on reducing the risk factors like lowering bad cholesterol and reducing blood pressure (BP). This chapter deals with the various protocols related to the risk factors of cardiovascular disease that include the procedures like obtaining electrocardiogram (ECG) and blood pressure (BP) signals and myocardial ischemia induction. These methods involve the determination of BP, ECG, plasma concentration, and the histopathological analysis of heart tissues, which are also described here. Key words ECG, BP, Cardiovascular, Heart, Myocardial ischemia

Aim: To investigate the cardioprotective potential of the extract/chemical compounds by using animal models. Introduction: The heart, blood and blood vessels are the three components of the cardiovascular system, in which the heart creates the force to drive blood through the vessels, while the mechanical properties of the blood vessel wall and heart muscle, as well as the fluid properties of blood, determine the blood flow behaviour and magnitude [1]. Any imbalance in these causes cardiovascular disease (CVD), which is generally termed for coronary heart disease, rheumatic and congenital heart diseases, cerebrovascular disease, venous thromboembolism and peripheral arterial disease, and the change in lifestyle increases the risk factors for CVD, which has become an ever-growing issue in the world [2]. Apart from the risk factors that include smoking, hypertension, low-density lipoprotein (LDL), abdominal obesity, impaired endothelial function, vascular inflammation, etc., metabolic diseases like diabetes and age also enhance the prevalence for cardiac and vascular diseases that include heart failure, atherosclerosis, stroke, renal failure and myocardial infarction [2–4]. An imbalance observed between coronary blood supply

Karuppusamy Arunachalam and Sreeja Puthanpura Sasidharan (eds.), Bioassays in Experimental and Preclinical Pharmacology, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-1233-0_8, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021

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and myocardial demand and resulting oxidative stress-induced lipid peroxidation are the main causes for heart failure. Therefore, treatment for reducing these risk factors, like reducing blood pressure (BP) and lowering bad cholesterol, enables the significant treatment to reduce heart failure [3, 5]. Principle: The interruption of coronary blood supply and related biochemical changes involving oxidative stress-induced lipid peroxidation, hyperlipidaemia and hyperglycaemia are observed as the main causes of myocardial infarction [5].

1

Obtaining Electrocardiogram (ECG) and Blood Pressure (BP) Signals Protocol: 1. The animals are anaesthetized by sodium pentobarbital (60 mg/kg), administered by intraperitoneal (i.p.) route. 2. Surgery is started after anaesthesia reaches the depth required. The rats are tracheostomized using polyethylene (PE) cannulas and a connected respirator (SAR-840, USA) to allow adequate ventilation (duration 38 breaths per minute [bpm]). 3. The femoral artery is catheterized using a cannula made with a polyethylene (PE) tube to obtain the blood pressure (BP) signal. 4. To obtain the electrocardiogram (ECG) signal, stainless steel hypodermic needles are used as sensors, positioned so as to allow the measurement of the potential difference related to the Dietary Inflammatory Index (DII) derivation. 5. The PE transducer and the ECG cable are connected to a system of signal conditioners. 6. The signals obtained from this system are sampled in real-time at a frequency of 1200 Hz by an analogue-digital converter board of 12-bit resolution (Daqboard 2000, USA).

2

Myocardial Ischemia Induction 1. The procedure for the induction of myocardial ischemia by coronary ligation in rats is performed according to the previously described technique [6, 7]. 2. After thoracotomy in the fourth left intercostal space, the heart is eviscerated and ligation is performed in the left anterior descending coronary artery, between the pulmonary artery

2 Myocardial Ischemia Induction

59

cone and the left atrium, using an atraumatic needle and cotton thread. 3. The heart is then quickly repositioned to the rib cage and the previously prepared suture is completed to close the chest. 4. The animals in the control group (submitted to fictitious surgery) are submitted to all steps of the surgical procedure described above, except for the ligation of the coronary artery. 2.1 ECG and BP Analysis

1. The digital signals are analysed by visual inspection of the record with the aid of the WinDaq/EX Playback and Analysis software (DATAQ Instruments, USA), using several compression factors for the signals. 2. In this phase, the analysis and classification of ECG variations after sampling 2-s segments are performed, and the values of systolic (SBP) and diastolic (DBP) blood pressure, heart rate (HR) and ECG intervals such as QT (interval between the beginning of the Q wave and the end of ECG T wave), RR (interval between two ECG R waves), PR (interval between the P wave and beginning of R wave) and QRS complex (beginning of Q wave until end of S wave) are obtained. 3. The QT interval is later corrected for heart rate using the formula: ðQTc ¼ QT=ðRR Þ 1=3Þ: 4. The QT interval can be corrected by the Fridericia formula for RR values less than 500 ms or by the Bazzett formula for RR values greater than 500 ms [8]. 5. Another parameter evaluated is the area under the curve of the ST segment, using the method of estimating the area by planimetry. 6. The area under the ST segment curve is demarcated in each set of sampled waves to demarcate the area under the curve; first, a vertical line is created starting from the point where the S wave inclination begins. Then, a horizontal line is drawn, from right to left, from the point where the T wave reaches the ECG bioelectric point. 7. The area estimate is then performed by planimetry, with the aid of the AUTOCAD® software. 8. The data are obtained as values absolute in time: before ligation (control), 1, 2, 3, 4, 5, 7, 10, 15, 20, 25 and 30 min after ligation. 9. Then, an analysis of the variation of the area is performed for each animal in relation to the control time.

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Animal Experiments on the Cardiovascular System

2.2 Determination of the Plasma Concentration of Sample Drug

1. At the end of the experiments, an aliquot of 0.5 mL of heparinized whole blood is collected from each animal to determine the plasma concentration of the sample drug using highperformance liquid chromatography (HPLC). 2. The blood samples are centrifuged at 1107  g and 250 μL aliquots of plasma obtained are stored in a freezer at 80  C until analysis. 3. Before using the HPLC method for determining the plasma concentration of sample drug, it is necessary to carry out bioanalytical validation in advance to ensure the reliability of the results. 4. Therefore, linearity, precision and accuracy parameters need to be studied. Calibration curves for determining linearity are obtained by analysing eight concentrations of the sample drug solution (Sigma, USA), each in sextuplicate, ranging from 40 to 1000 ng/mL. 5. Assess the precision and accuracy of the method using three different concentrations of sample drug in plasma, in triplicate. 6. With the validated method, the plasma concentration of sample drug is determined in the previously collected plasmas. 7. Plasma samples are thawed 24 h before analysis and mixed with metformin (internal standard) at a concentration of 3 μg/mL. 8. Then, 500 μL of acetonitrile is added to precipitate the proteins present in the plasma. 9. After homogenization in a vortex for 1 min and centrifugation at 14,881  g for 10 min, the supernatant needs to be filtered (0.45 μm) and evaporated at room temperature in vacuum desiccators. 10. After the complete evaporation of acetonitrile, the precipitate shall resuspend with the mobile phase (methanol:1% acetic acid in H2O, 50:50 (v/v)) and injected into the chromatograph. 11. Perform HPLC (Waters® 2695) at 254 nm in a photodiodearray detection (PDA) detector, using a C18 reverse-phase column and a mobile phase consisting of methanol and 1% acetic acid solution in water, with gradient elution (0–5 min: 55–90% methanol; 5–9 min: 90% methanol; 9.1–14 min: 55% methanol). The flow is 1.0 mL/min, and the injection volume is 75 μL.

2.3 Histopathological Analysis of the Heart

1. Assess the histopathological changes in the hearts of all animals in the groups. 2. For the assessment, experimental samples are collected and fixed in a 10% buffered formaldehyde solution (pH 7.2), with the objective of preserving the morphology and composition of the tissue until the slides are made.

References

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3. To make the slides, the hearts are subjected to dehydration in alcohol to remove all the water present in them. 4. After dehydration, do diaphanization in order to make the tissues translucent. For this, the tissues/organs are submitted to two xylol baths. Then, the hearts/tissues are embedded in paraffin, using two paraffin baths for its penetration into vessels and intercellular spaces. 5. The blocks of paraffin containing the impregnated tissues are then subjected to microtomy when two serial cuts of 4 μm thickness are obtained for each block. 6. The cut pieces are placed in a water bath so that the tapes are stretched and placed over the glass slides that are kept in an oven at 60  C for drying. 7. The slides are subjected to haematoxylin-eosin (HE) staining to provide a general analysis of histopathological changes. 8. The staining process consists of immersion of slides in xylol baths at first, for dewaxing, followed by immersion in alcohol and water baths for rehydration. 9. The slides are then subjected to the bath in haematoxylin dye (acid dye), and then they are washed in the water chain. 10. Then, differentiation is performed with the rapid passage of the slides in acid alcohol. 11. Again, the slides are washed under running water, immersed in the eosin dye (basic dye) and again wash under running water. 12. To complete the process, the blades are immersed in absolute alcohol baths quickly and taken to the oven at 60  C for a few minutes for drying. 13. After drying, the slides are immersed in xylol and assembled with Entellan (Merck) and coverslip. 14. Evaluations are performed using 10 and 40 objectives under the microscope Olympus BX50. 15. Photo documentation is carried out using a 40 objective Leica microscope, DM5000, coupled to the digital camera and computer.

