Luận văn phytochemical investigation antioxidant activities and hepatoprotective efficacy of indigofera tirunelvelica sanjappa against CCL4 induced wistar albino rats

PHYTOCHEMICAL INVESTIGATION, ANTIOXIDANT ACTIVITIES AND HEPATOPROTECTIVE EFFICACY OF INDIGOFERA TIRUNELVELICA SANJAPPA AGAINST CCL4 INDUCED WISTAR ALBINO RATS A THESIS SUBMITTED BY S SUBBURAYALU (Reg No 8176) BIOCHEMISTRY in partial fulfillment of the requirements for the award of degree of DOCTOR OF PHILOSOPHY MANONMANIAM SUNDARANAR UNIVERSITY TIRUNELVELI - 627012 TAMILNADU, INDIA DECEMBER – 2018 MANONMANIAM SUNDARANAR UNIVERSITY TIRUNELVELI - 627 012 CERTIFICATE The research work embodied in the present thesis entitled “PHYTOCHEMICAL INVESTIGATION, ANTIOXIDANT ACTIVITIES AND HEPATOPROTECTIVE EFFICACY OF INDIGOFERA TIRUNELVELICA SANJAPPA AGAINST CCL4 INDUCED WISTAR ALBINO RATS.” has been carried out in the BIOCHEMISTRY, Manonmaniam Sundaranar University, Tirunelveli The work reported herein is original and does not form part of any other thesis or dissertation on the basis of which a degree or award was conferred on an earlier occasion or to any other scholar I understand the University’s policy on plagiarism and declare that the thesis and publications are my own work, except where specifically acknowledged and has not been copied from other sources or been previously submitted for award or assessment S SUBBURAYALU (Reg.No 8176) RESEARCH SCHOLAR Dr.A.PALAVESAM Dr K.R T ASHA CO-SUPERVISOR SUPERVISOR Prof and Head Assistant professor Department of Animal Science Department of Biochemistry Manonmaniam Sundaranar University Government Arts College Tirunelveli Paramakudi ACKNOWLEDGEMENT I am most thankful to God Almighty for sustaining and keeping me in His grace and providential protection throughout my life I feel highly Privilege to acknowledge my sincere thanks to my research supervisor Dr.K.R.T.Asha, Assistant Professor, Department of Biochemistry, K.R College of Arts & Science, Government Arts College, Paramakudi, Ramnad Dist., for her encouraging and continuous support as a guide throughout the entire period of research investigation Her dynamic, vibrant personality and Immunse knowledge has been a source of insipiration to me for achieving more miles in life I deeply acknowledge the constant support, encouragement and invaluable guidance at every step of my project by Dr.A.Palavesam, Prof and Head, Department of Animal Science, Manonmaniam Sundaranar University, Tirunelveli for his assistance throughout this work were unparallel without which this work would not have been possible I would like to acknowledge my approbation and humility to Kalvi Thanthai Thiru K Ramasamy, Chairman,Thiru K.R Arunachalam, Managing Director , Dr.KNKSK.Chockalingam, Director, National Engineering College,Kovilpatti and Dr.An.Kannappan, Principal of K.R College of Arts & Science, Kovilpatti inspired me to start this research I express my heartful thanks to Dr.S.Chockalingam and Dr.R.Gopal, Formerly Principals of K.R College of Arts & Science, Kovilpatti for their advice and suggestions to carry out my work successfully I express my sincere thanks to the management of KRCAS for all their support in proving the lab facilities and library facilities to me throughout my studies I also thankful to the Staff Members, and Non Teaching Staff members of K.R College of Arts & Science, Kovilpatti inspired me to start this research My sincere thanks to my family members and all my friends who all behind the successful completion of this thesis (S.SUBBURAYALU) TABLE OF CONTENT INTRODUCTION 1.1 HERBAL MEDICINE 1.2 SECONDARY METABOLITES 1.3 ANTIOXIDANTS 1.4 LIVER DISEASE 1.4.1 Epidemiology 1.4.2 Hepatocytes 1.4.3 Hepatitis 1.4.4 Alcohol related liver diseases 1.4.5 Jaundice 1.4.6 Liver tumors 1.4.7 Reye’s syndrome 10 1.4.8 Hepatotoxins 10 1.5 STAGES IN THE LIVER DISEASES 10 1.5.1 Inflammation 10 1.5.2 Fibrosis 11 1.5.3 Cirrhosis 11 1.5.4 Liver failure 11 1.6 DRUG INDUCED LIVER DISEASES 12 1.6.1 Drugs that causes Liver Injury 12 1.7 LIVER FUNCTION TESTS 15 1.8 HERBS FOR LIVER DISEASES 16 1.9 EXTRACTION PROCEDURES:- 21 1.10 CHROMATOGRAPHIC TECHNIQUES 22 AIM OF THE WORK 24 REVIEW OF LITERATURE 25 3.1 LIVER DISEASES 25 3.2 HERBAL MEDICINE 32 3.3 QUALITY OF EXTRACTS 36 3.4 SECONDARY METABOLITES 39 3.5 ANTIOXIDANTS 45 3.6 GC-MS STUDIES 52 i 3.7 HEPATOPROTECTIVE PLANTS 57 3.7.1 Fabaceae Family plants for Hepatic diseases 75 MATERIALS AND METHODS 83 4.1 PLANT MATERIAL 83 4.1.1 Taxonomy 83 4.1.2 Habitat 83 4.1.3 Description 83 4.2 IDENTIFICATION AND AUTHENTICATION OF I TIRUNELVELICA 85 4.3 PHARMACOGNOSTIC METHODS 85 4.3.1 Physiological and Organoleptic Characteristic evaluation of I tirunelvelica 85 4.3.2 Fluorescence Analysis of I tirunelvelica 85 4.4 PHYSICOCHEMICAL PROPERTIES OF I TIRUNELVELICA 85 4.4.1 Determination of Foreign Matter 85 4.4.2 Determination of Moisture Content (Loss on Drying) 85 4.4.3 Determination of Total Ash 86 4.4.4 Determination of Acid Insoluble Ash 86 4.4.5 Determination of Water Soluble Ash 86 4.4.6 Determination of extractive values 86 4.4.7 Determination of Alcohol Soluble Extractive Value 87 4.4.8 Determination of Water Soluble Extractive Value 87 4.5 PHYTOCHEMICAL SCREENING 87 4.5.1 Extraction of plant materials- Cold extraction method 87 4.5.2 Behaviour of drug powder with various chemical reagents 87 4.6 QUANTITATIVE ANALYSIS OF PHYTOCHEMIAL CONSTITUENTS 89 4.6.1 Estimation of Total Alkaloids 89 4.6.2 Estimation of Total Flavonoids 89 4.6.3 Estimation of Phenol 90 4.7 IN-VITRO ANTIOXIDANT ASSAY 90 4.7.1 DPPH Radical Scavenging Assay 90 4.7.2 ABTS Radical Scavenging Assay 91 4.7.3 Ferric Reducing Antioxidant Power (FRAP) Assay 92 4.7.4Superoxide Radical Scavenging Activity 93 4.7.5 H2O2 Radical Scavenging Activity 94 ii 4.7.6 Hydroxyl Radical Scavenging Activity 94 4.8 STUDIES ON HEPATOPROTECTIVE SCREENING 95 4.8.1 In-vitro Hepatoprotective Studies 95 4.8.2 MTT ASSAY 95 4.9 IN-VIVO STUDIES 97 4.9.1 Experimental design 97 4.9.2 Experimental animals 97 4.9.3 Experimental Design for Toxicity Studies 98 4.9.4 Carbon Tetrachloride (CCl4) induced Hepatotoxicity 98 4.9.5 Experimental Design for Hepatoprotective Studies 99 4.9.6 Body Weight of the Animals 100 4.9.7 Determination of Red Blood Cells Count 100 4.9.8 Determination of White Blood Cells Count 100 4.9.9 Determination of Haemoglobin 100 4.9.10 Enumeration of Platelets Count 101 4.9.11 Determination of ESR 104 4.9.12 Determination of PCV 105 4.9.13 Estimation of Blood Glucose 105 4.9.14 Estimation of Protein 106 4.9.15 Estimation of Blood Urea 106 4.9.16 Estimation of Serum Uric Acid 106 4.9.17 Estimation of Creatinine 107 4.9.18 Estimation of Liver Glycogen 107 4.9.19 Estimation of Serum Bilirubin 108 4.9.20 Extraction of Lipids 108 4.9.21 Estimation of Serum Cholesterol 108 4.9.22 Estimation of Serum Triglycerides 109 4.9.23 Estimation of Phospholipids 109 4.9.24 Estimation of Free Fatty Acid 109 4.9.25 Assay of Serum HDL Cholesterol 110 4.9.26 Assay of Aspartate Transaminase (AST) 111 4.9.27 Estimation of Alanine Transaminase (ALT) 111 4.9.28 Estimation of Serum Alkaline Phosphatase 111 iii 4.9.29 Assay of Lactate Dehydrogenase (LDH) 112 4.9.30 Assay of Gamma- Glutamyl Transferase 112 4.9.31 Determination of Glucose- 6- Phosphatase 112 4.9.32 Assay of Glucose-6- Phosphatase Dehydrogenase 113 4.9.33 Assay of Hexokinase 113 4.9.34 Estimation of Lipid Peroxides 113 4.9.35 Assay of Reduced Glutathione 114 4.9.36 Assay of Superoxide Dismutase 114 4.9.37 Assay of Catalase 114 4.9.38 Assay of Glutathione Peroxidase (GPx) 115 4.9.39 Assay for Glutathione S Transferase (GST) 115 4.9.40 Determination of Ascorbic Acid (Vitamin -C) 115 4.9.41 Determination of D-Tocopherol (Vitamin E) 116 4.9.42 Activity of Na+/K+ Adenosine Triphosphatase 116 4.9.43 Activity of Magnesium Adenosine Triphosphatase 117 4.9.44 Activity of Calcium Adenosine Triphosphatase 117 4.9.45 Histopathological Studies 117 4.10 ISOLATION AND PREDICTION OF MARKER COMPOUND 117 4.11 BIOINFORMATIC STUDIES 118 4.11.1 Docking 118 4.11.2 Ligand preparation 118 4.11.3 Active site prediction 119 4.11.4 Docking protocol 119 4.12 STATISTICAL ANALYSIS 119 RESULTS AND DISCUSSION 121 5.1 PHYSIOLOGICAL AND ORGANOLEPTIC CHARACTERISTICS 121 5.2 PHYSICOCHEMICAL ANALYSIS 121 5.3 FLUORESCENCE ANALYSIS 123 5.4 PHYSICOCHEMICAL PARAMETERS 124 5.5 PRELIMINARY PHYTOCHEMICAL SCREENING 126 5.6 QUANTITATIVE ANALYSIS OF SECONDARY METABOLITES 128 5.7 ANALYSIS OF IN-VITRO ANTIOXIDANT ACTIVITY 131 5.7.1 DPPH free radical scavenging Activity 131 iv 5.7.2 ABTS free radical scavenging activity 134 5.7.3 FRAP Free Radical Scavenging Activity 138 5.7.4 SO Free Radical Scavenging Activity 141 5.7.5 H2O2 Free Radical Scavenging Activity 144 5.7.6 Hydroxyl Free Radical Scavenging Activity 147 5.8 IN-VITRO HEPATOPROTECTIVE SCREENING 150 5.8.1 MTT Assay 150 5.9 IN-VIVO TOXICITY STUDIES 154 5.9.1Effect of ItW-Et on weight of body, liver and kidney of Wistar Albino Rats 166 5.9.2 Effect of ItW-Et on hematological parameters of Wistar Albino Rats 166 5.9.3 Effect of ItW-Et on biochemical parameters of Wistar Albino Rats 167 5.9.4 Effect of ItW-Et on hepatic enzymes of Wistar Albino Rats 167 5.9.5 Effect of ItW-Et on histological study of Wistar Albino Rats 167 5.10 IN-VIVO HEPATOPROTECTIVE STUDIES 169 5.10.1 Effect of ItW-Et on the Body weight 200 5.10.2 Effect of ItW-Et on Hematological Parameters 202 5.10.3 Effect of ItW-Et on Biochemical Parameters 203 5.10.4 Effect of ItW-Et on Lipid Profile 207 5.10.5 Effect of ItW-Et on Hepatic enzymes 209 5.10.6 Effect of ItW-Et on Carbohydrates Metabolising Enzymes 211 5.10.7 Effect of ItW-Et on Antioxidants 213 5.10.8 Effect of ItW-Et on Non enzymatic Antioxidants 218 5.10.9 Effect of ItW-Et on Membranous ATPase enzymes 219 5.10.10 Effect of ItW-Et on histopathological studies of Liver Cells 220 5.11 MARKER COMPOUND ISOLATION 222 5.12 IN-SILICO ANALYSIS 228 5.12.1Docking studies of a Identified compounds from I tirunelvelica 228 SUMMARY 243 CONCLUSION 246 REFERENCES 247 v LIST OF TABLES Table 5.1: Physiological and organoleptic characteristics of whole plant dry powder of Indigofera tirunelvelica Sanjappa 121 Table 5.2: Fluorescence analysis of whole plant dry powder of Indigofera tirunelvelica Sanjappa treated with various reagents 122 Table 5.3: Fluorescence analysis of whole plant extracts of Indigofera tirunelvelica Sanjappa in different solvent systems 122 Table 5.4: Physiochemical analysis of whole plant of Indigofera tirunelvelica Sanjappa 124 Table 5.5: Extractive values of different extracts of whole plant dry powder of Indigofera tirunelvelica Sanjappa 125 Table 5.6: Preliminary Phytochemical screening of Indigofera tirunelvelica Sanjappa 126 Table 5.7: Quantitative analysis of important organic constituents of Indigofera tirunelvelica Sanjappa 128 Table 5.8: Effect of various concentrations of whole plant extracts of Indigofera tirunelvelica Sanjappa on the percentage of DPPH activity in different solvent systems 132 Table 5.9: Effect of Various Concentrations of Whole Plant Extracts of Indigofera tirunelvelica Sanjappa on the percentage of ABTS activity in Different Solvent Systems 136 Table 5.10: Effect of Various Concentrations of Whole Plant Extracts of Indigofera tirunelvelica Sanjappa on the percentage of FRAP activity in Different Solvent Systems 138 Table 5.11: Effect of Various Concentrations of Whole Plant Extracts of Indigofera tirunelvelica Sanjappa on the percentage of SO activity in Different Solvent Systems 141 Table 5.12: Effect of Various Concentrations of Whole Plant Extracts of Indigofera tirunelvelica Sanjappa on the percentage of H2O2 activity in Different Solvent Systems 144 Table 5.13: Effect of Various Concentrations of Whole Plant Extracts of Indigofera tirunelvelica Sanjappa on the percentage of Hydroxyl activity in Different Solvent Systems 147 Table 5.14: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Hep G2 cell line (MTT Assay) 151 vi Table 5.15: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on weight of body, liver and kidney of Wistar Albino Rats 154 Table 5.16: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Hematological and Biochemical parameters of Wistar Albino Rats 158 Table 5.17: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Hepatic enzymes of Wistar Albino Rats 162 Table 5.18: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on the Body weight (g) of CCl4 treated Wistar Albino Rats 169 Table 5.19: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Hematological Parameters of CCl4 treated Wistar Albino Rats 172 Table 5.20: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Biochemical Parameters of CCl4 treated Wistar Albino Rats 176 Table 5.21: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Lipid Profile of CCl4 treated Wistar Albino Rats 182 Table 5.22: Effect of ItW-Et on enzymes of CCl4 treated Wistar Albino Rats 185 Table 5.23: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Carbohydrates Metabolising Enzymes of CCl4 treated Wistar Albino Rats 188 Table 5.24: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Antioxidants of CCl4 treated Wistar Albino Rats 190 Table 5.25: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Non enzymatic Antioxidants of CCl4 treated Wistar Albino Rats 193 Table 5.26: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Membranous ATPase enzymes of CCl4 treated Wistar Albino Rats 196 Table 5.27: List of compounds Identified from ItW-Et 225 Table 5.28: Docking Score on the interaction of the high affinity potential of plant compounds with target protein - With Hepatitis B X (1QGT) 240 Table 5.29: Docking Score on the interaction of the high affinity potential of plant compounds with target protein - Heme Oxygenase I (1N3U) 241 vii Chapter VIII injury induced by carbon tetrachloride and paracetamol Journal of pharmacy and pharmacology, 55(10), 1413-1418 Hjerten S, and panh (1983) “Purification and characterization of Low affinity Ca2 ATPase from erythrocytes membrane” Bioche Biophys Acta 728:281-288 Hodnick WF, Ahmad S and Pardini RS 1998 Introducion of oxidative stress by redox