Pharmacological and Toxicological Screening of Vernonia Anthelmintica

Pharmacological and Toxicological Screening of Vernonia anthelmintica

Subia Jamil

DEPARTMENT OF PHARMACOLOGY FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCES UNIVERSITY OF KARACHI KARACHI-75270, PAKISTAN 2016

Pharmacological and Toxicological Screening of Vernonia anthelmintica

Subia Jamil

Thesis submitted for the partial fulfillment for the requirement of the Ph.D. degree in the Department of Pharmacology University of Karachi, Pakistan.

DEPARTMENT OF PHARMACOLOGY FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCES UNIVERSITY OF KARACHI KARACHI-75270, PAKISTAN 2016

In the name of Allah, the Most Beneficent, the most merciful

CERTIFICATE

This thesis is accepted in its present form by the Department of Pharmacology,

Faculty of Pharmacy and Pharmaceutical sciences, University of Karachi for the partial fulfillment of the requirements for the degree of Ph.D. in Pharmacology

Research Supervisor

______

Dr. Rafeeq Alam Khan Professor (Meritorious) Department of Pharmacology, Faculty of Pharmacy & Pharmaceutical sciences University of Karachi.

Dedicated to Mrs. Laila Zaidi, Mrs. Riaz Fatima (late), my mothers and Mr. Sabir Zaidi, my husband.

CONTENTS Acknowledgement List of tables I List of Figures III List of abbreviations V SUMMARY English IX Urdu X 1. INTRODUCTION 1.1 Description of Vernonia anthelmintica 2 1.2 Plant taxonomy 2 1.3 Importance of extraction 4 1.4 Herbal derived bioactive compounds 4 1.5 Components of Vernonia anthelmintica seeds 5 1.6 Toxicological screening 7 1.6.1 Hematology and coagulation 8 1.6.2 Hepatic profile 10 1.6.3 Renal profile and electrolytes 10 1.7 Pharmacological screening 12 1.7.1 Antibacterial 12 1.7.2 Cytotoxicity 13 1.7.3 Antioxidant 14 1.7.4 Inflammation 15 1.7.5 Nociception 16 1.7.6 Phytotoxic and insecticidal activity 17 1.7.7 Antileishmanicidal activity 18 1.7.8 Diabetes 18 1.7.9 Hyperlipidemia 20 1.8 Objective of the study 21

2. MATERIALS AND METHODS 2.1 Collection and identification of seed 23 2.2 Preparation of extract 23 2.3 Experimental design 23 2.4 Invitro studies 24 2.4.1 Qualitative phytochemical analysis 24 2.4.1.1 Wagner test for alkaloids 24 2.4.1.2 Ferric chloride test for cardiac glycosides 24 2.4.1.3 Benedict test for carbohydrates 24 2.4.1.4 Xanthoprotein test for proteins 25 2.4.1.5 Ferric chloride test for flavonoids 25 2.4.1.6 Foam test for saponins 25 2.4.1.7 Lead test for terpenoids 25 2.4.1.8 Salkowaski’s test for terpenoids 25 2.4.1.9 Spot test for fixed oils and fatty acids 25 2.4.2 Antibacterial assay 26 2.4.3 Cytotoxic assay 26 2.4.4 Brine shrimp lethality Assay 28 2.4.5 Antioxidant Assay 27 2.4.6 Antiglycation Assay 29 2.4.7 Phytotoxic Assay 30 2.4.8 Insecticidal Activity 30 2.4.9 Antileismanial Assay 31 2.5 Invivo studies 32 2.5.1 Animals 32 2.5.2 Acute oral toxicity 32 2.5.3 Sub chronic studies 33 2.5.3.1 Animal grouping and experimental design 33 2.5.4 Pharmacological models 34 2.5.4.1 Hyperlipidemic model (High fat high sµgar model) 34 2.5.4.2 Diabetic models 35

2.5.4.3 Inflammatory model 36 Carrageenan induced rat paw model 36 2.5.4.4 Analgesic models 36 Tail flick method 37 Hot plate method 37 2.6 Sample collection 37 2.7 Hematological analysis 38 2.8 Coagulation studies 38 2.9 Biochemical analysis 39 2.9.1 Lipid profile analysis 40 2.9.2 Blood glucose analysis 41 2.9.3 Liver function test 42 2.9.4 Renal function test 43 2.9.5 Electrolytes analysis 44 2.10 Histopathlogical examinations 45 2.11 Statistical analysis 47 3. RESULTS 3.1 Invitro studies 48 3.1.1 Qualitative phytochemical analysis 48 3.1.2 Antibacterial bioassay 49 3.1.3 Cytotoxic bioassay 49 3.1.4 Brine shrimp lethality test 52 3.1.5 Antioxidant bioassay 53 3.1.5.1 Radical scavenging activity of EEVA 54 3.1.6 Antiglycation bioassay 55 3.1.7 Phytotoxic bioassay 56 3.1.8 Insecticidal bioassay 57 3.1.9 Antileismanial bioassay 58 3.2 Invivo studies 59 3.2.1 Acute oral toxicity 59 3.2.2 Sub chronic studies 60

3.2.2.1 Effects on body weight 60 3.2.2.2 Effects on hematologic profile 61 3.2.2.3 Effects on Coagulation 62 3.2.2.4 Effects on glucose metabolism 63 3.2.2.5 Effects on lipid profile 64 3.2.2.6 Effects on liver function 65 3.2.2.7 Effects on renal function 66 3.2.2.8 Effects on electrolytes 67 3.3 Pharmacological models 68 3.3.1 Anti-inflammatory activity 68 3.3.1.1 Carrageenan induced rat paw model 69 3.3.2 Analgesic activity 70 3.3.2.1 Hot plate method 70 3.3.2.2 Tail flick method 71 3.3.3 Hyperlipidemic model ( High fat high sµgar model) 72 3.3.3.1 Antihyperlipidemic activity 72 3.3.3.2 Cardiac risk parameters 74 3.3.3.3 Anti-diabetic profile 78 3.3.3.4 Hepatic profile 80 3.3.4 Diabetic models 81 3.3.4.1 Normoglycemic model 81 3.3.4.2 Glucose induced hyperglycemic model 83 3.3.4.3 Alloxan induced diabetic model 85 3.4 Histopathological examinations 87 3.4.1 Heart 87 3.4.2 Kidney 91 3.4.3 Liver 95 4. DISCUSSION 100 5. CONCLUSION 120 6. REFERENCES 121

ACKNOWLEDGEMENTS

Almighty Allah is the one whose likehood towards knowledge and mankind compelled me to work for the safety and betterment of mankind. The increased use of herbal medicine in our population without authentic evidence made me to go for this piece of work. This thesis constitute of thorough screening of Vernonia anthelmintica seeds on pharmacological and toxicological grounds. It is my honor that I got the chance to express my greatest depth of gratitude to my research supervisor, Dr. Rafeeq Alam Khan, Professor Meritorious, Chairperson Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical sciences, University of Karachi, for his selfless support, thorough guidance and sincere advices. His new insights and valued experience helped me at every step of my work starting from selection of topic till the submission of thesis. I would like to express my appreciation to Prof. Dr. Rahela Najm, Professor, Department of Pharmacology for teaching us Ph.D. courses and all other cooperations she offered to me. No doubt she is always an inspiration to me. I am also thankful to Prof. Dr. Iqbal Azhar, Dean, Faculty of Pharmacy and pharmaceutical sciences, University of Karachi, Prof. Dr. Talat Mirza, Head of Department, Pathology, Dow University of health science and Prof. Dr. Iqbal Choudry, Head of H.E.J department for facilitating me in their capacities. I really want to mention thanks from the depth of my soul to my father, Mr. Jamil Akhter and mother Mrs. Riaz Fatima (late). Also wanted to thank Mrs. Laila Zaidi and Mr. Sabir Zaidi, who kept me motivated whenever I felt exhausted during my work. I also want to express my love to my children. My special thanks to Mr. Shadab Ahmed, lecturer, department of Pharmacology for his continued support and being a kind research fellow of mine. Sincere thanks are due for the technical staff specially Mr. Muhammad Kashif Ali, animal technician and Mr. Muhammad Ibrahim, computer technician Department of Pharmacology for the successful completion of my work. My heartily thanks to my friends and for all those who helped and motivated me in one or the other way and supported me whenever needed. Subia Jamil

LIST OF TABLES

Table-1 Cancer cell lines and their specifications 27 Table-2 Qualitative Phytochemical analysis of HEVA, EEVA AND WDVA 48 Table-3 Antibacterial bioassay of HEVA 49 Table-4 Antibacterial bioassay of EEVA 49 Table-5 MTT bioassay of HEVA and EEVA against 3T3 cell lines 50 Table-6 MTT bioassay of HEVA and EEVA against Hela cell lines 50 Table-7 MTT bioassay of HEVA and EEVA against PC3 cell lines 51 Table-8 MTT bioassay of HEVA and EEVA against MDA-MB-231cell lines 51 Table-9 MTT bioassay of HEVA and EEVA againstMCF-7 cell lines 51 Table-10 Brine shrimp lethality assay of HEVA 52 Table-11 Brine shrimp lethality assay of EEVA 52 Table-12 Antioxidant bioassay of HEVA and EEVA 53 Table-13 Antiglycation assay of HEVA and EEVA 55 Table-14 Phytotoxic bioassay of HEVA 56 Table-15 Phytotoxic bioassay of EEVA 56 Table-16 Insecticidal bioassay of HEVA 57 Table-17 Insecticidal bioassay of EEVA 57 Table-18 Leishmanicidal bioassay of HEVA and EEVA 58 Table-19 Oral acute toxicity of HEVA,EEVA and WDVA 59 Table-20 Effects of HEVA,EEVA and WDVA on body weight 60 Table-21 Effects of Vernonia anthelmintica on haematological profile 61 Table-22 Effects of Vernonia anthelmintica on coagulation 62 Table-23 Effects of Vernonia anthelmintica on diabetes marker 63 Table-24 Effects of Vernonia anthelmintica on lipid profile 64 Table-25 Effects of Vernonia anthelmintica on liver function 65 Table-26 Effects of Vernonia anthelmintica on renal function 66 Table-27 Effects of Vernonia anthelmintica on electrolytes 67 Table-28 Anti-inflammatory activity of Vernonia anthelmintica in rat paw 69 Table-29 Analgesic activity of Vernonia anthelmintica by hot plate method 70

Table-30 Analgesic activity of Vernonia anthelmintica by tail flick method 71 Table-31 Effects of Vernonia anthelmintica on hyperlipidemia in HFHS rat model 73 Effects of Vernonia anthelmintica on cardiac risk parameters in HFHS rat Table-32 75 model Effects of Vernonia anthelmintica on fasting blood glucose in HFHS rat Table-33 78 model Table-34 Effects of Vernonia anthelmintica on liver function in HFHS rat model 80 Effects of Vernonia anthelmintica on blood glucose levels of Table-35 82 normoglycemic rat Effects of Vernonia anthelmintica on blood glucose levels of Table-36 84 hyperglycemic rat Effects of Vernonia anthelmintica on blood glucose levels of alloxan Table-37 86 induced diabetic rat

LIST OF FIGURES pg Figure- 1 Vernonia anthelmintica plant grown at botanical garden Department 3 of Botany, University of Karachi. Figure-2 Vernonia anthelmintica seeds. 3 Figure-3 Percentage radical scavenging activity of ethanol extract of Vernonia 54 anthelmintica seed. Figure-4 Atherogenic index of HFHS rat model treated with extracts of 76 Vernonia anthelmintica. Figure-5 Cholesterol /HDL ratio of HFHS rat model treated with extracts of 76 Vernonia anthelmintica. Figure-6 LDL/HDL ratio of HFHS rat model treated with extracts of Vernonia 77 anthelmintica. Figure-8 Fasting blood glucose levels in HFHS rat model treated with extracts 79 of Vernonia anthelmintica. Figure-9 Cardiac tissue of control rabbit showing normal heart 87

Figure-10 Normal cardiac tissue of rabbit treated with 150 mg/Kg of HEVA 88 over 60 days Figure-11 Normal cardiac tissue of rabbit treated with 300 mg/kg of HEVA 88 over 60 days Figure-12 Normal cardiac tissue of rabbit treated with 150 mg/kg of EEVA over 89 60 days Figure-13 Normal cardiac tissue of rabbit treated with 300 mg/kg of EEVA over 89 60 days Figure-14 Normal cardiac tissue of rabbit treated with 150 mg/kg of WDVA 90 over 60 days Figure-15 Normal cardiac tissue of rabbit treated with 300 mg/kg of WDVA 90 over 60 days Figure-16 Renal tissue of control rabbit showing normal kidney 91