References 1. Kroeker CG (2018) Cardiovascular system: anatomy and physiology. In: Labrosse MR (ed) Cardiovascular mechanics. CRC Press, Boca Raton, FL, pp 1–17 2. Stewart J, Manmathan G, Wilkinson P (2017) Primary prevention of cardiovascular disease: a

review of contemporary guidance and literature. JRSM Cardiovasc Dis 6:2048004016687211 3. Dimmeler S (2011) Cardiovascular disease review series. EMBO Mol Med 3(12):697–697 4. Nabel EG (2003) Cardiovascular disease. N Engl J Med 349(1):60–72

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5. Ahmed R, Tanvir EM, Hossen M, Afroz R, Ahmmed I, Rumpa NE, Paul S, Gan SH, Sulaiman SA, Khalil M (2017) Antioxidant properties and cardioprotective mechanism of Malaysian propolis in rats. Evid Based Complement Alternat Med 2017:5370545 6. Fishbein MC, Maclean D, Maroko PR (1978) Experimental myocardial infarction in the rat. Am J Pathol 90:57–70

7. Selye H, Bajusz S, Gransso S, Mendell P (1960) Simple technique for the surgical occlusion of coronary vessels in the rat. Angiology 11:398–407 8. Indik JH, Pearson EC, Fried K, Woosley RL (2006) Bazett and Fridericia QT correction formulas interfere with measurement of druginduced changes in QT interval. Heart Rhythm 3(9):1003–1007

Chapter 9 Animal Experiments on Ulcerative Colitis Abstract This chapter demonstrated the methods to investigate the intestinal anti-inflammatory effect of the extract/ drugs on ulcerative colitis in rats induced by 2,4,6-trinitrobenzene sulphonic acid (TNBS) by intrarectal administration and determined the intestinal anti-inflammatory activity by tissue damage evaluations, biochemical, histological and immunostaining parameters and antioxidant properties. Furthermore, it also demonstrated the Dextran sodium sulfate (DSS)-induced colitis protocol in mice to evaluate the protective effect of extract/drugs for prolong days on clinical features (disease activity index), antioxidants, antiinflammatory and immunomodulatory activities in relation to the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), levels of proinflammatory cytokines and changes in both macroscopic and microscopic nature of the colonic mucosa. These methods and the colitis with recurrence method are useful for the evaluation of spectrum of activities such as reduction in suppression of inflammation, oxidative stress, modulating numerous signal transduction pathways and induction of apoptosis. Key words Ulcerative colitis, Proinflammatory cytokines, TNBS , DSS, Disease activity index, Immunomodulatory

Aim: To investigate the therapeutic property of a drug on ulcerative colitis using experimental animals. Introduction: Chronic gastrointestinal tract (CGT) disorders that include Crohn’s disease (CD) and ulcerative colitis (UC) are collectively termed as inflammatory bowel diseases (IBDs) and are characterized with inflammation and mucosal damage in the intestine [1]. Apart from the similar pathophysiological symptoms, the CD and UC differ from each other as the inflammation occurs normally in all layers of the bowel in the case of CD, while in UC it occurs in the mucosal areas [2]. Therefore, ulcerative colitis is considered as a chronic inflammatory gastrointestinal disorder with intestinal inflammation and damage to the intestinal mucosa, where the main clinical symptoms include diarrhoea, rectal bleeding, abdominal pain and weight loss. Also colorectal cancer is a serious complication on chronic condition [3]. Even though the pathogenesis of Karuppusamy Arunachalam and Sreeja Puthanpura Sasidharan (eds.), Bioassays in Experimental and Preclinical Pharmacology, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-1233-0_9, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021

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Animal Experiments on Ulcerative Colitis

IBDs is unknown, it is widely accepted that the dysfunctions in the immune response and an imbalance between the pro- and antiinflammatory cytokines, causes inflammation in this disease. Besides the expression of cyclooxygenase-2 (COX-2), induced nitric oxide synthase (iNOS) and an increase in reactive oxygen species also play an important role in the intestinal inflammation [1, 4]. Aminosalicylates, corticosteroids, immunosuppressive and anti-TNF-α (Tumor necrosis factor-alpha) drugs are the most commonly used therapeutic drugs for IBD, while these are also associated with many side effects such as fever, cramps, rash, kidney problems and abdominal pain [1]. Principle: The infiltration of neutrophil and other inflammatory cells into the intestine tissues, generation of inflammatory cytokines with induced nitric oxide synthase overexpression to form Nitric-oxide (NO) and also the augmented expression of COX-2 enzyme in intestine wholly generates oxidative stress, which results in the complete impairment of colon mucosa and the formation of lesions in intestinal tissues. These are observed as the main principle behind the models for UC [1]. Materials

1

l

Six complete cages anticoprophagic grid).

(polypropylene

cage,

l

Distilled water.

l

Analytical balance.

l

Beakers, tweezers and scissors.

l

Oral gavage.

l

TBNS.

l

Ethanol absolute.

l

Mesalazine.

l

Pipette and tips 1000 μL.

l

Cold saline 0.9%.

l

A 1 mL syringe with a needle attached to a probe.

cover

and

Ulcerative Colitis Induced by 2,4,6-Trinitrobenzenesulphonic Acid (TNBS) Protocol: 1. Use rats, weighing 180–220 g. 2. Set the animals in the laboratory (Cycle: light/dark for 12 h each).

1 Ulcerative Colitis Induced by 2,4,6-Trinitrobenzenesulphonic Acid (TNBS)

65

3. Randomize them into six groups of eight animals each. 4. Treat the animals orally (1.0 mL/100 g p.o.) as follows: – Group 1: Sham. – Group 2: Vehicle, 0.2% sodium carboxymethyl cellulose dissolved in distilled water. – Group 3: Extract/drug, 25 mg/kg, in Tween 80 2% dissolved in distilled water. – Group 4: Extract/drug 5 mg/kg, in Tween 80 2% dissolved in distilled water. – Group 5: Extract/drug 1 mg/kg, in Tween 80 2% dissolved in distilled water. – Group 6: Mesalazine 500 mg/kg, 0.2% sodium carboxymethyl cellulose dispersion in distilled water. 5. There are four pretreatments, once in 72, 48, 24, 2 h. 6. After the 24-h treatment, the animals are fasted from food for 18 h and water for 1 h before the last treatment (2-h treatment) and keep the animals in cages with anticoprophagic grid for 2 h, before induction. 7. Lightly anaesthetize the animal intraperitoneally with ketamine/xylazine solution (60 and 8 mg/kg, respectively). 8. Gently press the animal’s back with your fingertips to eliminate possible faeces residues that may be present in the colon. 9. Two hours after the last treatment, induce colitis by instilling 250 μL of TNBS via rectum (30 mg TNBS/250 μL 20% EtOH (Ethanol)) by inserting the cannula tube, which is approximately 8 cm for rats. Note: If there is any resistance, do not insert it, remove and restart the process so that there is no perforation on the intestine. 10. Slowly administer the solution and after the process, keep the animal upside down for 30 s. 11. After 24 h of induction, sacrifice the animal and remove 8 cm of the intestine from the anus. Note: For histological analysis, cut a segment of approximately 0.5 cm from the still closed intestine. 12. Afterwards, the animals colon is removed and opened for score determination according to Morris [5]: – 0: no damage. – 1: Localized hyperaemia, but without ulceration. – 2: Linear ulcers without significant inflammation. – 3: Linear ulcers with inflammation at one point. – 4: Two or more points of ulceration and inflammation.