active flavonids Adv Experi Med Biol 439: 131 – 150 Hosseinzadeh, S., Jafarikukhdan, A., Hosseini, A., & Armand, R (2015) The application of medicinal plants in traditional and modern medicine: a review of Thymus vulgaris International Journal of Clinical Medicine, 6(09), 635642 Hsouna, A B., Mongi, S., Culioli, G., Blache, Y., Ghlissi, Z., Chaabane, R., & Trigui, M (2016) Protective effects of ethyl acetate fraction of Lawsonia inermis fruits extract against carbon tetrachloride-induced oxidative damage in rat liver Toxicology and industrial health, 32(4), 694-706 Hu, J., Webster, D., Cao, J., & Shao, A (2018) The safety of green tea and green tea extracts consumption in adults–Results of a systematic review Regulatory Toxicology and Pharmacology Hu, L., Deng, L., Alsaiari, S., Zhang, D and Khashab, N.M., 2014 “Light-on” sensing of antioxidants using gold nanoclusters Analytical chemistry, 86(10), pp.4989-4994 Huber Meret, Daniella Triebwasser-Freese, Michael Reichelt, Sven Heiling, Christian Paetz, Jima N Chandran, Stefan Bartram, Bernd Schneider, Jonathan Gershenzon and Matthias Erb, 2015 Identification, quantification, spatiotemporal distribution and genetic variation of major latex secondary metabolites in the common dandelion (Taraxacum officinale Agg.) Phytochem., 115: 89-98 Huie, C.W., 2002 A review of modern sample-preparation techniques for the extraction and analysis of medicinal plants Analytical and bioanalytical chemistry, 373(1-2), pp.23-30 Hussain, F., Malik, A., Ayyaz, U., Shafique, H., Rana, Z., & Hussain, Z (2017) Efficient hepatoprotective activity of cranberry extract against CCl4-induced hepatotoxicity in Wistar albino rat model: Down-regulation of liver enzymes and strong antioxidant activity Asian Pacific journal of tropical medicine, 10(11), 1054-1058 Hussain, S I., Fatima, U., Sultana, T., Attuluri, S., Afifa, S A A., & Afia, A A (2017) Hepatoprotective activity of indian medicinal plant on CCl4 induced liver damage in wistar rats World Journal of Pharmacy and Pharmaceutical Sciences, 6(8(, 1860-1881 Ibrahim, Inas Rifaat, Mohamed Azmi Hassali, Fahad Saleem and Haydar F Al Tukmagi, 2016 A qualitative insight on complementary and alternative medicines used by hypertensive patients J Pharm Bioallied Sci., (4): 284 Jamuna Bai Aswathanarayan and Ravishankar Rai Vittal 2013 In-vitro evaluation of antioxidant and antibacterial activities of Rotula aquatica and Ancistrocladus heyneanus: Antioxidant and antimicrobial activity of medicinal plants Journal of Pharmacy Research (2): 313–317 Jegadeeswari, P., A Nishanthini, S Muthukumarasamy and V.R Mohan, 2014 Evaluation of antioxidant activity of Aristolochia krysagathra (Aristolochiaceae)-an important medicinal herb Int J Pharm., (1): 410416 Chapter VIII Jiménez-Arellanes, M A., Gutiérrez-Rebolledo, G A., Meckes-Fischer, M., & LnDíaz, R (2016) Medical plant extracts and natural compounds with a hepatoprotective effect against damage caused by antitubercular drugs: a review Asian Pacific journal of tropical medicine, 9(12), 1141-1149 Jones, R F (1961) Determination of packed cell volume by centrifugation Journal of clinical pathology, 14(2), 198 Joseph Mahimaidossa, Charles Antonyb and Alex Ramani Vincent, 2014 Phytochemical screening and bioactivity studies of Phyllanthus wightianus J Pharm Res., (1): 188–192 Joshi, B C., Prakash, A., & Kalia, A N (2015) Hepatoprotective potential of antioxidant potent fraction from Urtica dioica Linn.(whole plant) in CCl4 challenged rats Toxicology reports, 2, 1101-1110 Kadifkova-Panovska T, S Kulevanova and M Stefova, 2005 In-vitro antioxidant activity of some Teucrium sp (Lamiaceae) Acta Pharm., 55: 207–14 Kalsi, S., Verma, S K., Neha, A K., & Singh, N (2016) A Review on glycyrrhiza glabra (liquorice) and its pharmacological ac-tivities Int J Pharm Drug Anal., 4(5), 234-239 Kamel, I S., El-Sherbini, E S., Hassan, H A., & El-Ghareeb, M S (2017) Biochemical studies on hepatocellular carcinoma in male rats: the protective role of purslane seeds extract World Journal Of Pharmacy And Pharmaceutical Sciences, 6,(9), 41-56 Kamisan, F H., Yahya, F., Mamat, S S., Kamarolzaman, M F F., Mohtarrudin, N., Kek, T L., & Zakaria, Z A (2014) Effect of methanol extract of Dicranopteris linearis against carbon tetrachloride-induced acute liver injury in rats BMC complementary and alternative medicine, 14(1), 123 Karki, P., Parikh, S., Sharma, S K., Pradhan, B., & Shrestha, N R (2014) An echocardiographic evaluation of cardiac functions in patients with liver cirrhosis: A hospital based study Journal of Indian College of Cardiology, 4(1), 18-24 Kasote, D.M., Katyare, S.S., Hegde, M.V and Bae, H., 2015 Significance of antioxidant potential of plants and its relevance to therapeutic applications International journal of biological sciences, 11(8), p.982 Kaur Sarabjot and Poonam Mondal, 2014 Study of total phenolic and flavonoid content, antioxidant activity and antimicrobial properties of medicinal plants J Microbiol Exp., (1): 1-6 Khan, M R., & Siddique, F (2012) Antioxidant effects of Citharexylum spinosum in CCl4 induced nephrotoxicity in rat Experimental and toxicologic pathology, 64(4), 349-355 Khanal, Supreet, Niroj Shakya, Krishna Thapa, and Deepak Raj Pant, 2015 Phytochemical investigation of crude methanol extract of different species of Swertia from Nepal BMC Res Notes, (1): 821 Khatri, S., Kumar, M., Phougat, N., Chaudhary, R., & Kumar Chhillar, A (2016) Perspectives on phytochemicals as antibacterial agents: an outstanding contribution to modern therapeutics Mini reviews in medicinal chemistry, 16(4), 290-308 Khojasteh, S M B., & Khameneh, R J (2018) Sophora pachycarpa Root Extract Improves Testicular Damage in Carbon-Tetrachloride Intoxicated Rats Zahedan Journal of Research in Medical Sciences, 20(8) King J 1965 In : Practical Clinical Enzymology, Princeton MJ(Fol) Van D Nostrand Company, London 363 Chapter VIII Klein, S M., G Cohen and A I Cederbaum, 1991 Production of formaldehyde during metabolism of dimethyl sulphoxide by hydroxyl radical generating system Biochem., 20: 6006-6012 Kluwe WM 1981 Renal function test as indicators of kidney injuy in subacute toxicity studies Toxicol Appl Pashudhan 12: 2-4 Kokate CK 1989 Practical Pharmacognosy, II Edition, Nirali Prakashan, Pune, India, P.115-124 Kokoski, C J., R J Kokoski and F J Slama, 1958 Fluorescence of powdered vegetable drugs under ultraviolet radiation J Am Pharm Assoc (Baltim), 47 (10): 715-7 Kondeva-Burdina, M., Shkondrov, A., Simeonova, R., Vitcheva, V., Krasteva, I., & Ionkova, I (2018) In-vitro/in-vivo antioxidant and hepatoprotective potential of defatted extract and flavonoids isolated from Astragalus spruneri Boiss.