III

Figure-17 Normal renal tissue of rabbit treated with 150 mg/kg of HEVA over 92 60 day period Figure-18 Normal renal tissue of rabbit treated with 300 mg/kg of HEVA over 92 60 day period Figure-19 Normal renal tissue of rabbit treated with 150 mg/kg of EEVAover 93 60 day period Figure-20 Normal renal tissue of rabbit treated with 300 mg/kg of EEVA over 93 60 day period Figure-21 Normal renal tissue of rabbit treated with 150 mg/kg of WDVA over 94 60 day period. Figure-22 Normal renal tissue of rabbit treated with 300 mg/kg of WDVA over 94 60 day period Figure-23 Liver tissue of control rabbit. 96 Figure-24 Hepatic tissue of rabbit treated with 150 mg/kg of HEVA over 60 day 96 period showing mild signs of inflammation. Figure-25 Hepatic tissue of rabbit treated with 300 mg/kg of HEVA over 60 day 97 period showing mild to moderate signs of inflammation. Figure-26 Normal hepatic tissue of rabbit treated with 150 mg/kg of EEVA over 97 60 day period Figure-27 Hepatic tissue of rabbit treated with 300 mg/kg of EEVA over 60 day 98 period showing mild to moderate signs of steatosis. Figure-28 Normal hepatic tissue of rabbit treated with 150 mg/kg of WDVA 98 over 60 day period. Figure-29 Hepatic tissue of rabbit treated with 300 mg/kg of WDVA over 60 99 day period with mild to moderate signs of inflammation

LIST OF ABBREVIATIONS

A.I Atherogenic Index AGE Advanced glycation end products ALP Alanine phosphatase ALT Alaninie transaminase ANOVA Analysis of variance aPTT Activated partial thromboplastin time AST Aspartate transaminase ATCC American type culture collection ATP Adenosine triphosphate BR Bilirubin BRD Bilirubin direct BRT Bilirubin total CBC Complete blood count CHD Chronic heart disease CHOD-PAP Cholesterol oxidase/p-aminophenazone CHOL Cholesterol CLSI Clinical and laboratory standard institute DM Diabetes mellitus DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPPH 2, 2 diphenyl-1-picrylhydrazil DSA diazotized sulphanilic acid EDTA Ethylenediaminetetraacetic acid EEVA Ethanol extract of Vernonia anthelmintica ELISA Enzyme-linked immunosorbent assay FB Fibrinogen FBS Fetal bovine serum FCS Fetal Calf Serum GADPH Glyceraldehyde-3-phosphate dehydrogenase

GGT Gamma glutamyl transferase GI Gastrointestinal GLUT-4 Glucose transporter GPO-PAP Glycerophosphate oxidase-phenol aminophenazone Hb Hemoglobin HBA1c Hemoglobin A1c (glycated hemoglobin) HDL High density lipoprotein HEVA Hexane extract of Vernonia anthelmintica HFHS High fat high sugar IC50 Inhibitory concentration 50 IFCC international federation of clinical chemistry LCF Lipid clearing factor LD50 Lethal dose 50 LDL Low density lipoprotein LFT Liver function test MABA Micro-alamar blue assay MCH Mean corpuscular hemoglobin MCHC Mean corpuscular hemoglobin concentration MCV Mean corpuscular volume MIC Minimum inhibitory concentration MTT (3-(4, 5-dimethylthiazol-yl)-2,5-diphenyl tetrazolium bromide) NADH Nicotinamide adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate hydrogen NaN3 Sodium nitrite NF-Kb Nuclear factor kappa B O.D Optical density OGTT Oral glucose tolerance test PCV Packed cell volume PKC Protein kinase-C PLTs Platelets PT Prothrombin time

PTP-1B protein tyrosine phosphatase- 1B RBCs Red blood cells RFT Renal function test RNA Ribonucleic acid RNS Reactive nitrogen specie ROS Reactive oxygen species RSA Radical scavenging activity SD Standard deviation SEM Standard error to the mean SPSS Statistical package for social sciences STAT signal transducer and activator of transcription STZ Streptozocin TGs Triglycerides TNF-α Tumor necrosis factor- α TT Thrombin time VLDL Very low density lipoprotein WBCs White blood cells WDVA Water decoction of Vernonia anthelmintica

VII

SUMMARY

Importance of herbal medicine cannot be denied even in the modern era. Various active allopathic medicine came into being from plant origin. Vernonia anthelmintica is one such herbal remedy that has been used in traditional system of medicine. Many parts of this plant are being used by humans for curing various illnesses. Since the use of seeds of Vernonia anthelmintica is increasing in traditional practice it is the need of time to establish its safety and efficacy by thorough screening in terms of toxicity as well as pharmacological activity. Though few studies authenticate some pharmacological actions of this herb however, the safety profile is lacking even in these studies.

Present study is thus designed to evaluate the toxicological and pharmacological potential through in-vitro and in-vivo methods. In vitro experiments include phytochemistry of hexane, ethanol extract and water decoction of Vernonia anthelmintica seeds. Moreover bio-assays to evaluate antibacterial, cytotoxic, antioxidant, antiglycation, phytotoxic, insecticidal and antileishmanial potential of hexane and ethanol extracts were also undertaken. In-vivo methods include subchronic toxicity studies conducted on rabbits to elucidate its effects on hematology, coagulation, hepatic function, renal profile, electrolyte status and metabolism of lipids and glucose. Pharmacological disease models were also designed in Sprague drawly rat and albino mice to establish its role in hyperlipidemia, diabetes, inflammation and nociception.

The results revealed cytotoxic potential against selected cancer cell line, strong dose dependent antioxidant and phytotoxic potential and moderate antileishmanial potential in selected extracts. Considerable antiglycation potential was also observed in ethanol extract. Present study also revealed the safety of Vernonia anthelmintica on subchronic use. In vivo results showed no deleterious alteration in hematology, renal function, liver function and lipid/ glucose metabolism as compared to normal control. Subchronic use of Vernonia anthelmintica showed favorable coagulation profile by lowering fibrinogen levels. Furthermore Vernonia anthelmintica showed favorable effects in disease model of hyperlipidemia, diabetes, inflammation and nociception. Subchronic dosing also showed weight reducing potential of Vernonia anthelmintica.

Present results thus suggest the possible role of Vernonia anthelmintica in the safe treatment of obesity, diabetes, hyperlipidemia, inflammation, nociception, cancer and leishmaniasis. Furthermore its strong antioxidant potential can be used in preparing remedies to combat various diseases attributable to oxidative stress.

1. INTRODUCTION

Herbs are the sources of medicament right from the beginning of human civilization. As far as human health is concerned herbs proved to be nature’s gift to spend healthy and disease free life. Traditional medicine still holds a firm place even in the era of modern medicine.

According to statistics about 70 % of the population still depends on alternative medicine and people practiced these medicines as a first choice for chronic diseases like epilepsy, infertility, diabetes and psychological illnesses, more concisely in rural and tribal areas due to their easy availability, feasibility, cultural and spiritual beliefs. Integration of this alternative medicine with modern medicine generates the evidence based medicine (Shaikh and Hatcher, 2005).

The rapid progress of modern medicine failed to decline the fame of traditional medicine rather it was recognized worldwide as various proved to be highly efficacious. It is however worth mentioning that plant drugs used in traditional medicine needs detailed and thorough scientific studies to stay safe from toxicities (Shah et al., 2009).

A general perception about the herbal medicine is that they are low risk medicaments but published data contraindicate this myth as the use of herbal medicine is also associated with potential harm due to their intrinsic toxicities as well as because of impurities, adulteration, misidentification and effective interaction with other drugs. It is therefore the need of time to assess the safety and elucidate the toxicities of herbal medicine on scientific grounds (Jordan et al., 2010).

Herbal medicines are also called as phytomedicine or botanical medicine. It refers to utilization of plant seeds, berries, roots, leaves, bark or flower for treating diseases. Previous records has been pointed out that there are 8000 species of well- defined medicinal values in South Asia and are considered a vital part of conventional health care systems. Over 80 percent of Asian population is relying on these inexpensive and effective traditional medicines and are used against many diseases and infections. Even though in Pakistan, 2000 medicinal plant species are reported but, very few of them are being exploited (Aslam, 2002).

1.1 Description of Vernonia anthelmintica

Vernonia anthelmintica belongs to Astraceae (compositeae) family. Most frequently used synonym is Centratherum anthelminticum. Other synonyms are Baccharoides anthelmintica (L.) Moench, Vernonia anthelmintica (L.) Willd., Conyza anthelmintica L., Vernonia stenolepis Oliv., Dolosanthus sylvaticus Klatt (Bewley et al., 2006). Other names are Ascardia indica, Conyza ascardia, serratula anthelmintica (Nadkarni, 1996; Mashelkar 2008).

Vernonia anthelminticum commonly known as kalijiri is an important plant of subcontinent. It hasvalid role in alternative medicine for the treatment of differing illnesses. The seeds has prominent role as anthelmintic agent (Kirtikar and Basu, 1999).

The genus Vernonia has around one thousand species. The members of the genus are extensively used in medicine and food. Literature reported about 109 Vernonia species of medicinal importance. Vernonia anthelmintica is known for curing asthma, hiccough, inflammatory edema, sores, and itching of the eyes. Vernonia anthelmintica has also shown to have antidiabetic, anti-inflammatory and anticancer potential in various studies. Thus further studies are needed to elucidate its medicinal potential (Srivastava, 2014).

1.2 Plant Taxonomy

Vernonia anthelmintica is known to be an annual, upright plant that is usually 1.5 metres tall with pubescent and branched stem. Leaves are 5-9 cm in length and 2.5- 3.2 cm in width. Leaves are either lanceolate or elliptically lanceolate, crudely serrate and pubescent from both sides. Leaves are also found to be petiolate and tapered at the base. Heads of plant are 1.3-2 cm in diameter, subcorymbose having almost 40 flowers and linear bract near the top of peduncle. Achenes are about 4.5- 6 mm in length with oblong cylindrical shape (Mashelkar, 2008).

According to taxonomic classification Vernonia anthelmintica belongs to kingdom: plantae; division angiosperms; class; Eudicots, subclass: Asterids; order: Asterales; Family: Asteraceae; subfamily: Cichorioideae; tribe: Vernonieae; Genus: Vernonia

and species name is Vernonia Anthelmintica (Manvar and Desai, 2012). Vernonia anthelmintica is known with different names in different languages e.g. in Sanskrit, the most commonly used name is Somaraja, in hindi : kalijhiri; in Gujurati: Kalijiri; in English: purple fleabane; in Urdu it is called as janglijiri or kalijiri (Mashelkar, 2008).

Figure 2: Vernonia anthelmintica plant grown at botanical garden Department of Botany, University of Karachi

Figure 3: Vernonia anthelmintica seeds

1.3 Importance of Extraction

Selection of appropriate extraction method is the first most important step required for the elucidation of further separation and characterization. It lays an important as well as critical impact on the ﬁnal consequences and findings(Sasidharan et al., 2011). Depending upon the polar or non-polar nature of the extract, various solvents are used to extract the bioactive compounds from herbal products. When hydrophilic components are the targets, the usual solvents are methanol, ethanol or ethyl acetate whereas lipophilic components requires either dichloro-methane or its combination with methanol (1:1). However in some instances hexane is used in order to eliminate chlorophyll (Cos et al., 2006).

1.4 Herbal derived bioactive compounds

All compounds of natural systems can be broadly classified into primary and secondary metabolites. Primary metabolites are simple chemical substances such as sugars, amino acids, proteins and lipids and involved in basic growth and development of plant. However secondary metabolites are compounds involved in the survival of plant (Harborne, 1993). Amongst these secondary metabolites, some substances have great impact on biological systems and thus are termed as bioactive. Bioactive compounds referred to the compounds generated by plants and possess pharmacological and toxicological effects in human and animal systems. However nutrients in plants are not actually termed as bioactive compounds. Bioactive compounds in plants thus can be referred as secondary plant metabolites capable of producing pharmacological and toxicological effects in human and animal systems. Medicinal herbs are reported to contain higher concentration of potent bioactive compounds than plants consumed as food (Bernhoft et al., 2010).

On the basis of biosynthetic pathways bioactive compounds can be classified as i) terpenes and terpenoids, (ii) alkaloids and (iii) phenolic compounds. Most of the bioactive compounds belongs to one of these classes however each have specific structure and properties depending on the biosynthetic pathway they have taken(Croteau et al., 2000). Four known biosynthetic pathways of secondary metabolites

are i) shikimic pathway, ii)malonic pathway, iii) mevalonic pathway, iv) non- mevalonate pathway (Taiz and Zeiger, 2006).

Alkaloids are formed through shikimic acid pathway and tricarboxylic acid pathway by aromatic and aliphatic amino acids respectively. Phenolic compounds take shikmic and malonic acid pathway for their biosynthesis. Mevalonic and non- mevalonic acid pathways are used by terpenes to be biosynthesized (Azmir et al., 2013).

1.5 Components of Vernonia anthelmintica seeds

Phenols constitute a very vast group of bioactive plant metabolites. They are known to have intense importance due to their bioactive potential. Extraction methods play a key role in extraction of bioactive components. Single solvent failed to recover all phenolic components from herb, thus combination of solvent is usually applied in order to get optimum recovery of phenols. However alcoholic solvents have advantage that in dried samples they can penetrate into the cell membranes and enhance the extraction of intercellular material. Ani. V reported that methanol acetone extracts showed highest whereas aqueous extracts showed lowest phenolic components (Ani and Naidu, 2011).