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– 5: Two or more points of ulceration and inflammation where there is an injured area >1 cm of the length of the colon, with or without the presence of necrosis. Calculations: 1. Vehicle (Tween 80, 2%). Dissolve Tween 80, 2%, in distilled water Tween 80—2 g Distilled water—100 mL – Weigh the necessary amount in (inside) a small beaker. Add the water gradually until the dispersion is complete (homogenize with the help of a glass stick). Make up to the desired volume, mix and stock. – For example to prepare 25 mL of the vehicle, to administer approximately 2 mL/rat (200 g of weight on an average), weigh 0.5 g of Tween 80 and dissolve in 25 mL of distilled water. 2. Mesalazine. – Ten animals, around 220 g, receive 500 mg/kg of mesalazine (1.0 mL/100 g/p.o.), That is, 50 mg mesalazine/mL. – So there are a total of 2200 g of body weight, with 1 mL for every 100 g, being necessary therefore need 22 mL (so prepare 25 mL around) based on the calculation C1V1 ¼ C2V2 (refer to Chapter 2). – For this, weigh 1250 g of mesalazine and dissolve in 25 mL of sodium carboxymethylcellulose (0.2%). Mesalazine

500 mg

Carboxymethylcellulose

0.2%

Distilled water

1.0 mL

– Grind the tablets in a mortar with the pistil until a homogeneous fine powder is obtained. – Weigh the required amount of the mesalazine tablet powder so that you have the required amount of mesalazine and weigh sodium carboxymethylcellulose (0.2%), transfer to a mortar and grind well. Gradually add the necessary water, homogenizing by grinding until complete dissolution. 3. Extract/drug 25 mg/kg. – Ten animals, around 220 g, receive 25 mg/kg of extract (1.0 mL/100 g/p.o.), that is, 2.5 mg extract/mL. – So 22 mL (around 25 mL) is necessary for a total of 2200 g of body weight, with 1 mL for every 100 g.

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67

– Thus 25  2.5 mg ¼ 625 mg, that is, 0.625 g in 25 mL of Tween 80, 2% dispersion (Refer to Chapter 2 for the calculation). Extract/drug

0.625 g

Tween 80, 2%

25 mL

– Weigh the necessary amount of the extract and transfer it to a mortar and grind well. Gradually add the necessary water, homogenize by grinding until complete incorporation, with each addition. 4. Extract/drug 5 mg/kg. – Ten animals, around 220 g, receive 5 mg/kg of extract (1.0 mL/100 g/p.o.), that is, 0.5 mg extract/mL. – So there are a total of 2200 g of body weight, administered 1 mL for every 100 g, then 22 mL is necessary (prepare 25 mL). – Thus 25  0.5 mg ¼ 125 mg, that is, 0.125 g in 25 mL of the 2% Tween 80 dispersion. Extract/drug

0.125 g

Tween 80, 2%

25 mL

– Weigh the necessary amount of extract and transfer it to a mortar and grind well. Gradually add the necessary water, homogenizing by grinding until complete dissolution, with each addition. 5. Extract/drug 1 mg/kg. – Ten animals, around 220 g, receive 1 mg/kg of extract (1.0 mL/100 g/ p.o.), in 1.0 mg extract/mL. – So approximately there are a total of 2200 g of body weight, administered with 1 mL for every 100 g, if necessary then 22 mL (25 mL is prepared). – Thus, 25  1 mg ¼ 25 mg, that is, 0.025 g in 25 mL of the 2% Tween 80 dispersion. Extract/drug

0.025 g

Tween 80, 2%

25 mL

– Weigh the necessary amount of the extract and transfer it to a mortar and grind well. Gradually add the necessary water, homogenize by grinding until completely dissolved, with each addition.

68

2

Animal Experiments on Ulcerative Colitis

Chronic Ulcerative Colitis Induced by Dextran Sodium Sulphate(DSS) 2% [6] Principle: Chronic ulcerative colitis induced by DSS in mouse model uncovered a few highlights that are found in people, incorporating aggravation that begins in the distal colon and afterwards includes the proximal colon. A dysplasia that looks like the clinical course of human ulcerative colitis happens every now and then in the interminable period of DSS colitis. Taking care of mice with 1–5% DSS disintegrated in water is a reason for weight reduction, shortening the digestive tract, mucosal ulcers and invasion of provocative granulocytes [7]. Protocol: 1. Mice 6–8 weeks of age are randomly assigned to six experimental groups (n ¼ 10). 2. The groups are treated daily with vehicle (distilled water, 0.1 mL/10 g), extract (25, 100 and 400 mg/kg) and mesalazine (500 mg/kg). 3. Chronic ulcerative colitis (UC) is induced by administering multiple cycles of DSS (40,000–50,000 MW; Sigma). 4. The animals of the sham group received only distilled water ad libitum on days 1–5, 8–12, 15–19, 22–26, 29–33 and 36–40. 5. The rats are euthanized by CO2 inhalation followed by cervical dislocation on the 43rd day.

2.1 Determination of the Disease Activity Index

1. To determine the disease activity index (DAI), the animals are evaluated daily for changes in body weight and stool consistency. 2. The collection of faeces is made in only one animal, which is kept separated in a previously cleaned cage for 15–30 min, and the faecal content is collected and analysed for the bloodstain (hidden) in the faeces. 3. The criteria for calculating the disease activity index score is made according to that described by Walsh et al. [8]. 4. Elucidate the probable mechanisms involved in the intestinal anti-inflammatory action of extract/drug in mice by determining the antioxidant activity (GSH, SOD and CAT), nitric oxide concentration (NO), the concentration of pro- and antiinflammatory cytokines, expression of NF-κβ, p65, p-p65, p-JNK, JNK (c-Jun N-terminal kinases), Erk1/2, p-Erk1/2, iNOS, COX-2, p-AMPK and genes for TNF-α, IFN-γ (Interferon gamma), IL-2, IL-4 (Interleukin 4), IL-5 (Interleukin 5), IL-6, IL-10 (Interleukin 10), IL-12, IL-13 (Interleukin 13), IL-17 (Interleukin 17), IL-23, MCP-1, TGF- β1 (Transforming growth factor beta 1), claudin and occludin.

3 Colitis with Recurrence (Chronic)

2.2 Histopathological Analyses

69

1. The distal colons are collected and fixed for 24 h in 4% formaldehyde in phosphate-buffered saline (PBS, pH ¼ 7.4), dehydrated with alcohol in increasing grades, placed in xylene and embedded in paraffin. 2. Sections (3 μm) of the colon are stained with haematoxylin and eosin (H&E) and Periodic Acid-Schiff (PAS) at room temperature. Five areas are chosen at random in each section and examined under a 100 objective. 3. The following parameters are analysed in slides stained with H&E: cellular infiltrates, necrosis, oedema, activation of glandular cells and morphology of the epithelium. 4. In the case of PAS, the amounts of mucus-secreting cells are evaluated. 5. In each field, microscopic colon scoring is performed in a double-blind manner independently by two evaluators. The mucosal edema, cell damage, accumulation of inflammatory cells and goblet cell abundance are analysed. 6. To analyse the infiltration of inflammatory cells or the structure of the junctions, the sections are incubated with anti-CD4 (1:200 dilution; Santa Cruz Biotechnology, Inc., Dallas, USA) or anti-occludin (1:200 dilutions; Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA), followed by secondary antibodies conjugated with AlexaFluor 488 (dilution 1:1500) or AlexaFluor 555 (dilution 1:1500) from Invitrogen. 7. The immunostained antigens are visualized using a Leica DFC425 fluorescence microscope [Leica Microsystems (Schweiz) AG, SZ] and staining with occludin is observed with the aid of a laser scanning confocal microscope (C1, Nikon, Japan).

3

Colitis with Recurrence (Chronic) Protocol: 1. In this 3-week protocol, colitis is first induced using 10 mg of TNBS in 50% ethanol, as previously described, and after 14 days the animals have received a second dose of 10 mg of TNBS in an attempt to mimic the common relapses of intestinal inflammatory disease in humans [9]. 2. The animals are divided into six groups: two groups receive daily oral doses of the extract with greater effect, dissolved in distilled water. 3. The positive control group animals receive 100 mg/kg of sulfasalazine dissolved in the same vehicle solution, while the remaining control group (sham group) receives only the vehicle but without inducing colitis.