(Fabaceae) Food and Chemical Toxicology, 111, 631-640 Kuan-Hung Lin, Yan-Yin Yang, Chi-Ming Yang, Meng-Yuan Huang, Hsiao-Feng Lo, Kuang-Chuan Liu, Hwei-Shen Lin and Pi-Yu Chao, 2013 Antioxidant activity of herbaceous plant extracts protect against hydrogen peroxideinduced DNA damage in human lymphocytes BMC Res Notes, 6: 490 Kumar S, Kumar V and Om Prakash 2011 Microscopic evaluation and physiochemical analysis of Dillenia indica leaf Asian Pac J Trop Biomed 1(5): 337–340 Kumar Singh Amrit, Paul, Attrey Dharam, Tanveer Nave 2013 Bioactivity guided extraction of Seabuckthorn (Hippophae rhamnoides L ssp turkestanica) leaves Journal of Scientific and Industrial Research 72(05): 307 – 311 Kumaran K 2007 Estimation of total flavonoid content in propolis by twocomplementary colorimetric methods J of Food and Drug Analysis 10:178–182 Kuriakose, J., Raisa, H L., Vysakh, A., Eldhose, B., & Latha, M S (2017) Terminalia bellirica (Gaertn.) Roxb fruit mitigates CCl4 induced oxidative stress and hepatotoxicity in rats Biomedicine & Pharmacotherapy, 93, 327333 Laouar, A., Klibet, F., Bourogaa, E., Benamara, A., Boumendjel, A., Chefrour, A., & Messarah, M (2017) Potential antioxidant properties and hepatoprotective effects of Juniperus phoenicea berries against CCl4 induced hepatic damage in rats Asian pacific journal of tropical medicine, 10(3), 263-269 Larson RA 1998 The antioxidants of higher plants Phytochemistry 27(4):969–978 Latif Abdul and Sumbul Rehman, 2014 Standardization of a Herbal MedicineSwertia Chirayita Linn Pharmacophore., (1): 98 - 108 Lemon, S M., Ott, J J., Van Damme, P., & Shouval, D (2018) Type A viral hepatitis: A summary and update on the molecular virology, epidemiology, pathogenesis and prevention Journal of hepatology, 68(1), 167-184 Leyte-Lugo, Martha, R Emily Britton, H Daniel Foil, R Adam Brown, A Daniel Todd, José Rivera-Chávez, H Nicholas Oberlies and B Nadja Cech, 2017 Secondary metabolites from the leaves of the medicinal plant golden seal (Hydrastis canadensis) Phytochem Lett., 20: 54-60 Lowry OH, Rosebrough NJ, Farr AL and Randall RJ 1951 Protein measurement with Folin Phenol reagent J Biol Chem 193: 265-275 Maehly AC and Chance B 1954 In: Methods of Biochemical Analysis, Vol I, Glick D, editors, New York, Interscience 357 Chapter VIII Maggi, P., Biagio, A., Rusconi, S., Cicalini, S., D’Abbraccio, M., d’Ettorre, G & Squillace, N (2017) Cardiovascular risk and dyslipidemia among persons living with HIV: a review BMC infectious diseases, 17(1), 551 Mahmood, Z., Tasleem, F., Hameed, N., Jafrey, R., Azhar, I., Imam, S., & Mahmoo, Z A (2017) Hepatoprotective effect of Peltophorum pterocarpum leaves extracts and pure compound against carbon tetra chloride induced liver injury in rats Medical Research Archives, 5(4) Mahmoodzadeh, Y., Mazani, M., & Rezagholizadeh, L (2017) Hepatoprotective effect of methanolic Tanacetum parthenium extract on CCl4-induced liver damage in rats Toxicology reports, 4, 455-462 Maiti Bodhisattwa, B P Nagori and Rambir Singh, 2017 Recent trends in herbal drugs: a review Int J Drug Res Technol., (1): 17 -25 Malick CP and Singh MB 1980 Estimation of Phenol In: Plant Enzymology and histoenzymology New Delhi Kalyani Publishers 286 Malloy HT and Evelyn KA 1937 The determination of Bilirubin J Biol Chem 119:481 Manibusan, M.K., Odin, M and Eastmond, D.A., 2007 Postulated carbon tetrachloride mode of action: a review Journal of Environmental Science and Health Part C, 25(3), pp.185-209 Mannervik, B The isozymes of Glutatione Transferase Adv Enzymol Relat Areas Mol Biol 57, 357-417 (1985) Mansuy, D., Fontecave, M and Chottard, J.C., 1980 A heme model study of carbon tetrachloride metabolism: mechanisms of phosgene and carbon dioxide formation Biochemical and biophysical research communications, 95(4), pp.1536-1542 Maria Debbarma, A Nazir Pala, Munesh Kumar and W Rainer Bussmann, 2017 Traditional knowledge of medicinal plants in tribes of Tripura in Northeast India Afr J Tradit Complement Altern Med., 14 (4): 156-168 Marrelli Mariangela, Stefania La Grotteria, Fabrizio Araniti and Filomena Conforti, 2017 Investigation of the potential health benefits as lipase inhibitor and antioxidant of Leopoldia comosa (L.) Parl.: Variability of chemical composition of wild and cultivated bulbs Plant Food Hum Nutr., 72 (3): 274–279 Martin DW, Mayes PA and Rodwell YW 1981 Harper's Review of Biochemistry 18th Ed Lange Medical, CA, 61 Mathew, S and T E Abraham, 2006 In-vitro antioxidant activities and scavenging effects of Cinnamomum verum leaf extract assayed by different methodologies J Food Chem Toxicol., 44: 198-206 Mathews, Miya Gugulethu, Idris Ajayi Oyemitana, Oyedeji Oyehan Opeoluwa, Oluwafemi Samuel Oluwatobi, N Nkeh-Chungag Benedicta, Songca Sandile Phindile and Oyedejia Adebola Omowumi, 2016 Phytochemical screening, anti-inflammatory and analgesic properties of Pentanisia prunelloides from the Eastern Cape Province, South Africa Afr J Tradit Complement Altern Med., 13 (6): 179-185 McGregor, D., & Lang, M (1996) Carbon tetrachloride: genetic effects and other modes of action Mutation research, 366(3), 181-195 Menard DB, Gisselbrecht C, Marty M, Reyes F, Dhumeaux D 1980 Anti-neoplastic agents and the liver Gastroenterology 78:142-164 Chapter VIII Menpara Disha, and Sumitra Chanda, 2014 Phytochemical and pharmacognostic evaluation of leaves of Pongamia pinnata L (Fabaceae) Pharmacog Commn., (2): Misra HP and Fridovich I 1972 The role of superoxide anion in the autooxidation of epinephrine and a simple assay for SOD J Biol Chem 247: 3170-3175 Moossavi, M., Hoshyar, R., Hemmati, M., Farahi, A., & Javdani, H (2017) An invivo study on the hepato-protective effects of Crocus sativus, Ziziphus jujuba and Berberis vulgaris against acute acetaminophen and rifampicin-induced hepatotoxicity Clinical Phytoscience, 2(1), 16 Morales MA, Jabbagy AJ, and Terenizi HR 1973 Mutations affecting accumulation of Glycogen Neurospora News Letters 20:24-25 Moron MS, Depierre JW and Mannervik B 1979 Levels of glutathione, glutathione reductase and glutathione-S-transferase activities in rat lung and liver Biochim and Biophys Acta 5820: 60-68 Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK and Olson AJ 1998 Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function J Comp Chem 19:1639–1662 Mosmann, T., 1983 Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays J Immunol Methods., 65: 55-63 Nafiu Mikhail Olugbemiro and Anofi Omotayo Tom Ashafa, 2017 Antioxidant and inhibitory effects of saponin extracts from Dianthus basuticus Burtt Davy on key enzymes implicated in type diabetes in-vitro Pharmacogn Mag., 13 (52): 576 Nafiu, M O., Hamid, A A., Muritala, H F., & Adeyemi, S B (2017) Preparation, Standardization, and Quality Control of Medicinal Plants in Africa In Medicinal Spices and Vegetables from Africa (pp 171-204) Nagaraja, Y P., & Krishna, V (2016) Hepatoprotective effect of the aqueous extract and 5-hydroxy, 7, 8, 2′ trimethoxy flavone of Andrographis alata Nees in carbon tetrachloride treated rats Achievements in the Life Sciences, 10(1), 510 Naik, S R., Thakare, V N., & Patil, S R (2011) Protective effect of curcumin on experimentally induced inflammation, hepatotoxicity and cardiotoxicity in rats: evidence of its antioxidant property Experimental and Toxicologic Pathology, 63(5), 419-431 Nakamura Y, Watanabe S, Miyake N, Kohno H and Osawa T 2003 Evaluation as novel radical