The most studied phenyl propanoid derived components are termed as flavonoids. Flavonoids are under thorough research due to their extensive beneficial effects on human systems (Dixon and Pasinetti, 2010). Previous literature provides clear indications of flavonoids as free radical scavenger, antioxidant, coronary heart disease healer and anti-proliferative agent in various carcinomas. Certain reports also claimed flavonoids to have anti-human immunodeficiency virus activity. Thus increasing interest has been established to explore new flavonoids from dietary and herbal sources (Yao et al., 2004).

Flavonoids are also known for their antibacterial, antiviral and antiprotozoan potential with the added advantage that it is not severely deleterious to animal cells. Even modern medicine is taking use of flavonoids to treat various diseases because of their reported activity in inhibiting certain enzymes, stimulating

neurotransmitters and hormones and in scavenging free radicals(Havsteen, 2002). Seeds of Vernonia anthelmintica have reported to have flavonoids namely 2′,3,4,4′‐ tetrahydroxychalcone, butin and 5,6,7,4′‐tetrahydroxyflavone (Tian, et al. 2004).

Alkaloids are naturally occurring chemical constituents that are mostly nitrogenous basic atoms. Important alkaloids present in Vernonia anthelmintica seeds are elemanolide dimers namely vernodalidimers A, B and vernonine(Liu, et al. 2010). However seed oil of Vernonia anthelmintica constitute of trivernolin as a major component and 1,3‐divernolin and vernolic acid as minor constituents (Krewson and Luddy, 1964). Vernonia anthelmintica seeds contains various bioactive compounds that accounts for their pharmacological activities. These are epoxy vernolic acids, essential oils, steroids, glycosides, phenolics, flavonoids ad triterpenes (Toyang and Verpoorte, 2013, Ratnam et al., 2014). Ethanol extract of Vernonia anthelmintica seeds on phytochemical evaluation were reported to contain alkaloids, flavonoids, steroids, triterpenes and polyphenols (Otari et al., 2010).

Other components such as palmitic, oleic, myristic, stearic, monohydroxy-oleic, linoleic, vernolic acids and certain resins have been reported in previous studies (Chopra, 1933).

Foods and herbs containing stanol or sterol esters are reported to decrease serum cholesterol levels. Kata and colleagues claimed in a meta-analysis that 2g per day intake of stanols or sterols may reduce the LDL i.e. low density lipoproteins and these effects are additive with food and antihyperlipidemic medications. In the similar study they reported that adding sterols to statin is more beneficial as compared to increasing statin. There exist some concerns about increase in phytosterol levels in blood however, reported evidence is enough to facilitate the use of stanols and sterols for lowering serum LDL cholesterol levels especially in the individuals with high risks and predispositions to atherosclerosis and coronary heart disease. (Katan et al., 2003). Vernonia anthelmintica is known to possess few sterols namely sterols-4α-methyl vernosterol (Akihisa et al., 1992), (24a/S)-

stigmasta-5,22-dien-3β-ol and (24a/S)-stigmasta-7,22-dien-3β-ol (Mehta et al., 2005).

1.6 Toxicological screening

Herbal medicine are usually assumed safe but actually it may hold potential toxicity. The basis of this toxicity may be its misidentification, incorrect preparation or administration (Fennell et al., 2004). Plants under research should be examined for their toxicological profile as the toxins content may vary significantly among the various parts of plants. Even there are genetically determined differences in toxin content of same species cultivated at different places.

Various environmental and growth factors determine these variations such as soil, climate etc. it is also worth mentioning that apart from intrinsic toxins plant can carry deleterious compounds from external sources. Pharmacological as well as toxicological screening is the vital part of drug development (Ahmad et al., 2004).

Herbal medicine in the form of extract, pure compound or some of its derivative is a source of new drug discovery. Most of the natural products that are used in traditional medicine holds valid and strong scientific evidence with reference to their pharmacological activities. However lacking stays in the area of the toxicities to their consumers (Dias and Takahashi, 1994). Though because of the long term use of herbal medicine for some of its pharmacological indication it is anticipated that it is safe with low toxic potential, but various researches has indicated the adverse effects of herbal medicine in traditional use (Ertekin et al., 2005).

Therefore it is clear that no matter a plant has been in traditional use since year would not guarantee its safety in a long run. Thus data of acute and chronic toxicity should be established before its evidence based use in humans and animals. Hence the assessment of acute and chronic toxicity is the foremost step for drug development. Regardless of the extensive known pharmacological benefits of Vernonia anthelmintica, the detailed studies on the chronic use of this herbal medicine is lacking in the scientific literature.

Current study has undertaken the acute and sub-chronic toxicity studies of Vernonia anthelmintica in order to establish the safe use of drug. The effects of different extracts are being studied after chronic dosing and effects on hematology, liver, kidney and metabolism were evaluated. Histopathological analysis was also conducted to authenticate the biochemical changes in the specific organs.

Biochemical analysis holds an important role in definite diagnosis as well as in the assessment of risks involved in starting therapy in order to improve clinical outcomes. Biomarkers refer to a characteristic that is measured objectively and serves as an indicator of biological, pathological or pharmacological response to a therapeutic intervention (Vasan, 2006).

1.6.1 Hematology and Coagulation

Blood is an excellent pathological predictor that indicates the status of the exposed animals to a particular drug or toxin. Hematology gives the clear picture of number and morphology of blood cells namely erythrocytes, leucocytes and thrombocytes and hence serves as important markers for the diagnosis as well as monitoring of certain illness(Etim et al., 2014). Blood is a vital circulatory tissue which is constituted of plasma and suspended cells and plays a key role in normal physiology and homeostasis (Isaac et al., 2013).

Basic hematological screening includes red blood cells, white blood cells, MCV(mean corpuscular volume), MCH (mean corpuscular hemoglobin) and MCHC (mean corpuscular hemoglobin concentration), platelet count, and leucocyte count (Oyawoye and Ogunkunle, 2004). Red blood cells holds hemoglobin that transports oxygen. Thus the main function of RBCs is the transport of oxygen and carbon dioxide in the body. Thus reduced levels of erythrocytes indicates the hypoxic condition of tissues (Isaac, et al. 2013)

White blood cells serves as a body’s self-defense system that is involved to combat with infection and defend the host when foreign pathogens attacks the body by means of phagocytosis, antibody generation, transport and distribution within immune system Thus, decreased levels of white blood cells predispose animals and

humans to infections. While increased levels showed efficient antibody production in the process of phagocytosis and thus high resistance against infectious diseases (Soetan et al., 2013).

Platelets serves in clotting of blood. Decreased platelets in blood is the indication of compromised clotting system and patient with such condition is prone to internal and external bleeding tendency in case of injury (Purves et al., 2003).

Packed cell volume (PCV) also referred to as hematocrit is the percentage of RBCs in blood and as RBCs are involved in transportation increased PCV is a sign of efficient transport function (Isaac, et al. 2013)

Previous studies clearly indicate the importance of these hematological indices in the diagnosis of various diseases specially anemia. Packed cell volume, hemoglobin and mean corpuscular hemoglobin are major parameters to evaluate the status of circulatory erythrocytes and ability of bone marrow to produce RBCs in mammals (Peters et al., 2011). However increased PCV indicates either increased RBCs or decreased plasma volume. Decreased MCHC and MCH levels are the indication of anemia (Chineke et al., 2006).

The effects of Vernonia anthelmintica on hematology on sub-chronic dosing is lacking in the scientific literature. Hence present study has undertaken to evaluate the effects of different extracts of Vernonia anthelmintica on hematology in order to establish its safety on hematological system in rabbits.

Hemostasis is a well-organized natural physiological system responsible for maintaining the integrity of a vessel (Hook and Abrams, 2012). Though platelet mediated plug formation is the first step following endovascular injury (Levi, 2005) However platelet are not the sole players for maintaining hemostasis as it requires various coagulation factors that generate fibrin meshwork by activating coagulation cascade. This fibrin meshwork plays important part in aggregation of platelets and stabilization of blood clot (Furie, 2008).

Hyper-coagulation increases the risk of thrombotic events. The resulted thromboembolism may hinder the blood flow to the affected organs progressing to

ischemia and eventually cell death (Bruno et al., 2001). Coagulation is reported to be associated with the cardiovascular and cerebral diseases including thrombotic events, stroke and myocardial infarcts (Eisenreich and Rauch, 2011).

Bioactive compounds derived from herbal drugs are reported to effect coagulation by interfering with platelet aggregation and coagulation cascade (Cordier and Steenkamp, 2012). No previous study has been implicated to evaluate the effect of Vernonia anthelmintica on coagulation and hemostasis. Thus present study has undertaken the effect of different extracts of Vernonia anthelmintica on coagulation.

1.6.2 Hepatic Profile

Liver function test constitute the set of biochemical parameters that explain the status of liver. LFT includes liver enzymes namely AST, ALT, GGT and ALP. Bilirubin, albumin and coagulation studies aids in the diagnosis of liver damage. Serum aminotransferases are found to be increased in case of cytolytic damage due to certain drugs or other etiologies. Increase in ALP along with GGT is clear indication of cholestasis of hepatic origin.

Albumin and prothrombin elevation indicates functional compromise of liver. Increase in different fractions of bilirubin (conjugated and unconjugated) also gives a good means to differentiate hepatic and extra hepatic disease (Moreno et al., 2007).

The effect of Vernonia anthelmintica on liver is lacking in the previous literature thus present study is designed to assess the effects of different extracts of Vernonia anthelmintica on liver after sub chronic use in rabbits.

1.6.3 Renal profile

Kidney is a vital organ that is responsible for excretion of metabolic wastes and regulation of concentration of varying substances in serum. Renal function parameters are meant to diagnose the renal pathologies and their severities. Most frequently used biomarkers that serve for the assessment of renal function in

routine analysis are urea , uric acid, creatinine and electrolytes (Gowda et al., 2010).

Urea is a chief nitrogenous product formed by the catabolism of protein and amino acids. In kidneys it is filtered through glomeruli and part of it is reabsorbed with water (Banks, 2013).Though urea clearance is not the good indicator of glomerular filtration as its production depends on other non-renal factors but it is useful in differentiating acute renal failure with chronic one as blood urea nitrogen ration is increased in the later (Rosner and Bolton, 2006).

Uric acid is metabolic product of purines that are supposed to be the building blocks of cellular energy stores e.g. ATP, DNA and RNA. Uricase is the liver enzyme present in most of the mammals that convert urate into Allantoin and hence the uric acid levels are found to be reduced in them. However human uricase enzyme is not functional leading to high and more fluctuating levels of serum uric acid (Wu et al.,

1992).

Uric acid is not an inert biochemical parameter rather it have both wanted and unwanted effects. Uric acid has a benefit of being acting as antioxidant and serving positively in certain conditions of oxidative stress where as it can also stimulate proliferation of vascular smooth muscle and causes endothelial dysfunction. Thus on a darker side it has proved to have detrimental role and may cause development of hypertension, renal disease and cardiovascular disease (Johnson et al., 2003)

Creatinine is generated by the breakdown of creatine phosphate in muscles. Its production depends on muscle mass (Zuo et al., 2008). It is an excellent biomarker for assessing kidney function. Serum creatinine is also used to calculate glomerular filtration rate (Greg Miller et al., 2005). Increased serum creatinine level is the indicator of renal damage. In case of chronic renal failure as well as in uremia ultimate decrease in creatinine excretion from both tubules and glomeruli (Burtis et al., 2012). It was also reported that creatinine levels may vary depending upon extent of muscle function, muscle composition, diet composition and health condition (Banfi and Del Fabbro, 2006) . Apart from renal damage the elevated levels may be due to paralysis, anemia, hyperthyroidism and leukemia whereas

decreased levels are also seen in congestive heart failure, glomerulonephritis, shock, acute tubular necrosis, polycystic kidney and dehydration (Burtis et al., 2012).

Electrolyte analysis is frequently used to assess acid base imbalance. It is also used to monitor the effects of treatment that how it is affecting bodily organ function. This analysis may include estimation of sodium, potassium, chloride and bicarbonates for diagnosis and treatment of endocrine, renal, acid base, water balance and many other situations (James and Mitchel, 2006).

Serum total proteins are referred to two basic protein types namely albumin and globulin. Both of these have different roles in homeostasis. Albumin is known for maintenance of osmotic pressure and prevention of fluid from leaking into interstitium whereas globulins serve as building blocks of immune system. The levels of total protein depends on protein intake, its synthesis in liver and its removal by kidneys. Hence high levels may be generally due to inflammation, infection and increased dietary intake whereas low levels indicate liver disease, renal disease, malnutrition or malabsorption (Busher, 1990).

No scientific data till date authenticate the effects of Vernonia anthelmintica on renal profile hence present study is designed to estimate the safety of this herbal medicine on renal profile.