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4. The treatment is started 24 h after the first administration of TNBS and is continued until the day of death of the animals. 5. An additional group treated with vehicle and colitis receiving only the first dose of TNBS (control group without recurrence) is included as a reference. 6. Bodyweight, the occurrence of diarrhoea and total feed intake is recorded for each group daily. 7. The number used is 7 animals for the sham group, 7 for the control without recurrence and 21 for the other groups. 8. One-third of the animals in each group is sacrificed 1, 2 and 3 weeks after the first dose of TNBS, while the 7 animals in the control group without recurrence is killed only after 3 weeks. 3.1 Injury Assessment

1. The colic segment is obtained by laparotomy with the adhesion of adjacent organs. The colon is washed, placed on a plate on ice, clean the mesentery layer and other unwanted parts, opened by longitudinal cut. 2. The colon length is measured, weighed and the size of the lesion is determined by measuring using a ruler. 3. After this, the colon is divided longitudinally into different fragments for further analysis.

3.2 Macroscopic Score

1. For each animal, a 10-cm distal portion of the colon is removed and cut longitudinally and gently clean in physiological saline to remove faecal matter. 2. Macroscopic inflammation scores are assigned based on clinical characteristics of the colon using an arbitrary scale ranging from 0 to 4 as follows: 0 (no macroscopic changes), 1 (mucosal erythema only), 2 (mild mucosal oedema, mild bleeding or small erosions), 3 (moderate oedema, mild haemorrhagic ulcers or erosions) and 4 (severe ulceration, oedema and tissue necrosis).

3.3 FullThickness Organ Culture

1. The colon is opened longitudinally starting from the anus with surgical scissors. 2. Residual intestinal bacteria are removed by washing the colon three times with ice-cold phosphate-buffered saline (PBS) containing 20 mg/mL gentamicin. 3. With the aid of a 3 mm skin biopsy puncture instrument, approximately 4–6 circular samples of proximal and distal parts of the colon are obtained. 4. Each biopsy is transferred to separate wells of a 48-well plate containing 500 mL of supplemented RPMI 1640 culture medium.

3 Colitis with Recurrence (Chronic)

71

5. The samples are incubated for 12–24 h at 37  C in a cell culture incubator with humidified air with 5% CO2. 6. Supernatants are collected and frozen at 20  C for later cytokine measurements. 7. To check the antioxidant action of extracts/drug in a model of ulcerative colitis induced by TNBS, lipid peroxidase (LPO), superoxide dismutase (SOD) and reduced glutathione (GSH), that are link to the oxidative stress are measured (refer to Chapter 15; [1]). 3.4 Histological Analysis

3.5 Haematoxylin and Eosin Stain

The colon removed after perfusion with PBS (pH 7.4) plus 1 M sodium EDTA, and then fixed in 4% formalin solution for 24 h, dehydrated at growing concentrations of ethanol, xylol and paraffin-embedded in histological processor and sectioned into 3 μm sections using a microtome. 1. After making the slides, dewax the paraffin slides in an oven at 60  C for 2 h, followed by immersion in xylol and lowering concentrations of ethanol. 2. Afterwards, the slides are rehydrated and stained with haematoxylin and eosin. Morphological analyses of histopathological slides for the parameters like epithelial damage, oedema, cellular infiltration and necrosis using a 100 magnification optical microscope. 3. H&E staining is performed to examine the morphological alterations in the colon. The scoring system on the severity of inflammation for mucosa, submucosa, intestinal glands and muscular is carried out. (a) Mucosa: – 0: intact mucosa, without oedema and leukocyte, infiltrate. – 1: intact mucosa, presence of some erythrocytes and leukocytes in the lamina propria. – 2: the presence of epithelial alteration, but still maintaining cellular monolayer; the presence of some erythrocytes and leukocytes in the lamina propria. – 3: the presence of epithelial damage, presenting discontinuity of the epithelial monolayer; the presence of several erythrocytes and leukocytes in the lamina propria. – 4: the presence of epithelial damage, showing complete destruction of the epithelial layer; the presence of several erythrocytes and leukocytes in the lamina propria.

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Animal Experiments on Ulcerative Colitis

(b) Intestinal glands: – 0: basal mucus production. – 1: 25% increase in mucus production and typical glandular morphology. – 2: 50% increase in mucus production and increased glandular lumen. – 3: 75% increase in mucus production and increased glandular lumen. – 4: 100% increase in mucus production and increased glandular lumen. (c) Submucosa: – 0: Intact tissue, without oedema and leukocyte, infiltrate. – 1: Intact tissue, presence of some erythrocytes and leukocytes in the lamina propria. – 2: Presence of tissue alteration, with increased area and moderate destruction of collagen fibres; the presence of some erythrocytes and leukocytes in the connective tissue and presence of increased lumen of the blood vessels. – 3: Presence of tissue alteration, with increased area and destruction of collagen fibres; the presence of several erythrocytes and leukocytes in the connective tissue and presence of increased lumen of the blood vessels. – 4: Presence of tissue alteration, with increased area and intense destruction of collagen fibres; intense erythrocyte and leukocyte infiltrate in the connective tissue and presence of several blood vessels with an increased lumen. (d) Muscular: – 0: Intact tissue, without oedema and leukocyte, infiltrate. – 1: Intact tissue, presence of some erythrocytes and leukocytes. – 2: Presence of focal points of tissue necrosis, presence of some erythrocytes and leukocytes. – 3: Presence of several points of tissue necrosis, presence of some erythrocytes and leukocytes. – 4: Muscle tissue completely altered, characterized by necrotic destruction; the presence of several erythrocytes and leukocytes.

References

3.6 Periodic AcidSchiff (PAS) Stain

73

1. Following the paraffin dewaxing and rehydration process, slides are incubated for 15 min in 1% periodic acid solution, washed in running tap water for 5 min and incubated in Schiff Reagent for 1 h in a darkish and refrigerated environment. 2. After the incubation period, slides are removed and washed under running tap water for 10 min, dried at room temperature and stain the mucus-secreting cells and analysed under an optical microscope at 100 magnification.

References 1. Arunachalam K, Damazo AS, Macho A, da Silva Lima JC, Pavan E, de Freitas Figueiredo F, Arunachalam K, Damazo AS, Macho A, da Silva Lima JC, Pavan E, de Freitas Figueiredo F, Oliveira DM, Cechinel Filho V, Wagner TM, de Oliveira Martins DT (2020) Piper umbellatum L.(Piperaceae): phytochemical profiles of the hydroethanolic leaf extract and intestinal anti-inflammatory mechanisms on 2, 4, 6 trinitrobenzene sulfonic acid induced ulcerative colitis in rats. J Ethnopharmacol 2020:112707 2. Francescone R, Hou V, Grivennikov SI (2015) Cytokines, IBD, and colitis-associated cancer. Inflamm Bowel Dis 21(2):409–418 3. Amirshahrokhi K (2019) Febuxostat attenuates ulcerative colitis by the inhibition of NF-κB, proinflammatory cytokines, and oxidative stress in mice. Int Immunopharmacol 76:105884 4. Neurath MF (2017) Current and emerging therapeutic targets for IBD. Nat Rev Gastroenterol Hepatol 14(5):269 5. Morris GP, Beck PL, Herridge MS, Depew WT, Szewczuk MR, Wallace JL (1989) Hapten-

induced model of chronic inflammation and ulceration in the rat colon. J Gastroenterol 96:795–803 6. Waldner MJ, Neurath MF (2009) Chemically induced mouse models of colitis. Curr Protoc Pharmacol 46(1):5–55 7. Taghipour N, Molaei M, Mosaffa N, RostamiNejad M, Aghdaei HA, Anissian A, Azimzadeh P, Zali MR (2016) An experimental model of colitis induced by dextran sulfate sodium from acute progresses to chronicity in C57BL/6: correlation between conditions of mice and the environment. Gastroenterol Hepatol Bed Bench 9(1):45 8. Walsh AJ, Bryant RV, Travis SP (2016) Current best practice for disease activity assessment in IBD. Nat Rev Gastroenterol Hepatol 13 (10):567 9. Cheng H, Xia B, Guo Q, Zhang L, Wang F, Jiang L, Wang Z, Zhang Y, Li C (2007) Sinomenine attenuates 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis in mice. Int Immunopharmacol 7(5):604–611

Chapter 10 Experiments of Antibacterial Activities Abstract Bacterial infections are considered as serious threat to health even if having many drugs and the research for novel ones are increasing due to the complications with the emergence of drug-resistant microbes. This chapter specifies procedures to establish the Minimum Inhibitory Concentration (MIC) against a panel of bacterial strains using the broth microdilution method on Gram-positive and Gram-negative bacteria with the aid of in vitro antibacterial tests. The antibacterial tests determine the Minimum Bactericidal Concentration (MBC) of the extracts that presented MIC, analysis a combined therapy study using checkerboard and time kill methodologies and the antibacterial resistance analysed through biofilm induction assay. Investigation of systemic infection studies is through in vivo tests on rats. The mechanism of antibacterial action evaluated through the assays like antibiosis (antibiofilm), outer membrane permeability, efflux of potassium and nucleotide leakage tests. As well as investigated through DNA supercoiling gyrase, Topoisomerase IV decatenation assays, inhibition of macromolecular synthesis and damage tests of the membrane (DNA, RNA and cell wall). Key words Antibacterial activity, Topo isomerase, DNA, Outer membrane, MIC, MBC, Antibiosis (antibiofilm), Nucleotide leakage, Checkboard, Time kill method

Materials: l

Agar Agar.

l

Alamar Blue.

l

Amoxicillin.

l

Amphotericin B.

l

Ascorbic acid.

l

Blood agar.

l

Blood agar plate.

l

Brain and Heart Infusion Broth (BHI).

l

Brain Infusion Agar (BHI).

l

Capillary tubes.

l

Chloramphenicol.

l

Ciprofloxacino.