scavenging antioxidants J Agric Food Chem 51: 3309 – 3312 Nan, J X., Park, E J., Kim, Y C., Ko, G., & Sohn, D H (2002) Scutellaria baicalensis inhibits liver fibrosis induced by bile duct ligation or carbon tetrachloride in rats Journal of pharmacy and pharmacology, 54(4), 555-563 Nandita Das, MD Ekramul Islam, Nusrat Jahan, Mohammad Saiful Islam, Alam Khan, MD Rafikul Islam and MST Shahnaj Parvin 2014 Antioxidant activities of ethanol extract and fractions of Crescentia cujete leaves and stem bark and the involvement of phenolic compounds BMC Complementary and Alternative Medicine 14:45 Natelson S, Scott ML and Beffa D 1951 A rapid method for the estimation of urea in biological fluids by means of the reaction between diacetyl and urea Am J Chem Patol 21: 275-81 Chapter VIII Netala, S Ilvia, Reethika Penmetsa, Snehitha Nakka and Bhagya Lakshmi Polisetty, 2015 Pharmacognostic study of Callistemon citrinus L bark Int J Pharm Pharm Sci., (1): 427-430 Nevin, K G., & Vijayammal, P L (2005) Effect of Aerva lanata against hepatotoxicity of carbon tetrachloride in rats Environmental Toxicology and Pharmacology, 20(3), 471-477 Nurain Ismaila O., O Clement Bewaji, S Jarrett Johnson, D Robertson Davenport and Yang Zhang, 2016 Potential of three ethnomedicinal plants as antisickling agents Mol Pharm., 14 (1): 172-182 Ogeturk, M., Kus, I., Colakoglu, N., Zararsiz, I., Ilhan, N., & Sarsilmaz, M (2005) Caffeic acid phenethyl ester protects kidneys against carbon tetrachloride toxicity in rats Journal of ethnopharmacology, 97(2), 273-280 Ohkawa H, Ohishi N and Yagi K 1979 Assay of lipid peroxides in animal tissues for thiobarbituric acid reaction Annual Biochem 95: 351-358 Okaiyeto, K., Nwodo, U U., Mabinya, L V., & Okoh, A I (2018) A review on some medicinal plants with hepatoprotective effects Pharmacognosy Reviews, 12(24), 186 Olson H, Betton G, Robinson D, Thomas K, Monro A, Kolaja G, Lilly P, Sanders J, Sipes G, Bracken W, Dorato M, Van Deun K, Smith P, Berger B, and Heller A 2000 Concordance of the toxicity of pharmaceuticals in humans and in animals Regul Toxicol Pharmacol 32(1):56-67 Om Satyam Chaturvedi, Amit Joshi and Bal Krishna Dubey 2012 Pharmacognostical, Phytochemical evaluation and antiinflammatory activity of stem of Kalanchoe pinnata pers International Journal of Pharmaceutical sciences and Research 3(4): 1133-1140 Owen RW, Giacosa A, Hull WE, Haubner R, Spiegelhalder B and Bartsch H 2000 The antioxidant/anticancer potential of phenolic compounds isolated from olive oil European Journal of Cancer 36(10):1235–1247 Oyetunji, O A., Babatunde, I R., Chia, S L., Abraham, O A., Adewale, F B., Eweoya, O., & Williams, F E (2015) Ameliorative Effects of Adansonia Digitata Leaf Extract on Carbon Tetrachloride (CCL 4) Induced Testicular Toxicity in Adult Male Wistar Rats Anatomy Journal of Africa, 4(1), 481487 Padmaja, V., & Kumari, K M (2017) Protective Effect of Alysicarpus Monilifer L., Against CCl4 induced Hepatotoxicity in Albino Rats International Journal of Trend in Scientific Research and Development, 1(Issue-4) Pandavadra Minal and Sumitra Chanda, 2014 Development of quality control parameters for the standardization of Limonia acidissima L leaf and stem Asian Pac J Trop Biomed., 7: S244-S248 Panigrahy, Suchitra Kumari, Awanish Kumar and Renu Bhatt, 2017 Antioxidant potentials of successive solvent extracts from the unexplored Hedhychium coronarium rhizome J Food Sci Technol., 54 (10): 3297-3306 Parekh AC and Jung DH 1970 Determination of Cholesterol with ferric acetate, Uranium Acetate and sulphuric acid-ferrous sulphate reagents Anal Chem 42: 1523-1529 Paritha, I.A and Devi, C.S Effect of α-tocopherol on lipid peroxidation in isoproterenol induced myocardial infarction in rats Ind J Biochem Biophys 1997; 41: 369-376 Chapter VIII Parks, E., & Traber, M G (2000) Mechanisms of vitamin E regulation: research over the past decade and focus on the future Antioxidants and Redox Signaling, 2(3), 405-412 Parola, M., & Robino, G (2001) Oxidative stress-related molecules and liver fibrosis Journal of hepatology, 35(2), 297-306 Patrick-Iwuanyanwu, K C., Wegwu, M O., & Ayalogu, E O (2007) Prevention of CCl-induced liver damage by ginger, garlic and vitamin E Pak J Biol Sci, 10(4), 617-621 Petreikov, M., Dai, N., Granot, D., & Schaffer, A A (2001) Characterization of native and yeast-expressed tomato fruit fructokinase enzymes Phytochemistry, 58(6), 841-847 Popova Milena, Efstathia Giannopoulou, Krystyna Skalicka-Woźniak, Konstantia Graikou, Jaroslaw Widelski, Vassya Bankova and Haralabos Kalofonos, 2017 Characterization and biological evaluation of propolis from Poland Molecules, 22: 1159 Prabakaran, D., & Ashokkumar, N (2012) Antihyperglycemic effect of esculetin modulated carbohydrate metabolic enzymes activities in streptozotocin induced diabetic rats Journal of Functional Foods, 4(4), 776-783 Priyadarshi Apurva, Rajesh Kumari, Anil Kumar Sharma and Mohal Lal Jaiswal, 2016 Preliminary pharmacognostic and phytochemical investigation of Blepharis sindica- T Anders seeds Ancient Sci Life, 36 (2): 78 Pulido, R., L Bravo and F Saura-Calixto, 2000 Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay J Agric Food Chem., 48: 3396–3402 Qureshi, Muhammad Nasimullah, Guenther Stecher and Guenther Karl Bonn, 2016 Quantification of polyphenolic compounds and flavonoids in Achillea millefolium and Equisetum arvense Pak J Pharm Sci., 29: Rainsford, K D (2016) Side effects and toxicology of the salicylates In Aspirin and related drugs (pp 481-713) CRC Press Raj Kumar, Somen Acharya, Sharad Rai, O P Chaurasia and R B Srivastava 2014 Evaluation of antioxidant activities and total phenolic content of methanolic extract of Rhodiola heterodanta roots J Pharmacog Phytochem., (6): 189194 Rao, A L., Aminabee, S., & Eswariah, M C (2017) Evaluation of Hepatoprotective Activity of Indigofera barberi in Rats against Paracetamol Induced Hepatic Injury Advances in Investigational Pharmacology and Therapeutic Medicine 1:1-9 Re, R., N Pellegrini, A Proteggente, A Pannala, M Yang and C Rice-Evans, 1999 Antioxidant activity applying an improved ABTS radical cation decolorizing assay Free Radical Biol Med., 26: 1231-1237 Recknagel, R O., & Malamed, S (1958) The osmotic nature of mitochondrial swelling produced by carbon tetrachloride and inorganic phosphate Journal of Biological Chemistry, 232(2), 705-714 Recknagel, R O., Glende Jr, E A., Dolak, J A., & Waller, R L (1989) Mechanisms of carbon tetrachloride toxicity Pharmacology & therapeutics, 43(1), 139154 Reddy, V P., & Urooj, A (2017) Evaluation of hepatoprotective activity of Morus indica Linn against toxicity induced by carbon tetrachloride in rats Int J Pharmaceut Sci Res, 8, 845-51 Chapter VIII Reyes-Gordillo, K., Segovia, J., Shibayama, M., Tsutsumi, V., Vergara, P., Moreno, M G., & Muriel, P (2008) Curcumin prevents and reverses cirrhosis induced by bile duct obstruction or CCl4 in rats: role of TGF-β modulation and oxidative stress Fundamental & clinical pharmacology, 22(4), 417-427 Reynolds, J C (2016) The Netter