1.7 Pharmacological screening 1.7.1 Antibacterial Herbal medicine has shown promising role in the management of varying microbial infections because of the sustained adverse drug effects of synthetic antimicrobials. Thus exploration of safe, effective and economical medicinal agents of plant origin is the need of time in order to serve as alternatives for treating resistant microbial pathogens (Alam and Qureshi, 2010) reported the antimicrobial potential of Vernonia anthelmintica against gram positive as well as gram negative pathogens with the MIC of <1mg- <10 mg by disc diffusion method. Whereas only one fungal strain T.rubrum was found to be sensitive to Vernonia anthelmintica with MIC<50 mg. According to a previous study , the ethyl acetate extract of Vernonia

anthelmintica displayed strong and concentration dependent activity against the gram negative bacteria Klebsiella pneumonia and Ecoli whereas ethanol extract exhibited weak activity against all the test pathogens at 250 and 500 µg/disc (Ratnam et al., 2014).However different fractions of Vernonia anthelmintica seeds showed no antibacterial potential against Ecoli, Bsubtillis, s. Flexenari, S. aureus and P. aeruginosa at the tested concentration of 3 ug/ml (Syed, 2012).

1.7.2 Cytotoxicity

Current therapeutic approaches are addressing the role of herbs and spices in the detection of potent cytotoxic agents in order develop effective anticancer agents(Lai and Roy, 2004). Acute toxicity test showed no signs of mortality on oral administration of ethanol extract of Vernonia anthelmintica up to a dose of 5000 m/kg (Otari et al., 2010) Previous comparative studies on various herbal crude drugs reported that Vernonia anthelmintica possessed cytotoxic potential with LC50 value of 1.20ug/ml in brine shrimp lethality test (Kumar et al., 2009). Brine shrimp lethality test of acetone fraction of Vernonia anthelmitica seeds revealed no significant cytotoxicity whereas methanol and aqueous methanol fractions showed cytotoxic potential at very high doses (Syed, 2012).

MTT assay is a rapid and précised method used for the detection of living cells and the generation of signal proportionate the extent of activation of cell. This in-vitro quantitative assay is an efficient tool for detection of cytotoxic potential of drugs (Mosmann, 1983). Previous literature revealed experimentation of selected fractions of Vernonia anthelmintica on MTT assay. The chloroform fraction when tested for cytotoxicity by MTT assay revealed the activity against A-549,PC-3, MCF-7 and WRL-68 cell lines with IC50 values 31.42 ± 5.4, 22.61 ± 1.7, 8.1±0.9, and 54.93±8.3 µg/ml respectively. The proposed mechanism for this action is dose dependent inhibition of TNF- α (Arya et al., 2012a); Manvar and Desai 2012) in a literature view also reported that chloroform and acetone fractions that are devoid of fats showed anticancer activity using MTT assay against 5 tested cancer cell lines namely human cell A-375, mice melanoma cell B-16, breast cancer cells BCF-7, gastric cancer BGC-823 and hepatic carcinoma cells

HepG-2 of human origin (Manvar and Desai, 2012;Deng and Zhou, 2001). Antiproliferative activity against HL-60 cells and A-375 cells of dimers isolated from Vernonia anthelmintica was also reported (Liu et al., 2010b).

Literature revealed the anti-cancer potential of vernodalidimers isolated from Vernonia anthelmintica, Vernoanthelcin A-1 is reported to have effects on estrogen synthesis in ovarian granulosa cells of human origin (Srivastava, 2014).

1.7.3 Antioxidant

Antioxidants are needed by human body to nullify the effects of reactive oxygen species (ROS) in times of inflammation due to activation of immune cells. Patients with ongoing inflammation present with low levels of antioxidants either due to less intake or high consumption in situations of overwhelming ROS generation by activated immune cells. Thus antioxidants are known to impede the disease related oxidative stress (Mangge et al., 2014).

It is therefore logical to support the patient with exogenous antioxidants in situations where endogenous antioxidants are overwhelmed. Thus the need of finding new antioxidants from dietary and herbal origin is justified in order to prevent from chronic illnesses like cancer, cardiac heart disease and varying neurodegenerative diseases. In certain concentration free radicals are needed by the body as a part of human host defense mechanism. ROS (reactive oxygen species) and RNS (reactive nitrogen species) for example are two free radicals required by human body for the integrity and maturation of cell and cellular structures. The human defense system for example phagocytes; neutrophils and monocytes etc., all need free radicals in sufficient amount to eradicate the invading pathogen (Dröge, 2002).

It can be summarized that these free radicals at certain concentration are vital to normal human physiology However when free radicals and oxidants generated in excess they may cause a condition termed as oxidative stress. Oxidative stress refer to the condition of imbalance between generation and neutralization of free radicals and oxidants and may lead to the deleterious effects on cell membranes, proteins,

lipoproteins and even DNA. These deleterious effects on cellular structures are the key factors in the pathogenesis of varying diseases (Young and Woodside, 2001).

DPPH (2, 2 diphenyl-1-picrylhydrazil) assay is used for the evaluation of antioxidant potential of crude and raw samples. It has advantage over expensive methods as it is quick, simple and cheap in-vitro assay employed for the evaluation of compounds having ability to scavenge free radical scavenging or act as a hydrogen donor(Chen et al., 2013). Previous studies revealed the antioxidant potential of ethanol extract of Vernonia anthelmintica seeds. However further studies were recommended for other fractions and extracts of seeds (Santosh et al., 2013).

1.7.4 Inflammation

Inflammation is a general phenomenon that becomes active in the cases of infections, injuries or exposure to pathogens and contaminants that can be collectively or individually a sign of homeostatic dysfunction. Inflammation is mediated by elicitation of innate immune receptors against pathogens or injured cells and coordinated by immune signaling molecules and cytokines. Unfortunate is the condition where inflammation persists for longer times without getting its beneficial objective and hence inflammatory cells overwhelm an organ system resulting in damage to the host cells. This damage to host cells appears in the form of various chronic illnesses such as colitis, pancreatitis, ulceration, diabetes, cardiovascular diseases, arthritis and hepatitis (Ashley et al., 2012).

The Carrageenan test is a sensitive tool to elucidate the anti-inflammatory potential of oral anti-inflammatory agents. It provides an excellent acute phase inflammation model (Di Rosa, Giroud et al. 1971). Carrageenan is a mucopolysaccharide extract derived from alga chondrus crispus discovered by British pharmacist Stanford in 1862. It is made up of complex group of polysaccharides constituted of galactose related repeated monomers and are of three main types: lambda, kappa and iota. All have their own thermally reversible gelling characteristics. Lambda type is unique in the way as it does not gel at room temperature and can be used for induction of inflammatory response (Morris, 2003).

Carrageenan induced paw edema is a method used for measuring edema caused by 0.1 ml of 1% solution of carrageenan into the plantar tissues of hind paw of the rat. Within first 3-4 hours, peak edema develops. Drugs to be evaluated for anti- inflammatory potential can be analyzed by checking for inhibition of edema by pre- treatment of the animals by unit oral dose (Winter et al., 1962). Carrageenan induces acute, non-immune, well studied and easily reproducible inflammation with immediate appearance of basic signs such as edema, redness and hyperalgesia following injection. These inflammatory responses are the result of release of pro- inflammatory mediators e.g. histamine, bradykinin, tachykinins, complement and reactive nitrogen and oxygen species (Morris, 2003).

Previous studies on petroleum ether and methanol extracts of Vernonia anthelmintica revealed their anti-inflammatory potential (Ashok et al., 2010a). However data for hexane extract and water decoction of Vernonia anthelmintica is lacking in previous literature.

1.7.5 Nociception

Pain is a symptom of many disease conditions and its control is regarded as one of the most important therapeutic urgency. It is a disagreeable sensory or impassive experience that is associated with tissue injury. In spite of the fact that it is a defensive alarming signal but the discomfort it generates is often intolerable and becomes the cause of various side effects (Hasan et al., 2010).

Analgesics are the class of drugs used to combat or reduce pain. Non-steroidal anti- inflammatory drugs as well as opiates that are considered to be classical analgesics were actually originated from natural products. Though various synthetic analgesics have been developed and used to treat pain but it is worth mentioning that these agents are not devoid of serious side effects like GI bleeding , ulceration, respiratory distress, nausea and drowsiness (Mate et al., 2008).

Hence the search of new bioactive compounds derived from herbal sources having analgesic potential is the need of time. The laboratory anti-nociceptive models usually measure analgesic potential by simply measuring the reaction time to a

painful stimulus (Rang et al., 2006). Tail immersion method and hot plate model are two anti-nociceptive models used to study analgesics having central mode of action (Woolfe and MacDonald, 1944). Sensory nerves causes stimulation of nociceptors by applying thermal stimuli(Bachhav et al., 2009). Previous studies indicated the anti-nociceptive potential in methanol extracts of Vernonia anthelmintica and shown to have both central and peripheral mechanism of analgesia (Jahan et al., 2014). Petroleum ether and alcohol extracts also showed analgesic activity in animal model (Purnima et al., 2009a). Present study has undertaken three extracts; hexane, ethanol and water decoction for the elucidation of their anti-nociceptive potential.

1.7.6 Phytotoxic and insecticidal activity

Phytotoxicity refers to the toxic effect imposed by a compound on plant growth. Herbal drugs are also known to have phytotoxic potential. Lemna growth assays measures the phytotoxicity of pesticides and environmental toxins to higher plants.(Wang, 1990). Annual postharvest losses are resulting due to insect damage, microbial deterioration, and many other environmental factors such as humidity, aeration, temperature, and cleanliness of the massive storage and are expected to be 10–25% of production throughout the world. In recent years the research on the use of herbal insecticides has increased many folds. The major advantages of finding insecticides of plant origin are their comparatively less toxicity to mammalian life and compatibility with other systems of insect control (de Souza et al., 2010).

Herbal drugs and their extracts has a potential to pose as potent insecticides. According to a review claim ninety nine families, Two hundred and six genera and three hundred and forty six specie of medicinal plants possess insecticidal potential (Sukumar et al., 1991). The reported basic mechanisms of insecticidal potential of herbal extracts includes growth inhibition, anti-feedant, direct toxicity and mortality suppression of reproductive behavior and fertility (Jbilou et al., 2006). It is therefore need of time to investigate environmental friendly, biodegradable and cheap herbal insecticides (Rehman et al., 2015).

1.7.7 Antileishmanicidal activity

Lieshmaniasis is an insect transmitted infection considered as serious public health problem due to significant morbidity and mortality posed by it (Amato et al., 2008). It is caused by two protozoan parasites, trypansoma cruzi and trypansoma brucei(Cuervo et al., 2010). Reported cases claims that this parasite kills thousands and debilitate millions per year. On the basis of location and severity of infection it is classified as cutaneous lieshmaniasis i.e. localized form of long term ulceration, mucocutaneous lieshmaniasis i.e. more invasive and involves chronic involvement of mucous linings and visceral lieshmaniasis i.e. the most severe of all types as in this parasite detaches itself from inoculation site and spread in liver, spleen and bone marrow and ultimately death due to immunosuppression if left untreated (Peacock et al., 2007).

Drugs used to treat leishmaniasis unfortunately are toxic and vaccines against these infections have shown very low protection profile in field environment (Ouakad et al., 2007). Thus it is the need of time to discover and develop new and less toxic anti-infective agents against this parasite (Pitzer et al., 1998). Plant derived products have a strong potential to be tested as antileishmanial agents. Present study thus undertaken in-vitro antileishmanial bioassay to assess the leishmanicidal potential of Vernonia anthelmintica.

1.7.8 Diabetes

Diabetes mellitus is considered as one of the most progressing cause of morbidity and mortality. Worldwide epidemics claims about 2.5- 3 % of world’s population is victim of this disease furthermore in some countries this proportion has inclined to 7% or more. It is the state of chronic progressing metabolic disorder accompanied by various complications (Seghrouchni et al., 2002). Guariguata and colleagues estimated the global burden of diabetes and concluded that 382 million people had diabetes in the year 2013 and it is supposed to be increased to 592 million by 2035. They also claimed these diabetic patients belongs to low and middle socioeconomic countries (Guariguata et al., 2014).

Diabetes is a condition of sustained increase in blood glucose levels due to defects in insulin secretion, its utilization or both. Whatever would be the cause the sustained hyperglycemia may results in the malfunctioning of various body organs especially eyes, kidneys, nerves, heart and blood vessels and appears as retinopathy, nephropathy, neuropathy, cardiac complications and hypertension (Association, 2010). Muscle and adipose tissues are the sites involved in the pathogenesis of insulin resistance. As a result of increased caloric intake compared to body energy outlay, substrate induced increment in citric acid cycle takes place that generates increased mitochondrial NADH and ROS Reactive oxygen species (Maddux et al., 2001).

Apart from conventional basis of pathogenesis for diabetes and its complications the role of oxidative stress as the major cause of their sequel has been established. It has been postulated that sustained hyperglycemia and free fatty acid resulted in overproduction of superoxide, nitric oxide, PKC and NF-kB that in turn overexpress NADPH. The summation effect of all these changes may lead to generation of strong oxidant peroxynitrite and it resulted in damage of DNA. Damage of DNA leads to activation of certain nuclear enzymes that decreases the formation of GADPH and thus decrease the expression of GLUT4 in adipocytes and muscle causing insulin resistance, endothelial malfunction and decreased production and secretion of insulin in β pancreatic cells (Ceriello and Motz, 2004).