Karuppusamy Arunachalam and Sreeja Puthanpura Sasidharan (eds.), Bioassays in Experimental and Preclinical Pharmacology, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-1233-0_10, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021

75

76

Experiments of Antibacterial Activities l

Clarithromycin.

l

Cystine–lactose–electrolyte-deficient Agar.

l

Cyclophosphamide.

l

Defibrinated lamb blood.

l

Dextrose Potato Broth.

l

Dimethyl sulfoxide (DMSO).

l

ELISA kit.

l

Eppendorf tubes.

l

Ethyl Alcohol P.A. (ethanol).

l

Foetal bovine serum (SBF).

l

Glycerol.

l

Griess reagent (modified) G4410.

l

Heparin.

l

Hydrated Aluminium Chloride.

l

Iron sulphate II (ferrous sulphate).

l

MacConkey Agar.

l

Methyl alcohol P.A. (methanol).

l

Mice.

l

Mueller Hinton Agar.

l

Mueller Hinton Broth (MHB).

l

Plate counter.

l

Polymyxin B.

l

Potassium chloride.

l

Potassium ferrocyanide.

l

Potassium phosphate.

l

Quercetin.

l

RPMI-1640 with L-glutamine without bicarbonate.

l

Rutin.

l

Sabouraud Agar glycosis 4%.

l

Sabouraud broth.

l

Salmonella Shigella Agar (SS AGAR).

l

SkimMilk.

l

Skirrow supplement.

l

Sodium carbonate.

l

Sodium Chloride.

l

Sodium hydroxide.

l

Sodium nitroprusside (NPS).

1 Antibacterial Activity by In Vitro Assays: Bacterial Strains

1

l

Sterile PBS.

l

Nutrient agar.

l

Syringes with needle (1 mL).

l

Trichloroacetic acid (TCA).

l

Trimethoprim.

l

Tryptic soy broth (TSB).

l

Vancomycin.

77

Antibacterial Activity by In Vitro Assays: Bacterial Strains Aim: The efficacies of plant extract/drugs against Gram-positive and Gram-negative bacteria determined by using models of acute bacterial infection. Introduction: Among various infectious diseases, bacterial infections are considered a serious health threat that extends to economic and social complications. Even though many antibacterial drugs have developed, the complications on bacterial infections remain a big challenge due to the outbreaks of drug-resistant or multidrug-resistant microbial strains, new bacterial mutations, lack of a suitable vaccine, hospital-related infections and so on, and this becomes an unresolved problem as well as trouble to health services. Hence these complications due to microbial infections remain major reason of mortality or morbidity in patients [1–3]. Therefore, research in the antibacterial therapy is looking for special or new ways on drug/ vaccine development with synergistic effect on multidrug-resistant bacteria. Principle: Antibacterial mechanism of action commonly described with major four modes of actions which includes the involvement of inhibition/regulation of enzymes which are involved in cell membrane biosynthesis, nucleic acid metabolic process and repair and protein synthesis. The final mechanism of action involves the destruction of membrane structure [4]. 1. Microbiological tests are performed using the following strains of reference from the American Type Culture Collection (ATCC), representative of Gram-positive and Gram-negative fermenting and non-fermenting bacterial groups: – Staphylococcus aureus subsp. aureus (ATCC 29213TM). – Staphylococcus aureus subsp. aureus (ATCC 25923TM).

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Experiments of Antibacterial Activities

– Staphylococcus aureus subsp. aureus (ATCC 33591TM). – Staphylococcus aureus subsp. aureus (ATCC 33592TM). – Staphylococcus aureus subsp. aureus (ATCC 6538TM). – Staphylococcus epidermidis (ATCC 12228TM). – Escherichia coli (ATCC 25922TM). – Pseudomonas aeruginosa (ATCC 25619TM). – Acinetobacter baumannii (ATCC 19606TM). – Enterobacter aerogenes (ATCC 13048TM). – Klebsiella pneumoniae 4352TM).

subsp.

pneumoniae

(ATCC

2. Five routine strains of methicillin-resistant Staphylococcus aureus (MRSA) 1,485,279, MRSA 1605677, MRSA 1664534, MRSA 1688441 and MRSA 1830466. 3. The strains S. aureus (ATCC 29213TM) and E. coli (ATCC 25922TM) are employed in the testing validation stage, such as quality control, as recommended by documents of Clinical & Laboratory Standards Institute M07-A9 and M100-S24 [5, 6]. 4. During the text, the following terms are adopted to refer to the strains mentioned above, respectively: – MRSA 1485279. – MRSA 1605677. – MRSA 1664534. – MRSA 1688441. – MRSA 1830466. 1.1 Determination of the Minimum Inhibitory Concentration (MIC)

Protocol: 1. MIC is determined using the Mueller Hinton broth microdilution method (MHB) according to the recommendations of the Clinical and Laboratory Standards Institute [5], with minor adjustments, compared to representative reference samples of Gram-positive and Gram negative bacteria as well as routine, listed in the previous item. 2. A concentration gradient of the samples is established according to [7], whose study proposes that medicinal plants used traditionally against infections, they may show some activity when MIC FICI 2 Antagonism.

1.4 Bacterial Kinetics or Growth Curve or TimeKill Assay

Protocol: 1. The analysis of the growth curve is performed according to Sutton [9] to determine the influence of extract on the multiplication rate of S. aureus (ATCC 29213), S. aureus (ATCC 25923) and MRSA 1485279 strains. 2. 5  105 CFU/mL are exposed to extract and combinations with antibiotics in MHB broth (BP concentrations ranging from 1 to 4 MIC). 3. The MHB broth containing only bacterial inoculum is used as growth control.

1 Antibacterial Activity by In Vitro Assays: Bacterial Strains

81

4. The optical density reading at 630 nm is performed before incubation (t ¼ 0) and every 60 min until 10 h of incubation at 37  C. 5. The growth curve is constructed with the variation of optical density as a function of time. 1.5 Testing for Bacterial Cell Viability

Protocol: 1. The viability of bacterial cells in the presence of extract is determined according to a protocol by Kim et al. [10] with modifications. 2. One millilitre of bacterial cells are subcultured on nutrient agar, resuspended in sterile 0.9% saline and then standardized at an optical density of 0.7 at 600 nm. 3. Next, the cells are added to 19 mL of phosphate buffer pH ¼ 7.1 (50 mM) sterile with 1, 2 or 4 MIC of extract. 4. The controls are cells without extract in the presence of DMSO equivalent used in the solubilization and chloramphenicol and/or ampicillin in the concentration equivalent to MIC, as well as the association of these with extract in the combination that inhibited bacterial growth by the ‘checkerboard’ test. 5. At times equivalent to 1, 4 and 6 h of incubation at 37  C, an aliquot in the order of 105 dilution is inoculated in Petri dishes containing TSA agar (Tryptic Soy Agar) and incubated at 37  C for 20 h. 6. The result is expressed graphically as CFU/mL  105. 7. The ratio between the number of bacteria and the number of hours of incubation represented the generation time bacterial in the presence of extract, antibiotics and association.

1.6 In Vitro Antibiofilm Activity

Protocol: 1. The quantification of biofilm production is performed as described by Costa et al. [11]. 2. In a flat-bottom polystyrene microplate with 96 wells, 200 μL of test solution are added in concentrations equivalent to MIC, 2 MIC and 4 extract MIC as well as combinations of this with the ampicillin and chloramphenicol antibiotics with the bacterial inoculum at 108 CFU/mL being added at 2% (v/v). 3. Then, the microplates are incubated at 37  C for 24 h. 4. After this period, the contents of the plates are discarded, and each well is carefully wash with pH 7.1 phosphate buffer for 3 times to remove non-adhere cells.