Collection of Medical Illustrations: Digestive System: Part I-The Upper Digestive Tract E-Book Elsevier Health Sciences Rezvanian, M., Ooi, Z T., Jamal, J A., Husain, K., Jalil, J., Yaacob, Z., & Ghani, A A (2016) Pharmacognostic and Chromatographic Analysis of Malaysian Piper Nigrum Linn Fruits Indian Journal of Pharmaceutical Sciences, 78(3), 334-343 Riaz, M., M Shahid, A Jamil and M Saqib, 2017 In-vitro antioxidant potential of selected aphrodisiac medicinal plants J Biol Regul Homeost Agents, 31 (2): 419-424 Rosalki SB and Rau D 1972 Serum gamma glutamyl transpeptidase activity in alcoholism Clin Chem Acta 39: 41-47 Rotruck JT, Pope AL, Ganther H, Swanson AB, Hafeman DG and Hoeksira WG 1973 Selenium: Biochemical role as a component of glutathione peroxidase Science 1790: 588-90 Ruch, R J., S J Cheng and J E Klavning, 1989 Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea Carcinogens, 10: 1003-1008 Russo GL 2007 Ins and outs of dietary phytochemicals in cancer chemoprevention Biochem Pharmacol 74:533–544 Russo, M W., Hoofnagle, J H., Gu, J., Fontana, R J., Barnhart, H., Kleiner, D E., & Bonkovsky, H L (2014) Spectrum of statin hepatotoxicity: Experience of the drug-induced liver injury network Hepatology, 60(2), 679-686 Saad, B., Zaid, H., Shanak, S., & Kadan, S (2017) Introduction to Medicinal Plant Safety and Efficacy In Anti-diabetes and Anti-obesity Medicinal Plants and Phytochemicals (pp 21-55) Springer, Cham Sabu MC and Ramadasan Kuttan 2002 Anti-diabetic activity of medicinal plants and its relationship with their antioxidant property Journal of Ethnopharmacology 81(2): 155–160 Sadeghi Zahra, Kimia Kuhestani, Vahideh Abdollahi and Adeel Mahmood, 2014 Ethno pharmacological studies of indigenous medicinal plants of Saravan region, Baluchistan, Iran J Ethnopharmacol., 153: 111-118 Sahoo Atish Kumar, Jagnehswar Dandapat, Umesh Chandra Dash and Satish Kanhar, 2017 Features and outcomes of drugs for combination therapy as multitargets strategy to combat Alzheimer's disease J Ethnopharmacol., 215: 4273 Saklani Sarla, Abhay Prakash Mishra, Harish Chandra, Maria Stefanova Atanassova, Milan Stankovic, Bhawana Sati and Mohammsad Ali Shariati, 2017 Comparative evaluation of polyphenol contents and antioxidant activities between ethanol extract of Vitex negundo and Vitex trifolia L leaves by different methods Plants, (4): 45 Sanjappa, M., 1995 Revision of the genus Indigofera in India Fascicles of Flora of India, 21, pp.1-160 Sanjib Bhattacharya and Kamaruz Zuman M 2009 Pharmacognostical evaluation of Zathoxylum nitidum Bark International Journal of PharmTech Research 1(2): 292-298 Chapter VIII Sarah Bendaikha, Méredith Gadaut, Dominique Harakat and Alabdul Magid 2014 Acylated flavonol glycosides from the flower of Elaeagnus angustifolia L Phytochemistry 103: 129–136 Sardar, Andleeb Anwar, Anjum Perveen, Sidra Farid and Islam Ullah Khan 2017 Phytochemical screening, GC-MS analysis and in-vitro antioxidant activity of pollen of Centella asiatica (Linn.) urban a traditional medicinal plant Pak J Pharm Sci., 30: Sarges, P., Steinberg, J M., & Lewis, J H (2016) Drug-induced liver injury: highlights from a review of the 2015 literature Drug safety, 39(9), 801-821 Sarin, S K., Kumar, M., Lau, G K., Abbas, Z., Chan, H L Y., Chen, C J., & Dokmeci, A K (2016) Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update Hepatology international, 10(1), 1-98 Satapathy, S K., & Sanyal, A J (2015) Epidemiology and natural history of nonalcoholic fatty liver disease In Seminars in liver disease: Thieme Medical Publishers., 35 (03), 221-235 Sathasivam, S and A Manickam, 1996 Biochemical methods New age international (P) Limited, Publishers, II (ed.), New Delhi 152-160 Sayiner, M., Koenig, A., Henry, L., & Younossi, Z M (2016) Epidemiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in the United States and the rest of the world Clinics in liver disease, 20(2), 205-214 Schmidt, H.H., Stocker, R., Vollbracht, C., Paulsen, G., Riley, D., Daiber, A and Cuadrado, A., 2015 Antioxidants in translational medicine Antioxidants & redox signaling, 23(14), pp.1130-1143 Scott, W R P., P H Hünenberger, I G Tironi, A E Mark, S R Billeter, J Fennen, A E Torda, T Huber, P Krüger and W F Gunsteren, 1999 The GROMOS Biomolecular Simulation Program Package J Phys Chem A., 103: 3596– 3607 Shabir Lone, H., A Khursheed Bhat, M Haroon Bhat, Rabiya Majeed, Rajneesh Anand, Abid Hamid and A Mohd Khurood, 2014 Essential oil composition of Senecio graciliflorus DC: Comparative analysis of different parts and evaluation of antioxidant and cytotoxic activities Phytomed., 21 (6): 919– 925 Shahverdi, A.R., Abdolpour, F., Monsef-Esfahani, H.R and Farsam, H., 2007 A TLC bioautographic assay for the detection of nitrofurantoin resistance reversal compound Journal of Chromatography B, 850(1-2), pp.528-530 Shahwar Durre, Naeem Ahmad, Asma Yasmeen, Muhammad Akmal Khan, Sami Ullah and Rahman Atta-ur, 2015 Bioactive constituents from Croton sparsiflorus Morong Nat Prod Res., 29: 274-276 Sharma, B K., Ramashanker, S G., Rahaman, L., Nath, N., & Kaipeng, D L (2016) Plant based folk treatments from North East India for jaundice.(An overview) Journal of Medicinal Plants, 4(5), 234-247 Sharma, V., & Agrawal, R C (2017) In-vivo antioxidant and hepatoprotective potential of Glycyrrhiza glabra extract on carbon tetra chloride (CCl4) induced oxidative-stress mediated hepatotoxicity International Journal of Research in Medical Sciences, 2(1), 314-320 Sherif, Z A., Saeed, A., Ghavimi, S., Nouraie, S M., Laiyemo, A O., Brim, H., & Ashktorab, H (2016) Global epidemiology of nonalcoholic fatty liver disease and perspectives on US minority populations Digestive diseases and sciences, 61(5), 1214-1225 Chapter VIII Sheu, M.T., Jhan, H.J., Hsieh, C.M., Wang, C.J and Ho, H.O., 2015 Efficacy of antioxidants as a complementary and alternative medicine (CAM) in combination with the chemotherapeutic agent doxorubicin Integrative cancer therapies, 14(2), pp.184-195 Shikov Alexander, N., N Andrey Tsitsilin, N Olga Pozharitskaya, G Valery Makarov and Michael Heinrich, 2017 Traditional and current food use of wild plants listed in the Russian Pharmacopoeia Front Pharmacol., Shimizu, H., Daly, J W., & Creveling, C R (1969) A radioisotopic method for measuring the formation of adenosine 3′, 5′-cyclic monophosphate in incubated slices of brain Journal of neurochemistry, 16(12), 1609-1619 Shirwaikar A, Rajendran K, Dinesh Kumar C, Bodla R Antidiabetic activity of aqueous extract of Annona squamosa in streptozotocin-nicotinamide type diabetic rats J Ethnopharmacol 2004;91:171-5 Shrestha Savitri, Vasuki Srinivas Kaushik, Ravi Shankara Birur Eshwarappa, Sundara Rajan Subaramaihha, Latha Muuaiah Ramanna and Dhananjaya Bhadrapura Lakkappa, 2014 Pharmacognostic studies of insect gall of Quercus infectoria Olivier (Fagaceae) Asian Pac J Trop Dis., (1): 35-39 Singh Anupama, Vikas Anand Saharan and Anil Bhandari, 2014 Pharmacognostic standardization with various plant parts of Desmostachya bipinnata Pharm Biol., 52 (3): 298-307 