Various in-vivo and in-vitro studies stay in favor of this claim that antioxidants have a positive role in improving insulin sensitivity and available antioxidants like vitamin E, C and glutathione showed promising effects in decreasing insulin resistance (Paolisso and Giugliano, 1996).Whatever would be the pathogenesis of diabetes mellitus it usually concluded as decrease in insulin secretion/ sensitivity or both. Current oral hypoglycemic agents are not devoid of side effects (Howlett and Bailey, 1999). The exploration of antidiabetic agents from herbal medicine is thus justified.

Glycation is the fundamental molecular origin of numerous complications of diabetes e.g cardiovascular complications, nephropathy, retinopathy and

neuropathy. The major problem arises when non enzymatic reaction between carbonyl part of reducing sugars and amino groups of proteins takes place resulting in the generation of insoluble advance glycation end products. It will deposit on long lived proteins with subsequent compromised physiological functions (Odjakova et al., 2012).

One of the major reason behind the detoriation of proteins is glycation of non- enzymatic nature and it will eventually leads to different complications because of the generation of non-reversible advanced glycation end products. So herbal medicine holding antiglycation activities showed promising role in treatment of diabetes mellitus (Perera et al., 2013).

Previous studies generates the clues that seeds of Vernonia anthelmintica showed antidiabetic and anti hyperlipidemic potential in STZ induced rat model (Fatima et al., 2010). Arya and colleagues claimed that methanol fraction of Vernonia anthelmintica exhibited strong antidiabetic effects and suggested it as potential candidate for developing nutraceutical for the treatment of type 2 diabetes mellitus and complications associated with it like inflammation, oxidative stress and dyslipidemia (Arya et al., 2012b).

Published data is lacking for in-vitro antiglycation assay of Vernonia anthelmintica seed as well as non-alcoholic fraction also need to be evaluated for their antidiabetic potential. Present study thus undertaken normoglycemic as well as hyperglycemic models to elucidate the acute antidiabetic action of different extracts of Vernonia anthelmintica and antiglycation assay of HEVA and EEVA.

1.7.9 Hyperlipidemia

Hyperlipidemia or more accurately dyslipidemia is known to be the greatest threat that contributes to the occurrence and aggravation of coronary heart disease (Neil et al., 1990) The primary causes of death worldwide are CHD, stroke, hyperlipidemia and atherosclerosis (Smith et al., 1993).

Hyperlipidemia is defined as elevated levels of all lipids and lipoproteins (total cholesterol (TC), low density lipoprotein (LDL) and very low density lipoproteins

(VLDL). The only exception is high density lipoproteins (HDL) that are supposed to be decreased. Among these, the most common etiologies of ischemic heart disease are hypercholesterolemia and hypertriglyceridemia (Jackson and Beaglehole, 1995).

Apart from medication diet plays an important role in controlling lipid levels in blood. Various studies claimed that uncontrolled use of high fat diet is the reason behind insulin resistance that results in diabetes mellitus, its associated complications and oxidative stress (Park et al., 2011).

Present study has undertaken high fat high sugar (HFHS) rat model of hyperlipidemia as it is found to be very close animal model to human disease etiology. Utilization of trans and saturated fatty acids are the key players for the induction of insulin resistance. Vanaspati ghee contains trans and saturated fatty acids. Similarly coconut oil also contains saturated fatty acids in high proportion(Munshi et al., 2014). High sugar utilization increases the body weight and decreases circulating leptin levels resulting in insulin resistance, increase serum insulin levels, hypertriglyceridemia and hence hyperglycemia (Elliott et al., 2002).

This study will elucidate the effects of different extracts of Vernonia anthelmintica on hyperlipidemia.

1.8 Objective of the study

Chemical, biological and physical assays are the vital procedures for the identification of herbal extracts. For the analysis of compounds, phytochemical screening is conducted whereas to find out the bioactive compounds extracts are subjected to different bioassays. Bioassay provides the preliminary screen to elucidate the pharmacological potential of extracts. Similarly biochemical evaluation of extracts may provide strong grounds to the pharmacological potential of certain drug. Though the previous literature provided effective clues of pharmacological potential of Vernonia anthelmintica, however detailed studies and comparison between different extracts is lacking from authentic scientific literature.

Following aims and objectives were undertaken to effectively explore the pharmacological importance and toxicological profile of different extracts of Vernonia anthelmintica.

1. Qualitative phytochemical analysis of Vernonia anthelmintica extracts. 2. In-vitro bioassays to elucidate antibacterial, cytotoxic, brine shrimp lethality, antioxidant, anti-glycation, phytotoxic, insecticidal and leishmanicidal potential of Vernonia anthelmintica extracts. 3. In-vivo assessment of acute toxicity of Vernonia anthelmintica extracts. 4. Assessment of effects of Vernonia anthelmintica extracts on hematology, coagulation, liver function, hepatic function, lipid metabolism, glucose metabolism and electrolytes on sub-chronic use. 5. In-vivo assessment of anti-hyperlipidemic, anti-hyperglycemic, anti- inflammatory, anti-nociceptive effects of different extracts of Vernonia anthelmintica on pharmacological animal model

2. MATERIALS AND METHOD

2.1 Collection and identification of seeds

The seeds of Vernonia anthelmintica were bought from herbal market and authenticated by growing it in Center of plant conservation, Karachi University and Botanic Garden, University of Karachi against Herbarium number: G.H.No 92629.

2.2 Preparation of extract

The seeds were cleaned manually for removal of impurities like straws and dirt. The seeds were ground to coarse powder in a mechanical grinder and soaked in n- hexane for 10 days with occasional shaking. On 11th day the solvent containing hexane soluble constituents was filtered through muslin cloth. The filtrate was loaded in rotary evaporator for evaporation of solvent at 50oC at high pressure to obtain yellowish green oily hexane extract of Vernonia anthelmintica referred to as HEVA (hexane extract of Vernonia anthelmintica). The residual after filtration was soaked again in ethanol to obtain brownish black gummy ethanol extract of Vernonia anthelmintica referred to as EEVA (ethanol extract of Vernonia anthelmintica) by the same methodology described for hexane extract. To prepare water decoction of Vernonia anthelmintica seeds (WDVA), its coarse powder was boiled in distill water each time before dosing.

2.3 Experimental design

Present study has undertaken both in-vivo and in-vitro methods for the estimation of pharmacological and toxicological profile of Vernonia anthelmintica.

In-vitro methods include qualitative phytochemical analysis of Vernonia anthelmintica extracts and different bioassays including antibacterial, cytotoxic, brine shrimp lethality, antioxidant, antiglycation, phytotoxic insecticidal, antileishmanial assay of all extracts.

In-vivo methods embraced effects of different extracts on acute and chronic use of Vernonia anthelmintica extracts. Acute toxicity testing was conducted by Lorke’s method whereas chronic effects of the studied extracts on weight of animal,

hematology, coagulation liver, kidney and metabolism were also evaluated. Pharmacological disease models were also designed to study the role of these extracts on diabetes, inflammation, nociception and hyperlipidemia.

2.4 In-vitro studies

2.4.1 Qualitative phytochemical analysis

Following Phytochemical tests were employed to analyze the phytochemical constituents of HEVA, EEVA and WDVA.

2.4.1.1 Wagner test for Alkaloids

Small quantity of HEVA, EEVA and WDVA was dissolved in Methanol (2ml) and few drops of Hcl (1%) was added to it. The mixture was then heated and cooled and few drops of Wagner reagent is added. Formation of precipitate or turbidity confirms the presence of alkaloids in the sample (Rajesh, et al. 2013).

2.4.1.2 Ferric chloride test for Cardiac glycosides

Small quntities of HEVA, EEVA and WDVA were dissolved in 1 % glacial acetic acid. Few drops of ferric chloride solution was added to this mixture and 0.5 ml of concentrated suphuric acid was added along the walls of test tubes. Appearance of brown ring at interface is indicative of presence of cardenolide (Rajesh, et al. 2013).

2.4.1.3 Benedict test for Carbohydrates

Small quantities of HEVA, EEVA and WDVA were allowed to mix with few drops Benedict solution which contains cupric citrate complex in alkaline solution. The mixture was observed for reddish brown precipitate after boiling it

in water bath. Presence of precipitate confirms the presence of carbohydrate in the sample (Yadav and Agarwala, 2011)

2.4.1.4 Xanthoprotein test for proteins

Small amount of each extract is taken in separate test tubes. Sulphuric acid 1 ml was added sliding from the sides of test tubes. Presence of yellow precipitate is the indication of presence of Protein in the sample (Raju et al., 2011).

2.4.1.5 Ferric chloride test for Flavonoids

Sequential addition of 0.5 ml ammonia solution and concentrated sulphuric acid to small amount of each extract already dissolved in distilled water may resulted in the appearance of yellow color indicative of flavonoids. On standing this color would disappear (Rajesh et al., 2013).

2.4.1.6 Foam test for Saponins

Small amount of each extract was dissolved in distilled water and this mixture was shaken vigorously to observe persistent froth. The froth was observed for emulsion on shaking with 3 drops of olive oil (Rajesh et al., 2013).

2.4.1.7 Lead test for tannins

Small amount of each extract was dissolved in distilled water in the separate test tubes and few drops of ferric chloride were added to the solution. The mixture may turn blue or green if tannins are present (Rajesh et al., 2013).

2.4.1.8 Salkowaski’s test for terpenoids

Small quantity of each extract is dissolved in 1 ml of chloroform and 1 ml of sulphuric acid. Terpenoids if present may appear as reddish brown discoloration at the interface (Rajesh et al., 2013).

2.4.1.9 Spot test for fixed oil and fatty acid

Place a spot of different extracts on separate filter papers. Oil staining o the filter paper is the indication of presence of fixed oil and fats (Rajesh et al., 2013).

2.4.2 Anti-bacterial assay

Antibacterial assay of HEVA and EEVA is performed by 96 well plate method using Alamar blue microtiter plate assay(Pettit et al., 2005). Six bacterial strains Escherichia coli, Bacillus subtilis, Shigella flexenari, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi were used to check antibacterial potential of Vernonia anthelmintica. Pathogens were grown in Mueller Hinton medium and inoculums were made to be adjusted at 0.5 Mc Farland turbidity index. Test compounds were prepared in DMSO in the concentration of 1:1 and different stock solutions were prepared. All micro-wells were dispensed with media. HEVA and EEVA were added to separate wells whereas control well was left without any test compound. All the plates were make up to the volume of 200 µl lastly 5×10 6 cells were added in each well including both control and test. Whole plate was covered with Para film and incubated for 18-20 hours t 37o C. Alamar blue dye was then added to each well and covered with aluminum foil and shaken at 80 rpm in a shaking incubator for 2-3 hours. Change in color of dye from blue to pink is the indication that bacterial strains grown in the wells. Absorbance was recorded at 570 and 600 nm in ELISA reader (Choudhary and Thomsen, 2001).Following strains were used for antibacterial activity of Vernonia anthelmintica: Bacterial strains Escherichia coli ATCC 2599 Bacillus subtilis ATCC 23857 Shigella flexenari ATCC 12022 Staphylococcus aureus NCTC 6571 Pseudomonas aeruginosa NCTC 10662 Salmonella typhi ATCC 14028

2.4.3 Cytotoxicity assay (MTT assay) MTT assay is a quick and efficient quantitative assay used for estimating the proliferating or surviving cells. Principle of reaction is the reduction of MTT (3- (4,5-dimethylthiazol-yl)-2,5-diphenyl tetrazolium bromide) by different

dehydrogenase enzymes existing in living cells. The cleavage of tetrazolium ring in active mitochondria results in generation of formazin crystals that are purple in color (Scudiero et al., 1988). These purple colored crystals are made to dissolve in DMSO and optical density is measured by spectrophotometer (ELISA reader). The assay was optimized and modified according to different cell lines. 3T3, Hela, PC3, MCF-7 and MDA-MB-231 cell lines are used to assess the cytotoxic potential of HEVA and EEVA. Cancer cell lines (table-1) were cultured in Dulbecco’s modified eagle medium containing 10% FBS (fetal bovine serum), 100 IU/ml of penicillin and 100 µg/ml of streptomycin, in 75 ml flask and placed in 5% CO2 incubator at temperature 37o C. On confluency, cells were made to harvested and plated in 96 well tissue culture plates of flat bottom in 100 µL medium according to seeding density shown below. Next day HEVA and EEVA were added in triplicate in the concentration of 50 µg/ml and incubated for 48 hours. After incubation extracts were removed and 200 µl MTT in the concentration of 0.5 µg/ml was added to each well and incubated at 37oC for 3 hours. 100 µl DMSO was thus used to dissolve purple colored formazan crystals formed by the reduction of MTT and absorbance was measured at 570nm (except for 3T3 line where it is 540 nm) using microplate reader (Spectra Max plus molecular device, CA, USA)(Mosmann, 1983;Carmichael et al., 1987). Doxorubicin was taken as positive control in all cell lines except for 3T3 cell lines where cyclohexinide was used as standard drug. The decrease in viable cells called as percent inhibition was calculated by following formula: Percent Inhibition = 100 - (mean of optical density of test compound - mean of optical density of negative control) / (mean of optical density of positive control - mean of optical density of negative control) x 100)

The extracts that had shown ≥ 50 % inhibition were further tested for IC50 value by making further dilution from stock solutions in order to get less than 50 % inhibition. The IC50 value was calculated using EZ-fit software.