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Experiments of Antibacterial Activities

5. The biofilms are fixed with 98% ethanol and stained with a violet crystal solution. 6. All tests are performed in hexaplicate, growth control is prepared using inoculum and sterile medium, method control is a diluent system used for sample and antibiotic (DMSO 1%— for extract and EtOH—for chloramphenicol) and negative control is used only the sterile medium. 7. The results are expressed as a percentage of inhibition of adherence of the microorganism, calculated according to the formula below, where OD is the optical density, measured in the presence of extract and/or antibiotics and ODcc is the measure of growth control. % adhesion inhibition ¼ 1  (ODassay/ ODcc)  100. 1.7 Potassium Efflux and Microscopic Observations [1]

Protocol: 1. Weigh 1 mg of the extract or isolated drug in an Eppendorf. 2. Solubilize it in 1 mL of sodium phosphate buffer (pH 7.2). 3. Cultivate Shigella flexneri, Enterococcus faecalis and S. aureus (or bacteria which showed a MIC up to 400 μg/mL) in BHI broth and extract for overnight at 35  C microbiological oven. 4. Wash and resuspend the cells until a cell concentration of 1  107 cells/mL is obtained in sodium phosphate buffer (pH 7.2). 5. Add 1 mL of the bacterial suspension at 2 MIC of the extract or isolate. 6. Incubate for different times (T0, T1 h, T2 h, T3 h and T4 h). After centrifugation, measure the amount of K+ released in the supernatant. 7. Negative control: bacteria + sodium phosphate buffer. 8. Make slides for microscopy of the bacterial sediment using Gram stain. 9. Proceed with the readings under an optical microscope. Calculations: 1. Extract 1: Stock solution ¼ 1 mg/mL. MIC ¼ 12.5  2 ¼ 25 μg. C1V1 ¼ C2V2. 1000  V1 ¼ 25  1. V1 ¼ 0.025 mL ¼ 25 μL for S. flexneri and E. faecalis 2. Extract 2: Stock solution ¼ 1 mg/mL. MIC ¼ 6.25  2 ¼ 12.5 μg.

1 Antibacterial Activity by In Vitro Assays: Bacterial Strains

83

C1V1 ¼ C2V2. 1000  V1 ¼ 12.5  1. V1 ¼ 0.0125 mL ¼ 13 μL for S. flexneri and E. faecalis 3. Extract 3: Stock solution ¼ 1 mg/mL (for this extract it is necessary to weigh two Eppendorfs). For S. flexneri: MIC ¼ 400  2 ¼ 800 μg. C1V1 ¼ C2V2. 1000  V1 ¼ 800  1. V1 ¼ 0.8 mL ¼ 800 μL. For S. flexneri: MIC ¼ 200  2 ¼ 400 μg. C1V1 ¼ C2V2. 1000  V1 ¼ 400  1. V1 ¼ 0.4 mL ¼ 400 μL. 4. Extract 4: Stock solution ¼ 1 mg/mL. For S. flexneri: MIC ¼ 200  2 ¼ 400 μg. C1V1 ¼ C2V2. 1000  V1 ¼ 400  1. V1 ¼ 0.4 mL ¼ 400 μL. For S. aureus: MIC ¼ 12.5  2 ¼ 25 μg. C1V1 ¼ C2V2. 1000  V1 ¼ 25  1. V1 ¼ 0.025 mL ¼ 25 μL. 5. Extract 5: Stock solution ¼ 1 mg/mL. MIC ¼ 100  2 ¼ 200 μg. C1V1 ¼ C2V2. 1000  V1 ¼ 200  1. V1 ¼ 0.2 mL ¼ 200 μL for S. aureus. 1.8 Nucleotide Leakage Assay [1]

Protocol: 1. Weigh 1 mg of the extract or isolated drug in an Eppendorf. 2. Solubilize the extract or drug in 1 mL of PBS (pH 7.4). 3. Growing in BHI broth, extract overnight S. flexneri, E. faecalis and S. aureus (or bacteria which showed a MIC μg to 400) at 35  C microbiological oven.

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4. Wash and resuspend the cells until a cell concentration of 1  107 cells/mL is obtained in PBS buffer (pH 7.4). 5. Add in 2 mL of the bacterial suspension and the MIC of the extract or isolated drug. 6. Incubate for different times (T0, T100 min (1 h and 40 min), T200 min (3 h and 20 min), T300 min (5 h) and T400 min (6 h and 40 min)). After centrifugation, determine the absorbance at 260 nm. 7. Negative control: bacteria + PBS buffer. 1.9 Outer Membrane Permeability Assay [1]

Protocol: 1. Weigh 1 mg of the extract or isolated drug in an Eppendorf. 2. Solubilize the extract or isolated compound in 1 mL of distilled water. 3. Cultivate overnight strains of S. flexneri or E. coli (or Gramnegative bacteria that showed a MIC up to 400 μg/mL) in BHI broth at 35  C in a microbiological oven. 4. Proceed with a serial dilution of the extract or isolate (400–3.25 10 μg/mL), ½ MIC of the test compound (FIXED) + 1 μg/mL erythromycin, ½ MIC of the test compound (FIXED) + 10 μg/mL rifampicin, in Muller-Hinton broth, in 96-well microplates. Obs. Each well should contain ½ of the MIC of the test compound. 5. The plate must be assembled according to the instructions below: (a) Plate 1: Lines A and B: Serial dilution of test compound only. Lines C and D: Serial dilution of erythromycin only. Lines E and F: Serial dilution of erythromycin + ½ MIC of the test compound (FIXED). Line G: Growth control. Line H: Sterility control (b) Plate 2: Lines A and B: Serial dilution of test compound only. Lines C and D: Serial dilution of rifampicin only. Lines E and F: Serial dilution of rifampicin + ½ MIC of the test compound (FIXED). Line G: Growth control. Line H: Sterility control. 6. Incubate in a biochemical oxygen demand (BOD) oven for 10 h at 37  C. 7. After 10 h of incubation, monitor the decrease in absorbance using an ELISA reader at 630 nm.

1 Antibacterial Activity by In Vitro Assays: Bacterial Strains

1.10 DNA Supercoiling Gyrase Assays and Topoisomerase IV (Topo IV) Decatenation Assays [12]

85

Protocol: 1. 1 U of an enzyme (gyrase or Topo IV, Inspiralis) converts 0.5 mg of relaxed pBR322 DNA (Deoxyribonucleic acid) to the supercoiled form (gyrase) of 24 or 200 ng kinetoplast DNA decatenates (Topo IV). 2. The enzymatic activity is detected by incubation for 45 min at 37  C, in a total reaction volume of 30 mL. 3. Standard reaction mixtures for gyrase supercoiling assays contained 35 mM Tris–HCl (pH 7.5), 24 mM KCl, 700 mM K-Glu, 4 mM MgCl2, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5% (w/v) glycerol and 0.1 mg/mL albumin. 4. The Topo IV activity is measured using a decatenation assay that monitors the ATP-dependent decoupling of DNA from minicircles containing 40 mM HEPES-KOH (pH 7.5) kDNA, 100 mM K-Glu, 10 mM magnesium ethyl, 10 mM DTT, 1 mM ATP and 50 mg/mL albumin. 5. Typically, the supercoiling assays contained 1 L of gyrase (corresponding to 20 ng of E. coli or S. aureus of the enzyme, respectively) and the assays contained 1 L decatenation of Topo IV (corresponding to 18.7 or 200 μg E. coli or S. aureus, respectively). 6. The reactions are stopped by adding an equal volume of stop buffer [40% sucrose, Tris–HCl 100 (pH 7.5), EDTA and 100 mM Bromophenol Blue], follow extraction with 1 volume of chloroform/isoamyl alcohol (24: 1). 7. Then, 20 mL of the aqueous phase of each sample is analysed in 1% agarose gels for 4 h at 80 V in Tris/EDTA acetate buffer and visualized after staining with ethidium bromide. 8. Before gel electrophoresis, the test samples supercoiling are submitted to a buffer exchange with 10 mM Tris–HCl (pH 8.0), performed by dialysis with MFTM-membrane filters (Millipore, 0.025 mm). 9. The aqueous phase of the test mixes is pipette to the membranes flotation in a Petri dish. 10. After 3 h, the test mixtures are removed from the membranes and mixed with 15 mL of loading buffer (50% water, 49.75% glycerol and 0.25% bromophenol Blue). The IC50 is defined as the concentration that causes 50% inhibition of super-spiration or the decatenation reaction. 11. The IC50 for inhibition of supercoiling and decatenation, respectively, can be assessed visually as the concentration of compound that leads to a 50% reduction in the supercoiling band or minicircle, respectively.