Singh, D P., & Mani, D (2015) Protective effect of Triphala Rasayana against paracetamol-induced hepato–renal toxicity in mice Journal of Ayurveda and integrative medicine, 6(3), 181 Singhal AK, Bhati VS and Singhal VK 2010 Pharmacognostic study of aerial parts of plant Geniosporum prostratum (`L) Benth J Sci Specul Res 1(1):19–24 Sistare, F D., Mattes, W B., & LeCluyse, E L (2017) The promise of new technologies to reduce, refine, or replace animal use while reducing risks of drug induced liver injury in pharmaceutical development ILAR journal, 57(2), 186-211 Sobeh, M., Mahmoud, M F., Abdelfattah, M A., El-Beshbishy, H A., El-Shazly, A M., & Wink, M (2017) Albizia harveyi: Phytochemical profiling, antioxidant, antidiabetic and hepatoprotective activities of the bark extract Medicinal Chemistry Research, 26(12), 3091-3105 Solan, H P (2017) Bioactive Components of Vigna species: Current Prospective Bull Env Pharmacol Life Sci, 6, 01-13 Sonibare, Mubo Adeola and Oluwaseun Victoria Olatubosun, 2015 Pharmacognostic and free radical scavenging evaluation of Cyathula prostata (Blume) L Pharmacog J., (2): 107-116 Sotelo-Félix, J I., Martinez-Fong, D., & De la Torre, P M (2002) Protective effect of carnosol on CCl4-induced acute liver damage in rats European journal of gastroenterology & hepatology, 14(9), 1001-1006 Spence TJ, Levels of translatable mRNA coding for rat liver Glucokinase.,J Biol Chem 1983 ;258 : 9143-9146 Sreepriya, M., & Bali, G (2006) Effects of administration of Embelin and Curcumin on lipid peroxidation, hepatic glutathione antioxidant defense and hematopoietic system during N-nitrosodiethylamine/Phenobarbital-induced hepatocarcinogenesis in Wistar rats Molecular and cellular biochemistry, 284(1-2), 49-55 Srilaxmi, P., Sareddy, G R., Setty, O H., & Babu, P P (2010) Protective efficacy of natansnin, a dibenzoyl glycoside from Salvinia natans against CCl induced Chapter VIII oxidative stress and cellular degeneration in rat liver BMC pharmacology, 10(1), 13 Stamp, N March 2003 Out of the Quagmire of Plant Defense Hypotheses The Quarterly Review of Biology 78 (1): 23-55 Ständer Sonja and Malcolm Greaves, 2016 Pruritus, Prurigo and Lichen Simplex Rook's Textbook of Dermatology John Wiley & Sons, Ltd, West Sussex, UK Stanger, B Z (2015) Cellular homeostasis and repair in the mammalian liver Annual review of physiology, 77, 179-200 Stewart P, Boonsiri P, Puthong S and Rojpibulstit P 2013 Antioxidant activity and ultrastructural changes in gastric cancer cell lines induced by Northeastern Thai edible folk plant extracts BMC Complementary and Alternative Medicine 13:60 Stickel, F., & Shouval, D (2015) Hepatotoxicity of herbal and dietary supplements: an update Archives of toxicology, 89(6), 851-865 Subburayalu, S., & Asha, K R T (2017) Phytochemical and antimicrobial screening of Indigofera tirunelvelica sanjappa: an important herb World Journal of Pharmacy and Pharmaceutical Sciences, 6(11), 883 – 892 Subhrajyoti Roya, Bibhabasu Hazrab, Nripendranath Mandalb and Tapas Kumar Chaudhuria, 2013 Assessment of the antioxidant and free radical scavenging activities of methanolic extract of Diplazium esculentum Int J Food Sci Technol., 16 (6): 1351-1370 Sujai Suneetha 1993 Histopathological Techniques, in Handbook of CMAI Medical Laboratory Technology by Robert H Carman, Christian Medical Association of India.Chapter 24 508-541 Surendra, H.B and Alpana, R 2007 Hepatoprotective properties of Bauhinia variegate bark extract Yakugaku Zasshi, 127(9), 1503-1507 Swanson, M A (1955) [83] Glucose-6H2O→ Glucose+ PO4 Methods in enzymology, 2, 541-543 Taddeo, Vito Alessandro, Salvatore Genovese, Philippe de Medina, Roberta Palmisano, Francesco Epifano and Serena Fiorito, 2017 Quantification of biologically active O-prenylated and unprenylated phenylpropanoids in dill (Anethum graveolens), anise (Pimpinella anisum) and wild celery (Angelica archangelica) J Pharm Biomed Anal., 134: 319-324 Taha Sherif, M and A Sohair Gadalla, 2017 Development of an efficient method for multi residue analysis of 160 pesticides in herbal plant by ethyl acetate hexane mixture with direct injection to GC-MS/MS Talanta., 174: 767-779 Téllez, L., Rodríguez-Santiago, E., & Albillos, A (2018) Fontan-Associated Liver Disease: A Review Annals of hepatology, 17(2), 192-204 Teschke, R., & Eickhoff, A (2015) Herbal hepatotoxicity in traditional and modern medicine: actual key issues and new encouraging steps Frontiers in pharmacology, 6, 72 Thapliyal Somesh, Vijay Juyal and Anil Bhandari, 2014 Pharmacognostic Screening of Hedychium spicatum Rhizomes Int J Pharmaceu Chem Sci., (4): 483488 Thirumalaikumaran R, Chamundeeswari D, Seethalakshmi S, and Gopal V 2013 Pharmacognostical, phytochemical and antioxidant studies of the aerial parts of Urena lobata L Basic Research Journal of Medicine and Clinical Sciences 2(2): 32-36 Chapter VIII Tirkey, N., Pilkhwal, S., Kuhad, A and Chopra, K., 2005 Hesperidin, a citrus bioflavonoid, decreases the oxidative stress produced by carbon tetrachloride in rat liver and kidney BMC pharmacology, 5(1), p.2 Tiwari, R., Latheef, S K., Ahmed, I., Iqbal, H., Bule, M H., Dhama, K., & Yatoo, M I (2018) Herbal Immunomodulators-A Remedial Panacea for Designing and Developing Effective Drugs and Medicines: Current Scenario and Future Prospects Current drug metabolism, 19(3), 264-301 Togo S, Akiyama H, Yamaguchi S, Ichikawa Y, Ike H and Shimada H 2002 Clinical evaluation of granisetron as an inhibitor of nausea and vomiting induced by oral anticancer drugs Oncol Rep 9:277-282 Torres-González, L., Muñoz-Espinosa, L E., Rivas-Estilla, A M., Trujillo-Murillo, K., Salazar-Aranda, R., Waksman De Torres, N., & Cordero-Pérez, P (2016) Protective effect of four Mexican plants against CCl4-induced damage on the Huh7 human hepatoma cell line Annals of hepatology, 10(1), 73-79 Tripathi Akhilesh, K and Seema Kohli, 2014 Pharmacognostical standardization and antidiabetic activity of Syzygium cumini (Linn.) barks (Myrtaceae) on streptozotocin-induced diabetic rats J Complement Integr Med., 11 (2): 7181 Trump, B.F., Berezesky, I.K., Sato, T., Liaho, K.U.,Phelps, P.C and De Claris, N Cell calcium, cell injury and cell death Environ Health Perspect 1984; 57: 281 - 287 Ullaha Farhat, Naveed Iqbal, Muhammad Ayaz, Abdul Sadiq, Ihsan Ullah, Sajjad Ahmad and Muhammad Imran, 2017 DPPH, ABTS free radical scavenging, antibacterial and phytochemical evaluation of crude methanolic extract and subsequent fractions of Chenopodium botrys aerial parts Pak J Pharm Sci., 30: Venukumar, M R., & Latha, M S (2002) Antioxidant activity ofcurculigo orchioides in carbon tetrachloride—induced hepatopathy in rats Indian Journal of Clinical Biochemistry, 17(2), 80-87 Vilstrup, H., Amodio, P., Bajaj, J., Cordoba, J., Ferenci, P., Mullen, K D., & Wong, P (2014) Hepatic encephalopathy in chronic liver disease: 2014 Practice Guideline by the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver Hepatology, 60(2), 715-735 Vinay Kanta, Madhuri Mehtab and Chandresh Varshneyac 2012 Antioxidant potential