Table 1: Cancer cell lines and their specification

Seeding density Cell line Cancer type ATCC no (cells/well) 3T3 Mouse fibroblast CRL-1658 500 PC3 Prostrate cancer CRL-1435 10,000 MCF-7 Breast cancer HTB-22 10,000 MDA-MB-231 Breast cancer HTB-26 8000

2.4.4 Brine Shrimp Lethality Assay The brine shrimp lethality bioassay was carried out on HEVA and EEVA using the standard procedure (Meyer et al., 1982b). A rectangular dish was used as a hatching tray and it is half filled with already filtered brine shrimp solution containing artificial sea water (38 g/L of distill water, pH 7.4). The eggs of Artemia salina (50 mg) was then sprinkled on it and left for hatching under incubation at 37ºC. Hatching period of eggs of Artemia Salina take 48 hrs. When placed in sea water artificially prepared for this purpose. EEVA and HEVA (20 mg each) were dissolved in 2 ml of ethanol and hexane respectively. From this solution 5, 50 and 500 µl was transferred to 3 different vials this process is carried out in triplicate (3 test/Concentration). The final concentration was 10,100 and 1000µg/ml respectively. The solvent used is allowed to evaporate overnight. After hatching, active nauplii that were free from their egg shells were collected from illuminated part of the hatching tray and used for the test. 10 mature Nauplii were then placed in each vial using a Pasteur pipette. Sea water was used to make up the volume to 5 ml and placed under illumination at 25-27º C for 24 hrs. Same method was repeated to prepare other vials containing solvent and cytotoxic drug Etoposide as negative and positive controls respectively. The vials were examined and the number of dead larvae in each bottle was counted after incubation period of 24 hrs. The death Percentage was calculated using simple mathematical equation (eq:1) and LD50 was analyzed using Finney program with 95% confidence Interval(Finney, 1971). Percentage of Death %= (Total nauplii-alive nauplii) × 100

2.4.5 Antioxidant Assay

DPPH solution is prepared by dissolving it in 100 % ethanol to get 300 µM solution. HEVA and EEVA were dissolved in DMSO (dimethyl sulfoxide) and 5 µl of these samples were added to 96 well plate and absorbance is noted at 515 nm. 95 µl of DPPH was added to each well. The plate were than incubated for 30 minutes at 37 ˚C after covering the plate in order to avoid any evaporation of solvent. Plate was subjected to microplate reader at 515 nm to get final absorbance. Control contained only DMSO while Gallic acid and N-acetyl cysteine served as the standard in this assay (Uddin et al., 2011). Following mathematical equation is used to calculate % radical scavenging activity (%RSA) % RSA = 100 - (O.D of sample/O.D of control x 100)

(O.D designates optical density)

2.4.6 Antiglycation assay

Antiglycation assay of HEVA and EEVA was performed using Bovine serum albumin –methylglyoxal glycation model described by Lee et al., with some modifications (Lee et al., 1998). 96 well plate was used to conduct this process in a way that each well was supplied with 50 µl Bovine Serum Albumin solution (10 mg/ml), 50µl Methyl Glyoxal (14mM) and 20 µl of HEVA and EEVA prepared in 1mM DMSO. 0.1 M Phosphate buffer (pH 7.4) containing NaN3 (3 mM) was added to make up the volume to 200 µl. Control wells contains DMSO instead of test samples and rutin was used as standard antiglycation agent (IC50 = 294± 1.50uM). All the test were performed in triplicates. After loading all components of test mixture plate was covered with paraffin sheet and then with lid and shaken on mechanical shaker and finally left in incubation at 37o C for 9 days. On completion of incubation days, glycation of proteins was assessed by measuring the specific fluorescence (excitation, 330 nm; emission, 420 nm) against blank by using microplate spectrophotometer (Spectra Max, Molecular Devices, CA, USA)

2.4.7 Phytotoxic assay

The lemna bioassay for inhibition and promotion of plant growth was used to assess the phytotoxic potential of HEVA and EEVA. Lemna minor belonging to lemnaceae family was used as a test weed. A new clone of test weed was cleaned in water and cultivated under favorable conditions for 1-2 days. Healthy fronds thus obtained were washed in water and transferred to nutrient solution. E- Medium was freshly prepared by mixing various constituents in 1 liter distilled water and pH is adjusted between 6 -7 by adding potassium hydroxide pellets (stock solution). Working E- medium was prepared by mixing 100 ml of stock solution and 900 ml of distilled water. 30 mg of HEVA and EEVA was dissolved in 1.5 ml of ethanol and it served as a stock solution. Three flasks are inoculated with 10, 100 and 1000 µl of test solutions for making 10, 100 and 1000 µg/ml concentration. Ethanol was allowed to evaporate overnight. 20 ml of working E- medium was added to flask and rosette of 2-3 fronds were planted in it. Positive (growth promoters) and negative (growth inhibitors) control flask were also prepared in the same manner. All the flask were placed in growth cabinet for seven days. Plants were examined daily during incubation and number of fronds present per flask were recorded. Percentage regulation was calculated with reference to negative control as follow (Erukainure et al., 2016) :

% Regulation = 100 - No. of fronds in test X 100 No. of fronds in negative control

Result was analyzed using criteria such that 0-39% inhibition would be considered as low activity, 40-59% inhibition as moderate activity, 60-69 % as good activity and above 70% as significant activity (Itokawa et al., 1982).

2.4.8 Insecticidal activity Tribolium castaneum and callosobruchus analis are the two test insects used for assessing insecticidal activity of HEVA and EEVA by Impregnated filter paper

method. Test samples were prepared by adding 200 mg of test sample with 3 ml of ethanol. The stored grain pests are reared in laboratory under controlled temperature and humidity in plastic bottles containing sterile growing media. Insects of uniform age and size are used for assay. It is a simple three day procedure. On day 1 filter paper were placed in 9 cm petri dishes and whole sample prepared was poured onto it using micropipette. Plates were left for evaporation of solvent for 24 hrs. Next day every dish was supplied with 10 insects of each species with the help of clear brush. Plates were thus incubated at 27 o C for next 24 hours with 50 % relative humidity in growth chamber. Survival of insects was assessed on day 3 by simply counting alive insect of each specie. Negative control contains only ethanol and insects whereas positive control contains permethrin at the concentration of 239.5 µg/ml and insects. Percentage inhibition was calculated by following formula (Choudhary and Thomsen 2001).

% mortality = 100 – No. of insect alive in test group × 100

No. of insects alive in control

2.4.9 Anti-leishmanial Assay

Leishmania promastigotes (Leishmani Major; ATCC 50155) were grown in bulk in modified NNN biphasic medium using normal saline. The parasites were cultured with RPMI 1640 medium along with 10 % fetal calf serum (FCS). Centrifugation of parasites at 2000 rpm for 10 minutes was conducted and then parasites were washed three times with normal saline at the same rpm and duration. The parasites thus obtained were diluted with freshly prepared culture medium to final concentration of 1 ×10 6 cells/ ml (Habtemariam, 2003). Medium was added in 96 well micro titer plate, 20 µl of HEVA and EEVA were added and serially diluted. 100 µl of parait culture was added in all wells. Negative control contains no test compound and positive control contains standard antileishmanial drugs (amphotericin B, and Pentamidine) in varying concentrations. All the tests done in duplicates. The plate was incubated between 22-25oC for 72 hours. The culture was examined in neubaure chamber and IC50 values of test extracts having

leishmanicidal activity were calculated by Ezifit software (Choudhary et al., 2005).

2.5. In-vivo studies

In-vivo studies embraced effects of different extracts on acute and chronic use of Vernonia anthelmintica extracts. Acute toxicity testing was conducted to evaluate the LD50 value whereas chronic effects of the studied extracts on coagulation, hematology, liver, kidney and metabolism were also evaluated to establish the safe use of drug. Pharmacological models were also prepared to study the role of these extracts on diabetes, inflammation, nociception and hyperlipidemia

2.5.1 Animals

Three different species of animal i.e. Rabbits, rats and mice were used to conduct the present in-vivo studies. Before conducting any study, all the animals were left under close monitoring in standard laboratory environment for at least one week. During this acclimatizing phase all animals were monitored for their general health and gross behaviors including edema, diarrhea, urination pattern, ulceration, skin and fur conditions and drowsiness or anxiety signs.

2.5.2 Acute oral toxicity

The mice were kept in cages and acclimatized in the standard environment of 12 hr. / 12 hr. light /dark cycles and standard temperature. Animals had free access to standard pellet diet and tap water. The studies are in accordance with the standard ethics of animal use and Lorke’s method was adopted to carry out Acute toxicity studies (Lorke, 1983). This method is divided into 2 phases. In the first phase nine albino mice were selected and divided into 3 groups constituting 3 mice each. The first group received HEVA orally at a dose of 1000 mg/kg; second group received the same extract at the dose of 100 mg/kg; whereas the third group received the extract at the dose of 10 mg/kg body weight. Animals were observed for 24 hours periodically to assess general sign and symptoms of gross toxicity including mortality. In the second phase 3 mice were selected and assigned to different groups containing one animal each. HEVA is administered orally to these groups

in the doses of 1600, 2900 and 5000 mg/kg. Same procedure was repeated for oral acute toxicity of EEVA and WDVA on fresh animals. Based on the results of both the phases LD50 was calculated as follow:

LD50 = √ (D0 × D100)

Where D0 = Highest dose producing mortality

2.5.3 Sub-chronic studies

2.5.3.1 Animal grouping and experimental design

Healthy albino rabbits of either sex with mean body weight of 1532 ± 90.59 grams were caged in steel cages. Environmental conditions were maintained at 23± 2˚C with 50-60 % humidity throughout the study span. Regular food was provided and water was freely accessible to experimental animals. Animals were assigned to following group

GROUP 1: Normal control treated with distill water.

GROUP 2: Treated with 150 mg/kg body weight HEVA

GROUP 3: Treated with 300 mg/kg body weight HEVA

GROUP 4: Treated with 150 mg/kg body weight EEVA

GROUP 5: Treated with 300mg/kg body weight EEVA

GROUP 6: Treated with 150 mg/kg body weight WDVA

GROUP 7: Treated with 300 mg/kg body weight WDVA

Extracts were given to animals every morning at the same time by oral route through orogastric tube for 60 days. Blood samples were drawn from each animal 60 days of dosing through cardiac puncture technique. Fasting blood glucose, lipid profiles, Liver function parameters, renal function tests and electrolytes were analyzed in serum samples, hematological parameters were assessed in blood samples and coagulation profile was analyzed in the blood.

2.5.4 Pharmacological models

Four different pharmacological models were also designed to evaluate the activities of different extracts on hyperlipidemia, diabetes, inflammation and pain.

2.5.4.1 Hyperlipidemic Model: (high fat high sugar model)

Forty eight Sprague drawly rats of either sex aged 13 weeks (175-250gms) were used in the experimentation. The animals were caged in 6 groups of eight animals each. Hyperlipidaemia was induced in rats by using high fat high sugar diet. Fat components consisted of locally available Vanaspati ghee and coconut oil in the ratio of 3:1 (v/v) whereas sugar contents contain 25 % fructose w/v ad libitum (Munshi et al., 2014). HFHS diet was given in the dose of 3 ml/kg to all groups orally except control for twelve weeks. The animals were weighed in the start and then periodically after every week. Animals were numbered weighed and randomly stratified into 6 groups of 8 animals each as follow:

Group 1: Received standard diet + water daily for 12 weeks and was labeled as normal control (NC), Group 2: Received standard diet + HFHS diet daily for 12 weeks and labeled as disease control (DC) , Group 3: Received standard diet + HFHS diet daily for 12 weeks + Atorvastatin in the dose of 2.1 mg/kg/day after 8 weeks and continued for next 4 weeks and labeled as standard control (SC), Group 4: Received standard diet + HFHS diet daily for 12 weeks + HEVA in the dose of 300 mg/kg/day after 8 weeks and continued for next 4 weeks and labeled as HEVA group, Group 5: Received standard diet + HFHS diet daily for 12 weeks + EEVA in the dose of 300 mg/kg/day after 8 weeks and continued for next 4 weeks and labeled as EEVA group. Group 6: Received standard diet + HFHS diet daily for 12 weeks + WDVA in the dose of 300 mg/kg/day after 8 weeks and continued for next 4 weeks and labeled as WDVA group.

At the end of experimental period 12 hr fasting blood sample of animals were drawn after decapitation and serum was assessed for fasting blood glucose, lipid profile ( LDL, HDL, VLDL, Total cholesterol, triglycerides and Cholesterol/ HDL ratio) and liver function tests (SGPT, SGOT, ALP, GGT and total bilirubin).

Cardiac risk parameters, atherogenic index and protection percentage were calculated using following formulae:

Atherogenic Index = Total cholesterol – HDL-C (Schulpis and Karikas, 1998)

HDL-C

Protection percentage = A.I of disease control – A.I of treated group × 100

A.I of disease control

2.5.4.2 Diabetic Model

One hundred and twenty Sprague Dawley rats of both sexes weighing 175- 250 g were used to assess the effects of Vernonia anthelmintica on blood glucose levels. The rats were randomly divided into 5 groups of eight animals for three sets of assessment i.e. normoglycemic blood glucose levels, glucose tolerance test and alloxan induced diabetic profile.