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12. IC50 values are the averages of at least three separate experiments. To test the effect of K-Glu on gyrase and Topo IV activity, buffers are mixed without this component. 1.11 Macromolecular Synthesis and Membrane Damage Assays (DNA, RNA and Protein)

Protocol: 1. Macromolecular synthesis (MMS) assays are performed on mutant E. coli cultures and on wild type cultures of S. aureus, as previously described [12]. 2. Briefly, E. coli-TolC5 CGSC5633 is obtained from a reputed institute. 3. This strain is an efflux mutant and is used to assess the overall mode of action of successful compounds. 4. The assays are performed using the radiolabelled precursors [methyl-3H] thymidine, [5-3H] uridine and L-[4,5-3H] leucine or L-[2,6-3H] phenylalanine to determine the effect of the representative compound REP323219 on DNA, RNA and protein synthesis, respectively. 5. For protein synthesis, the 10 min labelling reaction with L-[4.5-3H] leucine is followed by 5 min of chasing with 10 mM cold leucine to decrease the background due to bound leucine tRNA. 6. The effect of a selected compound (REP323370) on the cell wall and lipid synthesis is evaluated using radiolabelled precursors [3H] N-acetyl-D glucosamine and glycerol [1,3-3H], respectively.

2

Animal Experiment (In Vivo) on Antibacterial Activity Aim: The in vivo efficacies of plant extract against Staphylococcus aureus determined by using a mouse model of acute bacterial infection. Introduction: Among various infectious diseases, bacterial infections are considered a serious health threat that extends to economic and social complications. Even though many antibacterial drugs have developed, the complications on bacterial infections remain a big challenge due to the outbreaks of drug-resistant or multidrug-resistant microbial strains, new bacterial mutations, lack of a suitable vaccine or hospital-related infections and so on and this becomes an unresolved problem as well as trouble to health services. Hence these complications due to microbial infections remain major reason of mortality or morbidity in patients [1–3]. Therefore research in the antibacterial therapy is looking for special or new ways on drug/vaccine development with synergistic effect on multidrug-resistant bacteria.

2 Animal Experiment (In Vivo) on Antibacterial Activity

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Principle: The anti-bacterial mechanism involves the restrain of cell wall synthesis or depleting energy of cell through accumulating in the membranes of bacteria or changing the permeability of cell membrane resulting in the loss of cellular constituents or at some occasions causes the disruption to the cell membrane and alteration in the structure and function of cellular components, which are all result in mutation and thereby cell damage and death [1]. Among all, the anti-bacterial property is also attained through generating reactive oxygen species which oxidize the bacterial membrane and create lipid peroxide that increases the damage of bacterial cell wall via stimulating the oxidative protein and apoptotic genes [13]. 2.1 Systemic Bacterial Infection in Mice [1]

Protocol: 1. For the systemic infection experiments, the extract is used against two bacterial clinical isolates of S. aureus and E. coli. 2. Swiss albino male and female mice, weighing between 25 and 35 g need to allocate into ten groups of ten animals each. 3. The negative control group received distilled water (vehicle) orally and the positive control group received meropenem (20 mg/kg) subcutaneously as treatment. 4. In the test groups, different doses (0.01, 0.1, 1, 10, 50, 100, 200, 300 and 500 mg/kg) of the tested extracts is given orally. 5. The bacterial strains plate on nutrient agar, 24 h before the experiment. 6. The bacterial inoculum of S. aureus is adjusting to MacFarland 6 scale (21  108 CFU/mL), for E. coli the scale MacFarland 3 scale (9  108 CFU/mL) is used. 7. These bacterial concentrations are capable of inducing systemic infection and causing death in 100% of the animals in less than 14 days. 8. The bacterial infection is induced by the intraperitoneal administration (0.2 mL) of the bacterial suspension in BHI broth to the animals. 9. Treatment of the animals is done immediately and 4 h after inoculation of the animals, and they are observed for 14 days to record mortality.

2.2 Determination of In Vivo Antistaphylococcal Activity [14]

Protocol: 1. Test organisms are cultured overnight in BHI at 37  C. 2. The bacterial inoculum of Staphylococcus aureus is adjusted to scale 6 (21  108 CFU/mL) MacFarland, and these bacterial concentrations are capable of inducing systemic infection in the animals.

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3. The bacterial cells grown to the late exponential phase in BHI broth need to harvest and wash with sterile PBS (Phosphate Buffered Saline). 4. Swiss albino female mice allocated into five groups of six animals each. 5. The negative control group received distilled water (vehicle) orally and the positive control group received amoxicillin (20 mg/kg) subcutaneously as treatment. 6. In the test groups, different doses (50, 100 and 250 mg/kg) of the extracts is given orally. 7. The bacterial infection is induced by the intraperitoneal administration (0.2 mL) of the bacterial suspension in BHI broth. 8. Treatment of the animals needs to do after 4 h inoculation of the animals. 9. The animals observed for 14 days and the animals are checked for mortality percentage. 10. The number of surviving mice for each dosage is recorded for 14 days after the infection, and the mean effective dose sufficient to protect 50% of the mice (ED50) is determined from the final survival rates using GraphPad PRISM software. 2.3 Immunosuppressed Mice Infection Model [15]

Protocol: Treatment of mice with cyclophosphamide (CP), Staphylococcus aureus 1. Mice are injected with CP (200 or 350 mg/kg b.w.) intraperitoneally (i.p.) as indicated in the below. Group I: Sham. Group II: Vehicle (S. aureus 5  106 (0.2 mL)). Group III: Amoxicillin (25 mg/kg b.w. + S. aureus 5  106 (0.2 mL)). Groups IV–VI: Extract (5, 25, 100 mg/kg). 2. Bacteria are administered intravenously (i.v.), into lateral tail vein, four days after CP, at a dose of 5  106/mouse. 3. Bacterial cell numbers are determine colorimetrically at a wavelength of 600 nm according to previously prepared standards. Determination of S. aureus in the organs 4. Twenty four hours after infection, the mice sacrifice, the organs (spleens, livers and kidneys) isolated and homogenized using a plastic syringe piston and a plastic screen, in sterile PBS (1 g of wet tissue per 25 mL of PBS).

References

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5. Five- and fiftyfold dilutions of cell suspension applied onto blood agar plates and incubated overnight and the colonyforming units (CFU) are enumerate. 6. The number of colonies express as the number of CFU per milligram of the organ. Analysis of cell types in the circulating blood and bone marrow 7. Samples of blood take on day 0, just before administration of CP, 4 days after administration of CP, just before administration of bacteria (day 4) and at 24 h following infection (day 5). 8. The bone marrow isolated on days 0 and 5. 9. Blood and bone marrow smears are prepare and stain with May-Gru¨nwald and Giemsa reagents. 10. The preparations are review microscopically by a histologist at 1000 magnification. Determination of plasma TNF-α, IFN-γ and IL-6 levels 11. Cytokine assay in plasma after infection, experimental animals (five mice per group) are anaesthetize with sodium pentobarbital and bled from the retro-orbital plexus with heparin Pasteur pipettes. 12. The tubes are centrifuged and the plasma is separated and stored at 20  C until assayed. Concentrations of TNF-α, IFN-γ and IL-6 determined, using mouse cytokine ELISA kits. 13. Assays perform according to the manufacturer’s instructions, and the results express in pg/mL.