and total phenolic contents of seabuckthorn (Hippophae rhamnoides) pomace Free Radicals and Antioxidants 2(4): 79–86 Vinayaka KS, Prashith Kekuda TR, Nethravathi HR, Thippeswamy NB, and Sudharshan SJ 2009 Free radical scavenging and insecticidal activity of Elaeagnus kologa Schldl Drug Invention Today 1(1): 74-77 Vvedenskaya, I O., R T Rosen, J E Guido, D J Russell, K A Mills and N Vorsa, 2004 Characterization of flavonols in Cranberry (Vaccinium macrocarpon) powder J Agric Food Chem., 52: 188–195 Wagatsuma, T., Kuno, A., Angata, K., Tajiri, K., Takahashi, J., Korenaga, M., & Narimatsu, H (2018) Highly Sensitive Glycan Profiling of Hepatitis B Viral Particles and a Simple Method for Dane Particle Enrichment Analytical chemistry, 90(17), 10196-10203 Wang Jue, Tiejie Wang, Pu Xie, Guo Yin and Xiaofan Li, 2014 New phenanthrene derivatives with nitric oxide inhibitory and radical-scavenging activities from Pholidota imbricata Hook Nat Prod Res., 28: 251-256 Chapter VIII Wefers, H., & Sies, H (1988) The protection by ascorbate and glutathione against microsomal lipid peroxidation is dependent on vitamin E European Journal of Biochemistry, 174(2), 353-357 Wei Jiangchun, Xiaokui Huo, Zhenlong Yu, Xiangge Tian, Sa Deng, Chengpeng Sun, Lei Feng, Chao Wang, Xiaochi Ma and Jingming Jia, 2017 Phenolic acids from Balanophora involucrata and their bioactivities Fitoterapia, 121: 129135 Weiß, J., Rau, M., & Geier, A (2014) Non-alcoholic fatty liver disease: epidemiology, clinical course, investigation, and treatment Deutsches Ärzteblatt International, 111(26), 447 Whelton, P K., Carey, R M., & Aronow, W S (2018) Hypertension Guidelines Diabetes, 140, 90 Wilson JE 1995 Hexokinases Rev Physiol Biochem Pharmacol 126:65–198 Wintrobe MM, Lee GR, Boggs DR, Bithel TC, Athens JW, Foerester 1961 Clinical Hematology 5th ed, Philadelphia 326 Wong, R J., Aguilar, M., Cheung, R., Perumpail, R B., Harrison, S A., Younossi, Z M., & Ahmed, A (2015) Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States Gastroenterology, 148(3), 547-555 Wu, Ya-Nan, Wei Wang, Guo-Dong Yao, Xue-Feng Jiang, Yan Zhang and ShaoJiang Song, 2017 Antioxidant phenolic acids from the leaves of Armeniaca sibirica J Asian Nat Prod Res., 1-8 Xu, Y.J., Tappia, P.S., Neki, N.S and Dhalla, N.S., 2014 Prevention of diabetesinduced cardiovascular complications upon treatment with antioxidants Heart failure reviews, 19(1), pp.113-121 Yadav, S K (2016) 21 Role of Natural Anti-Oxidants to Manage Oxidative Stress Reshaping Agriculture and Nutrition Linkages for Food and Nutrition Security, 326, 185 Yang, M Y., Peng, C H., Chan, K C., Yang, Y S., Huang, C N., & Wang, C J (2009) The hypolipidemic effect of Hibiscus sabdariffa polyphenols via inhibiting lipogenesis and promoting hepatic lipid clearance Journal of agricultural and food chemistry, 58(2), 850-859 Yang, Y., Luk, J., & Sofair, A N (2018) Epidemiology of Chronic Liver Disease in the United States In Clinical Epidemiology of Chronic Liver Diseases (pp 57-74) Springer, Cham Yang, Y., Yuan, S X., Zhao, L H., Wang, C., Ni, J S., Wang, Z G., & Zhou, W P (2015) Ligand-directed stearic acid grafted chitosan micelles to increase therapeutic efficacy in hepatic cancer Molecular pharmaceutics, 12(2), 644652 Younossi, Z M., Koenig, A B., Abdelatif, D., Fazel, Y., Henry, L., & Wymer, M (2016) Global epidemiology of nonalcoholic fatty liver disease— meta-analytic assessment of prevalence, incidence, and outcomes Hepatology, 64(1), 73-84 Younossi, Z M., Otgonsuren, M., Henry, L., Venkatesan, C., Mishra, A., Erario, M., & Hunt, S (2015) Association of nonalcoholic fatty liver disease (NAFLD) with hepatocellular carcinoma (HCC) in the United States from 2004 to 2009 Hepatology, 62(6), 1723-1730 Yuan, H., Ma, Q., Ye, L and Piao, G., 2016 The traditional medicine and modern medicine from natural products Molecules, 21(5), p.559 Chapter VIII Yutana P, Patcharin N and Narumon S 2009 Antioxidant activity and xanthine oxidase inhibitor from Thai medicinal plants used for tonic and longevity Bangkok Thailand: Kasetsart University Annual Conference Zangar, R C., Benson, J M., Burnett, V L., & Springer, D L (2000) Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes Chemico-biological interactions, 125(3), 233-243 Zeb Anwar, Farhat Ullah, Muhammad Ayaz, Sajjad Ahmad and Abdul Sadiq, 2017 Demonstration of biological activities of extracts from Isodon rugosus Wall Ex Benth: Separation and identification of bioactive phytoconstituents by GC-MS analysis in the ethyl acetate extract BMC Complement Altern Med., 17: 1-16 Zidorn Christian, 2017 Guidelines for consistent characterization and documentation of plant source materials for studies in phytochemistry and phytopharmacology Phytochem., 139: Chapter VII CONCLUSION From the above affirmed results, it may be concluded that the physiochemical standards generated will be useful for the proper identification of a plant These kinds of results have been recognized as tools to measure the medicinal plant identity relationship under physiochemical to resolve the taxonomic controversies The study also helps to establish the botanical identity for this plant that could be made use for those who deal with the species and also in the quality assurance of the plant species With the support of phytochemical studies and in-vitro pharmacological activities the ethanolic whole plant extract of Indigofera tirunelvelica Sanjappa was selected and subjected to in-vivo pharmacological studies The ethanolic whole plant extract of Indigofera tirunelvelica Sanjappa at the dose level of 400mg/kg bw showed significant hepatoprotective activity These ethanolic extract of Indigofera tirunelvelica derivative also showed promising hepatoprotective activity by in-vivo model Further studies were focused on structural activity relationship of phytoconstituents isolated from the whole plant ethanolic extract of Indigofera tirunelvelica Sanjappa This scientific study revealed the efficacy of the lead molecules and it would definitely have patent application in future Hence, the ethanolic extract of whole plant of Indigofera tirunelvelica Sanjappa can be recommended for therapeutic exploration in medical field These observations will certainly through more lights for the further research in the field of phytochemistry and also in the clinical application of phytochemical constituents of ethanolic whole plant extract of Indigofera tirunelvelica Sanjappa 246 ... present thesis entitled ? ?PHYTOCHEMICAL INVESTIGATION, ANTIOXIDANT ACTIVITIES AND HEPATOPROTECTIVE EFFICACY OF INDIGOFERA TIRUNELVELICA SANJAPPA AGAINST CCL4 INDUCED WISTAR ALBINO RATS. ” has been carried... exploration and exploitation of the hepatoprotective activity of Indigofera tirunelvelica Sanjappa against CCL4 induced hepatoxicity in Wistar Albino Rats 23 Chapter II AIM OF THE WORK The aim of the... and Indirect Bilirubin of CCl4 treated Wistar Albino Rats 181 Figure 5.26: Effect of whole plant ethanol extract of I tirunelvelica (ItW-Et) on Lipid Profile of CCl4 treated Wistar Albino Rats