Animals are grouped in such a way that in each set, Group 1 received normal saline in the equivalent amount as that of treatment groups, Group II received Glibenclamide in the dose of 5 mg/kg and served as standard and Group III, IV and V received HEVA, EEVA and WDVA in the dose of 300 mg/kg respectively. For oral glucose tolerance test, after 30 minutes of receiving extracts and standard drug every animal in each group of OGTT had received oral glucose in the dose of 2 g/kg in form of aqueous solution. In Alloxan induced diseased diabetic model prior induction of diabetes in overnight fasted rats was undertaken by giving 150 mg/kg i.p. After 1 hr of alloxan administration animal were provided with food and water. Animals were kept in close observation and blood glucose levels were measured using glucometer by tail prick method. Only the diabetic rats with the blood glucose level between 180- 400 mg /dl were selected and assigned to above mentioned groups for experimentation.

Blood glucose levels were measured at 0, 30 min, 60 min, 120 min, 180 min and 240 min interval by tail prick method using one- call glucometer and recorded in mg/dl

2.5.4.3 Inflammatory model

Carrageenan induced rat paw model

Carrageenan induced rat paw model was used to assess the anti-inflammatory potential of Vernonia anthelmintica. Sixty four Sprague Dawley rats were divided into eight groups, of eight rats each. 0.1 ml of 1% carrageenan was injected to induce oedema as the sign of inflammation into the sub-plantar area of the right hind paw. Control group of rats was given normal saline in the equivalent volume as that of test groups and standard group of rats received ibuprofen in the dose of 100 mg/kg through oral route. The treatment groups of rats were subjected to the oral administration of 150 and 300 mg/kg of the hexane and ethanol extracts and water decoction of Vernonia anthelmintica seeds 1 hr before carrageenan administration in hind paw. The paw volume was thus measured using digital plethysmometer before administration of carrageenan referred to as Vo and 1, 2, 3, 4, 5, 6 and 24 h thereafter referred to as Vt. Paw edema is the marker of inflammation and it was calculated as increase in paw volume after treatment subtracted from baseline paw volume of respective animals. Percentage reduction in volume was also calculated to quantify and compare the inhibition of edema with respect to control. Following formula is used and results were expressed as percentage of inhibition of edema (Winter et al., 1962).

Percent reduction in volume = [(Vt–V0) control – (Vt–V0) treated] / (Vt–V0)

control×100.

2.5.4.4 Analgesic model

Analgesic activity of Vernonia anthelmintica was estimated by two different methods i.e. Tail flick method and hot plate method.

Tail flick method

Sixty four albino mice were divided into eight groups, of eight mice each. Control group of mice was given normal saline in the equivalent volume as that of test groups and standard group of mice received aspirin in the dose of 100 mg/kg through oral route. The treatment groups of mice were subjected to the oral administration of 150 and 300 mg/kg of the hexane and ethanol extracts and water decoction of Vernonia anthelmintica seeds 1 hr before experimentation. The latency time was measured in seconds at time 0, 30min, 60 min, 90 min and 120 min by dipping the tail of the animal in hot water (50oC) (Luiz et al., 1988).

Hot plate method

Sixty four Sprague Dawley rats were divided into eight groups, of eight rats each. Control group of mice was given normal saline in the equivalent volume as that of test groups and standard group of rats received aspirin in the dose of 100 mg/kg through oral route. The treatment groups of rats were subjected to the oral administration of 150 and 300 mg/kg of the hexane and ethanol extracts and water decoction of Vernonia anthelmintica seeds 1 hr. before experimentation. The latency time was measured in seconds at time 0, 30 min, 60 min, 90 min and 120 min by placing the animal on hot plate (50oC) (Eddy et al., 1950).

2.6 Sample Collection

Blood samples of rabbits were collected by cardiac puncture whereas rat’s blood samples were drawn after decapitation at scheduled time. Animal handling and sample collection according to CLSI approved guidelines H21-A5 (Adcock, 2008)

Plasma and serum were collected in tubes containing 3.8% tri sodium citrate and gel tubes and immediately subjected to centrifugation at 3000 rpm for 15 minutes and 2000 rpm for 10 minutes respectively using 14 K Humax (Human Germany). The plasma and serum thus separated were stored at – 20oC for determination of various coagulation and biochemical parameters. For analysis of hematologic

parameters blood samples were collected in EDTA tubes and were subsequently tested within two hours of sample collection by Humacount (Human Germany).

2.7 Hematological analysis

EDTA .K3 tubes were used for the collection of 2 ml blood for hematological examination. Humacount plus (Human Germany) is a fully automated device that was used for the analysis of Red blood cell count, White blood cell count, platelet count, haemoglobin.

2.8 Coagulation studies:

Purple top plasma tubes containing 3.8% sodium citrate were used for the collection of 2-3 ml of blood from control and treated animals through cardiac puncture. Plasma was centrifuged in 14 K Humax centrifuge at 1500 rpm for 10 minutes to separate plasma from rest of blood cells. Thrombin time (TT), prothrombin time (PT), Activated partial thromboplastin time (aPTT) and fibrinogen (FB) were analysed by Humaclot Duo. Standard reagent kits of Human Germany were used for this analysis.

Thrombin time:

It is a simple test to detect any interference with the conversion of fibrinogen to fibrin. In this procedure, undiluted plasma is facilitated to clot by the addition of thrombin. To measure TT 150 µl of plasma and control was added in separate pre- warmed test tubes and incubated for 3 minutes at 37o C. After incubation 150 µl of thrombin reagent was added and time required for clot formation was recorded.

Activated partial thromboplastin time:

With the exception of factor VII activated partial thromboplastin time is one of very important diagnostic tool to detect the deficiencies of all other clotting factor i.e. factor XII, XI, X, IX, VII, V, II, I and prekallikrein. aPTT is determined using Ellagic acid as activator. aPTT reagent containing plasma activator and phospholipid is added to plasma to replace platelet in clotting process. This mixture is activated through incubation and then re-calcified with calcium

chloride and clot formation is timed.to perform test 100 µl of plasma and control was poured to separate pre-warmed test tube and 100 reagent 1 containing ellagic acid was added to it. After gentle mixing and incubation for 3 minutes at 37o C,

100 µl of pre-warmed reagent 2 containing Cacl2 was added and time required for clot formation was recorded.

Fibrinogen:

Thrombin in high concentration was added to diluted plasma and clotting time was thus measured. A calibration curve was prepared by recording clotting in different dilutions of reference plasma of known fibrinogen concentration. The results were than compared with this calibration curve to obtain fibrinogen levels in mg/dl (Clauss, 1957). To perform test samples and control plasma was diluted with imidazole buffered saline in 1:8 proportion. 200 µl of these diluted samples and control were added in the test tubes and incubated for 3 minutes at 37ºC. After incubation 100 µl thrombin reagent was added and timer was started to record time required for clot formation. With the help of calibration these clotting time were converted into fibrinogen concentration in mg/dl.

Prothrombin Time:

This test is a quick test that uses thromboplastin-SI reagent to detect one stage prothrombin time. Thromboplastin (Tissue Factor) and calcium is used to measure one stage PT which in turns measures the clotting time of plasma. Addition of calcium and tissue factor in plasma generates activated factor X A which is responsible for the formation of thrombin and finally an insoluble fibrin clot. To perform test 100 µl of plasma /control was added to separate pre- warmed test tubes and incubated at 37oC for 3 minutes. After incubation 200 µl of thromboplastin reagent was added and time required for clot formation was recorded.

2.9 Biochemical Analysis

For biochemical analysis 7 ml of blood was collected in yellow top serum collection tube and set to centrifugation at 3000 rpm for 10 minutes to separate

serum from whole blood. This supernatant serum was aspirated using pipettes and stored in Eppendorf tubes for testing on Humalyzer 3000. It is automated chemistry analyser used for biochemical analysis using standard kits provided by Human Germany.

2.9.1 Lipid Profile analysis

Following parameters were analysed

Cholesterol:

CHOD-PAP Method is a colorimetric test that involves enzymes and lipid clearing factor. This method is used for estimation of cholesterol in serum. The reaction principle involves enzymatic hydrolysis and oxidation that leads to the determination of cholesterol. Quinoneimine is synthesized from H2O2 and 4- aminophenazone. Phenol and peroxidase facilitated this reaction and quinoneimine thus formed acts as an indicator in this reaction. 10 µl of sample/ standard was added to 1000 µl of enzyme reagent, mixed gently and incubated for five min at 37°C or ten minutes at 20-25°C. Absorbance of the sample and the standard was measured against the reagent blank within 60 minutes of preparation (Trinder 1969).

Triglycerides:

Triglycerides liquicolor was conducted through GPO-PAP method. It was an enzymatic colorimetric method that uses lipid clearing factor (LCF) for the estimation of Triglycerides. Enzymatic hydrolysis with lipase was used for the determination of triglycerides. Quinoneimine acted as an indicator that is formed by the reaction of 4- aminoantipyrine and 4- chlorophenol. This reaction is catalyzed by peroxidase. 10 µl of standard/ sample was mixed gently with 1000 µl of reagent and incubated for five min at 37°C or ten minutes at 20-25°C. Absorbance of the sample and the standard was measured against the reagent blank within 60 minutes of preparation (Trinder, 1969).

HDL- Cholesterol:

Different methodologies has been involved in selective precipitation and removal of LDL and VLDL which is followed by enzymatic estimation of HDL in the supernatant fraction of serum (Warnick et al., 1995). In this study Cholesterol liquicolor test using kits provided by human Germany was conducted to estimate HDL- cholesterol (Gordon et al., 1977, Friedewald et al., 1972). Phosphor- tungstic acid and magnesium chloride was used to precipitate chylomicrons, VLDL, and LDL. The supernatant fraction that contains HDL is estimated for HDL cholesterol with the standard kits supplied by Human Germany. 200 µl of sample was added to precipitant, mixed well and incubated for 10 minutes at room temperature and Centrifuged for 10 minutes at 4000 rpm. The 100 µl clear supernatant was separated from the tubes within 1 hour and determination of the cholesterol concentration using 1000 µl cholesterol liquicolor reagent was conducted.

LDL-Cholesterol/VLDL cholesterol

LDL-C is the low density lipoprotein and it is measured by kits provided by Human Germany. 10 µl of sample was added to 75 µl enzyme in test tube. After gentle mixing and incubation 250 µl substrate was added and incubated at 37 C. Absorbance was thus measured after 5 minutes. VLDL-C was calculated by dividing 5 with triglycerides levels. (Friedewald et al., 1972).

2.9.2 Blood glucose analysis

Glucose liquicolor is GOD-PAP method (Enzymatic Colorimetric Test for Glucose) Enzymatic oxidation of glucose in the presence of glucose oxidase generates hydrogen peroxide. This formed hydrogen peroxide reacts with phenol and 4 aminoantipyrine to a red violet coloured dye quinoneimine. This last reaction is catalysed by peroxidase(Trinder, 1969). 10 µl of sample/ standard was mixed with 1000 µl of reagent Mix, incubate for 10 minutes at 20-25°C. Measure the absorbance of the sample (∆A sample) and the Standard (∆A STD) respectively, against the reagent blank within 60 minutes.

2.9.3 Liver function tests

GPT (ALAT):

Gamma pyruvate transaminase or alanine aminotransferase is analyzed according to the recommendations of the expert panel of international federation of clinical chemistry (IFCC). Kinetic method for the determination of ALAT without pyridoxalphosphate activation is used in this study (Schumann et al., 2002)100 µl of sample was mixed with 1000 µl of working reagent (prepared by mixing 2 ml of substrate with one bottle of buffer) and absorbance was measured after 1 min.

GOT (ASAT)

Gamma oxalobutyric transaminase or aspartate transferase is analyzed by kinetic method for determination of ASAT without pyridoxalphosphate activation. This procedure is according to the recommendations of the expert panel of international federation of clinical chemistry (IFCC) (Schumann et al., 2002). 100 µl of sample was mixed with 1000 µl of working reagent (prepared by mixing 2 ml of substrate with one bottle of buffer) and absorbance was measured after 1 min.

GGT

Gamma glutamyl transferase is analysed by colorimetric method according to Persijin and vander slik that is standardized against recommended IFCC method (international federation of clinical chemistry). (Persijn and van der Slik, 1976). 100 µl of sample was mixed with 1000 µl of working reagent (prepared by mixing 2 ml of substrate with one bottle of buffer) and absorbance was measured after 1 min.

ALP

Alkaline phosphatase is analysed by optimized standard method according to recommendation of German clinical chemistry association using Diethanolamine buffer and DGKC orthophosphoric monoester phosphohydrolase (Keiding et al., 1974). 100 µl of sample was mixed with 1000 µl of working reagent (prepared by

mixing 2 ml of substrate with one bottle of buffer) and absorbance was measured after 1 min.