References 1. Arunachalam K, Asceˆncio SD, Soares IM, Aguiar RWS, da Silva LI, de Oliveira RG, Balogun SO, de Oliveira Martins DT (2016) Gallesia integrifolia (Spreng.) Harms: in vitro and in vivo antibacterial activities and mode of action. J Ethnopharmacol 184:128–137 2. Sirelkhatim A, Mahmud S, Seeni A, Kaus NHM, Ann LC, Bakhori SKM, Hasan H, Mohamad D (2015) Review on zinc oxide nanoparticles: antibacterial activity and toxicity mechanism. Nano-Micro Lett 7(3):219–242 3. Zorofchian Moghadamtousi S, Abdul Kadir H, Hassandarvish P, Tajik H, Abubakar S, Zandi K (2014) A review on antibacterial, antiviral, and antifungal activity of curcumin. Biomed Res Int 2014:186864 4. Kapoor G, Saigal S, Elongavan A (2017) Action and resistance mechanisms of

antibiotics: A guide for clinicians. Journal of anaesthesiology, clinical pharmacology, 33(3):300 5. CLSI C (2012) M100-S25: performance standards for antimicrobial susceptibility testing. Twenty-fifth informational supplement. CLSI, Annapolis Junction, MD 6. CLSI C (2014) Performance standards for antimicrobial susceptibility testing; twentyfourth informational supplement. M100-S24 January. CLSI, Annapolis Junction, MD 7. Fabry, W, Okemo, PO, Ansorg, R (1998) Antibacterial activity of East African medicinal plants. Journal of ethnopharmacology 60 (1):79–84. 8. Fadli M, Saad A, Sayadi S, Chevalier J, Mezrioui NE, Page`s JM, Hassani L (2012) Antibacterial activity of Thymus maroccanus and Thymus broussonetii essential oils against

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nosocomial infection-bacteria and their synergistic potential with antibiotics. Phytomedicine 19:464–471 9. Sutton S (2006) The Gram stain. Pharm Microbiol Forum Newlett 12(2):4 10. Kim DH, Kim MI, Park HG (2015) Recent advances in genetic technique of microbial report cells and their applications in cell arrays. Biomed Res Int 2015:182107 11. Costa GA, Rossatto FC, Medeiros AW, Correa APF, Brandelli A, Frazzon APG, MOTTA ADS (2018) Evaluation antibacterial and antibiofilm activity of the antimicrobial peptide P34 against Staphylococcus aureus and Enterococcus faecalis. An Acad Bras Cienc 90(1):73–84 12. Ribble W, Hill WE, Ochsner UA, Jarvis TC, Guiles JW, Janjic N, Bullard JM (2010) Discovery and analysis of 4H-pyridopyrimidines, a class of selective bacterial protein synthesis

inhibitors. Antimicrob Agents Chemother 54 (11):4648–4657 13. Sarker SR, Hossain MD, Polash SA, Takikawa M, Shubhra RD, Saha T, Islam Z, Hossain M, Hasan M, Takeoka S, Sarkerf SR (2019) Investigation of the antibacterial activity and in vivo cytotoxicity of biogenic silver nanoparticles as potent therapeutics. Front Bioeng Biotechnol 7:239 14. Ahsan M, Gonsales AV, Sartini S, Wahyudin E, Nainu F (2019) In vivo anti-staphylococcal activity of roselle (Hibiscus sabdariffa L.) calyx extract in Drosophila model of infection. J Herbmed Pharmacol 8(1):41–46 15. Girard D, Regan PA, Milisen WB, Retsema JA, Swindell AC (1996) Influence of immunosuppression on the pharmacokinetics and pharmacodynamics of azithromycin in infected mouse tissues. J Antimicrob Chemother 37:21–35

Chapter 11 Experiments of Antifungal Activities Abstract This chapter describes the protocols for evaluating the antifungal activity of extract/drugs against a broad spectrum of Candida species. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) are determined by the broth microdilution techniques while the synergistic antimicrobial activity of the tested complexes combined with two antifungal drugs (ketoconazole and amphotericin B) made by checkerboard and biofilm assays. The effect of extract/drugs on yeast cell morphology is studied by optical and transmission electron microscopy. Moreover, procedures of possible action on cell walls (sorbitol), cell membranes (ergosterol binding), the time-kill curve and biological activity on the yeast’s morphology are discussed in this chapter. The protocols thus help to investigate a great spectrum of antifungal pointing action of extract/drugs and its possible mode that confirming its high cytotoxic activity against fungal strains. Key words Candida, Ergosterol, Sorbitol, MFC, Biofilm, Time-kill curve

Aim: To investigate the antifungal activity of chemical substance/plant extract by in vitro and in vivo models. Introduction: Fungal infections are considered as ever-increasing global health problem because of their involvement in various human diseases and treatments. However, there are antifungal therapies with small molecules, monoclonal antibodies and radioimmunotherapy, and over last years the treatments are mostly concentrated on small molecules and monoclonal antibodies that inhibit the target molecules of fungal structure [1]. Hence over their widespread use, the percentage of drug-resistant fungal strains has increased by means of several mechanisms of resistance like overexpression of efflux pump proteins and biofilm formation, alteration in drug target and sterol biosynthesis and the reduction in the intercellular concentration of target enzyme [2]. This signifies the need for novel therapeutic approaches in fungal infection treatment takes into account the pathways of resistance.

Karuppusamy Arunachalam and Sreeja Puthanpura Sasidharan (eds.), Bioassays in Experimental and Preclinical Pharmacology, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-1233-0_11, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021

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Principle: The fungicide interferes with the fungal cell membrane results altering the structure, inhibit the synthesis of the cell membrane and increase the membrane permeability causing the loss of cell constituents and fungal death. As well as it causes inhibition to germination of spore, respiration at the cellular level and proliferation, thus reducing the fungal growth [3, 4].

1

Agar Disc Diffusion Method [5] Materials: (a) Fungal inoculum. l

Personal Protective Equipment Kits (PPEs).

l

Middle (Sabouraud).

l

Sterile handle.

l

Sterile Kahn tubes (distilled water or sterile saline).

l

Sterile syringe.

l

Sterile gauze.

l

Scissors.

l

Glove.

l

Alcohol 70%.

l

Fungi from the fridge.

(b) Culture medium. l

Materials for 4–150 mm Petri dishes.

l

Sabouraud dextrose broth + agar agar.

l

Ruler.

l

Autoclave.

l

Distilled water.

l

500 mL flask

l

Laminated paper.

l

Kraft paper.

l

Autoclave tape.

l

Cotton.

l

Vortex.

l

Micropipettes.

l

DMSO 2%.

l

Amphotericin B.

l

Medium.

1 Agar Disc Diffusion Method l

Alcohol.

l

Cecon sterile paper discs.

l

Fine tip histological anatomical forceps.

l

Bunsen burner.

l

Matchbox.

l

96-well plate

93

(c) Fungal cultures. l

Candida albicans, ATCC 60193.

l

Candida parapsilosis, ATCC 22019.

l

Candida tropicalis, ATCC 750.

l

Candida glabrata, ATCC 2001.

l

Candida cruzei, ATCC 6258.

l

Aspergillus fumigatus.

l

Sporothrix brasiliensis.

l

Fonsecaeae pedrosoi.

l

Cryptococcus neoformans.

l

Aspergillus niger.

Protocol: 1. The agar diffusion assay described by the Clinical and Laboratory Standards Institute (CLSI [6]), formerly National Committee for Clinical and Laboratory Standards (NCCLS) (CLSI M44-A [7]), is adopted. 2. Petri dishes are prepared with Sabouraud agar and inoculated on its surface, 4 mL of inoculum in the concentration of 0.5 McFarland. 3. Remove the excess with a sterile Pasteur pipette and dry it. 4. The testing extracts are dissolved in Dimethyl sulfoxide (DMSO) 2%, 50 mg/mL concentration is used to prepare the stock solution. 5. 5 μL of stock solution of the extract on a 0.5 cm paper disc is added. 6. For standard solution, the amphotericin B, at a concentration of 1.6 mg/mL of Dimethyl sulfoxide (DMSO) is used. 7. Tests are carried out in duplicate. 8. The disc-diffusion test is performed in Mueller-Hinton Agar medium supplemented with 2% glucose and 0.5 mg/mL methylene blue as well as in Mueller-Hinton medium without addition of glucose and methylene blue. 9. All strains tested are recently grown on potato dextrose agar.

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10. The plates are incubated in greenhouses at 35
C for 24 h. 11. On the test day, the inoculum is prepared using colonies of approximately 1.0 mm in diameter for each strain, which is suspended in 4.0 mL of sterile saline. 12. The turbidity of the suspension is adjusted to 0.5 on the McFarland scale. 13. The strains are inoculated into the plates containing medium and then the antifungal discs are added. 14. The plates are read visually after an incubation period of 24/72 h at 35
C and the zone of inhibition formed around the antifungal discs are measured and express in millimetres. 15. After reading and measuring the zone, in millimetres, the results are compared with the following criteria. For amphotericin B, zone >10 mm (Sensitive) and 10 mm (Sensitive) and 20 mm (Sensitive), halo 10–20 mm (Intermediate), zone 19 mm (Sensitive), zone 14–19 mm (Intermediate) and