Bilirubin Direct and Total

It is a photometric test to estimate direct and total bilirubin by modified Jendrassik /Grof Method. In this test bilirubin reacts with diazotized sulphanilic acid (DSA) to form red Azo dye. The concentration of bilirubin is proportional to the absorbance of the dye at 546 nm. Direct bilirubin is the water soluble glucuronide fraction and directly reacts with DSA whereas bilirubin conjugated to albumin represent the In-direct fraction of bilirubin that only reacts in the presence of accelerator. Total bilirubin was calculated simply by adding direct and indirect bilirubin (Mori, 1978).1000 µl of total/ direct bilirubin reagent was mixed with 1 drop of total/direct nitrite reagent and incubated for 5 minutes. 100 µl of sample was then added, mixed and incubated at room temperature for 10 to 30 minutes.

The absorbance of sample was measured against sample blank (∆A546).

2.9.4 Renal Function Tests

Total protein:

Total protein in serum is estimated by photometric colorimetric test by Biuret method (Gornall et al., 1949). Protein in alkaline solution when react with cupric ions it form purple complex. The absorbance of this complex is proportional to the protein concentration in the serum. 20 µl of sample/standard was added to 1000 µl of total protein reagent and mixed well. The mixture was incubated for 10 minutes at 20- 25 ˚C. The absorbance of the sample and standard was measured against the reagent blank wit

Creatinine

Creatinine is estimated by Jaffe- Method which is photometric colorimetric test for kinetic measurements method without deproteinisation (Bonsnes and Taussky, 1945). Creatinine when made to react with picric acid forms an orange red

complex in alkaline solution. The absorbance of this complex is proportional to the creatinine concentration in the sample. Sodium hydroxide was diluted with distil water in the ratio of 1:4. Picric acid was mixed with diluted sodium hydroxide in the ratio of 1:1 to prepare working reagent. 100 µl of sample/standard was mixed with 1000 µl of working reagent and mixed well. After 30 seconds, the absorbance of sample and standard was measured.

Uric acid

Uric acid in serum is measured by PAP-Method. It is an enzymatic colorimetric test for uric acid with lipid clearing factor (Barham and Trinder, 1972). Hydrogen peroxide is formed by the reaction of uric acid with uricase. This hydrogen peroxide reacts with 3,5 dichloro- 2 hydroxybenzene- sµlfonic acid (DHCHBS) and 4 amino phenazone (PAP) to give a red violet quinoneimine dye as indicator. This process id facilitated under the catalysis of peroxidase. 20 µl of sample/standard was mixed well with 1000 µl of reagent and incubated for 10 minutes at 20- 25 ˚C. The absorbance of the sample / standard was measured against the reagent blank within 15 minutes.

Urea

Urea liquicolor is an enzymatic colorimetric test that is employed for urea estimation in serum. Urea in the presence of water and urease generates ammonia and carbon dioxide. In a modified Berthelot reaction the ammonium ions react with hypochlorite and salicylate to form green colored dye. The absorbance is proportional to the urea content in the serum sample.(Berthelot, 1859). 10 µl of sample/standard was mixed to 1000 µl of enzyme reagent 1 and incubated for 5 min at 20- 25 ˚C. Measure the absorbance of the sample / standard against the reagent blank within 15 minutes.

2.9.5 Electrolytes analysis Calcium

Calcium is estimated in plasma by photometric test for calcium by CPC method (Barnett, 1973).Calcium ions react with o-cresolphthalien-complexone in an

alkaline pH to form a complex of purple color. The absorbance of this complex is thus detected and it estimates the concentration of calcium in the plasma. Reagents and buffers are mixed in equal quantity as per need to prepare working reagent. Mixture is allowed to stand for 30 minutes at room temperature before use. 20 µl of sample/ standard was mixed with 1000 µl working reagent and the absorbance of sample (∆Asample) and standard (∆Astd) against the reagent blank within 5 to 30 minutes was measured.

Sodium

Serum sodium is analyzed by photometric determination by Mg-Uranyl acetate method (color Test). Sodium when reacted to Mg- uranyl acetate when made to react with sodium forms yellow brown complex with Thioglycolic acid. The difference between reagent blank and analysis is directly proportional to sodium concentration (Trinder, 1951). 20 µl of sample/ standard was added to 1000 µl precipitating solution.Closed tubes were mixed well and allowed to stand for 5 minutes. Intensive shaking for at least 30 seconds was performed. It was then allowed to stand for 30 minutes and centrifuged at high speed for 5- 10 minutes. 20 µl of supernatant from standard/ sample was added to 1000 µl of reagent and mixed well. After 5-30 minutes, absorbance of reagent blank (∆ARB), the standard

(∆Astd) and the sample (∆Asample) were measured against distill water at 360-410 nm (Hg 366 or Hg 405).

2.10 Histopathological examination

Histopathological examination was performed using methods experimented by (Alnaqeeb et al., 1989). Test and control samples were selected at random to estimate histopathological variations among different groups. Specimens of liver, heart and kidney were removed from body and preserved in formalin (10%) for at-least 24 hrs before processing to prevent bacterial decomposition and autolysis. Shape, color, size and gross appearance of organs were recorded before cutting. Suitable parts were then cut from each sample and were preserved in cassettes. These portions were processed in automatic tissue processor (Gilford 101 system) and embedded by using steel mold trays containing paraffin. Tissue sectioning

was done by using rotary manual microtome that provided sections of 3-4 microns thickness from wax blocks. The tissue sections were mounted on slides and dried gently in oven at 60oC for 1 hr. Finally the tissue sections were sequentially stained by immersing the slides in following order:

Xylene-1 for 3-5 min. Xylene-2 for 3-5 minutes to remove wax. 100 % Alcohol for 1 minute 90% alcohol for 1 minute 70 % alcohol for 1 minute Sections were hydrated by rinsing in distill water Placed in hematoxylin for 5 minutes to stain nucleus De-stained in tap water for 5 minutes to remove extra stain Slides were agitated for 5- 10 minutes in acid alcohol (Hcl: alcohol in the ratio of 1:79) to remove stains from slides. Washed in tap water for 2 minutes Seen under microscope to ensure sufficient differentiations. De- stained in tap water or 1 minute. Cytoplasm stained in 1 % aqueous solution of eosin for 2 minutes Rinsed in tap water for 30 seconds Dehydrated in 85% alcohol for 30 seconds. Dehydrated in 100 % alcohol for 30 seconds. Slides were cleaned again in xylene for 30 seconds for removal of alcohol. Slides were mounted in DPX (distrene polyester xylene), covered, dried in fume hood for 90 minutes and labeled. Slides were examined with light microscope using different magnifications and morphology were recorded by taking permanent photographs.

2.11 Statistical analysis All the results of In-vivo studies were entered and analyzed by SPSS (Statistical Package for Social Sciences). Data was presented as mean ± S.E.M. one way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to analyse the results of chronic studies as well as diseased models i.e. Hyperlipidemia, inflammation, analgesia and diabetes. Values of P≤0.05 were taken as significant and p ≤0.005 as highly significant.

3. RESULTS

3.1 In-vitro studies 3.1.1Qualitative Phytochemical Analysis Table 2 shows the phytochemicals present in HEVA, EEVA and WDVA. All three extracts showed the presence of flavonoids and terpenoids. Saponins and proteins are absent in all three extracts. Alkaloids, tannins and fixed oils are present in HEVA and EEVA. Furthermore EEVA is positive for carbohydrates whereas HEVA showed the presence of cardiac glycosides also.

Table-2 Qualitative phytochemical analysis of HEVA, EEVA and WDVA

Secondary metabolites Test employed HEVA EEVA WDVA Alkaloids Wagner test + ve + ve - ve carbohydrates Benedict test -ve + ve - ve Cardiac glycosides Ferric chloride test + ve - ve + ve Flavonoids Ferric chloride test + ve + ve + ve Proteins Xanthoproteins test - ve - ve - ve Saponins Foam test - ve - ve - ve Tannins Lead test + ve green + ve blue - ve terpenoids Salkowaski’s test + ve + ve + ve Fixed oils and fatty acids spot test +ve -ve -ve

+ ve sign represent presence of secondary metabolite. - ve sign indicates absence of metabolite. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica.

3.1.2 Antibacterial bioassay Table-3 and Table-4 show the results of microplate Alamar blue assay (MABA) for the evaluation of antibacterial activity of hexane and ethanol extracts of Vernonia anthelmintica respectively. Table 1: depicted that HEVA shows non- significant inhibition against E. coli and S. flexanari while it shows no activity against bacillus subtilis, staphylococcus aureus, pseudomonas aerµgenosa and Salmonella typhi at tested concentration of 150 µg/ml.

Table 2; shows that that EEVA holds no inhibition against Ecoli and Salmonella typhi whereas non-significant inhibition against bacillus subtilis, S. flexanari, Staphylococcus aureus, pseudomonas aerµgenosa at tested concentration of 150 µg/ml. Ofloxacin was used as standard antimicrobial agents in tis assay. Table- 3 Antibacterial bioassay of HEVA Percent (%) Percent (%) inhibition Name of bacteria inhibition Ofloxocin Escherichia coli 16.86 89.49 Bacillus subtilis 0 87.50 Shigella flexenari 4.61 84.21 Staphylococcus aureus 0 90.17 Pseudomonas aerµginosa 0 90.05 Salmonella typhi 0 91.08

HEVA = Hexane extract of Vernonia anthelmintica.

Table-4 Antibacterial bioassay of EEVA Percent (%) Percent (%) inhibition Name of bacteria inhibition Ofloxocin Escherichia coli 0 77.25 Bacillus subtilis 8.98 80.00 Shigella flexenari 6.35 81.21 Staphylococcus aureus 6.42 80.50 Pseudomonas aerµginosa 10.05 73.78 Salmonella typhi 0 78.47

EEVA= Ethanol extract of Vernonia anthelmintica.

3.1.3 Cytotoxicity bioassay Table-5, table-6, table-7, table-8 and table-9 show the cytotoxic activity of hexane and ethanol extract of Vernonia anthelmintica against 3T3, Hela, PC3, MDA-MB- 231 and MCF-7 cancer cell lines respectively. EEVA shows significant activity against 3T3 cell line with the percent inhibition of 57.2 and IC 50 value of 38.6± 4.9 at the concentration of 50µg/ml, cyclohexinide was used as standard anticancer agent in this assay. However HEVA and EEVA show inhibition against all other cell lines also but none of them achieved significance at the concentration of 50 µg/ml. Doxorubicin was used as standard drug against all other cell lines except 3T3 cell line.

Table -5

MTT bioassay of HEVA and EEVA against 3T3 cell line

Sample Concentration % inhibition IC50 ± SD

HEVA 50 µg/ml 21.8 Inactive EEVA 50 µg/ml 57.2 38.6± 4.9 Cyclohexinide 50 uM 65.95 0.67± .17

HEVA = Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica.

Table-6

MTT bioassay of HEVA and EEVA against Hela cell line

Sample Concentration % inhibition IC50 ± SD HEVA 50 µg/ml 11.3 Inactive EEVA 50 µg/ml 36.5 Inactive Doxorubicin 50 uM 96.2 0.192 ± 0.03

HEVA = Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica.

Table-7 MTT bioassay of HEVA and EEVA against PC 3 cell line

Sample Concentration % inhibition IC50 ± SD HEVA 50 µg/ml 49.2 Inactive EEVA 50 µg/ml 36.545.6 Inactive Doxorubicin 50 uM 99.17 0.31±0.03

HEVA = Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica.

Table – 8 MTT bioassay of HEVA and EEVA against MDA-MB-231 cell line

Sample Concentration % inhibition IC50 ± SD HEVA 50 µg/ml 21.76 Inactive EEVA 50 µg/ml 29.13 Inactive Doxorubicin 50 uM 89.19 0.92 ± 0.1

HEVA = Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica.

Table-9 MTT bioassay of HEVA and EEVA against MCF 7 cell line

Concentration Sample % inhibition IC ± SD (mg/ml) 50 HEVA 50 µg/ml 27.25 Inactive EEVA 50 µg/ml 38.59 Inactive Doxorubicin 50 uM 89.19 0.92±0.1

HEVA = Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica.

3.1.4 Brine shrimp lethality bioassay The results of cytotoxicity of HEVA and EEVA by brine shrimp lethality assay are depicted in table 10 and table 11 respectively. It shows that hexane and ethanol extracts are moderately cytotoxic to brine shrimp with LC50 value 216.1 µg/ml and 104.1 µg/ml respectively at 24 hours.

Etoposide was used as a standard drug with LD50: 7.4625 µg/ml.

Table- 10 Brine shrimp lethality bioassay of HEVA

Dose LD50 No. of shrimps No. of survivors (µg/ml) (µg/ml) 10 30 30 100 30 20 216.110 1000 30 5

HEVA = Hexane extract of Vernonia anthelmintica.

Table-11

Brine shrimp lethality bioassay of EEVA Dose LD50 No. of shrimps No. of survivors (µg/ml) (µg/ml) 10 30 24 100 30 20 104.1659 1000 30 5

EEVA= Ethanol extract of Vernonia anthelmintica.

3.1.5 Antioxidant bioassay Table-12 shows DPPH assay of hexane and ethanol extracts of Vernonia anthelmintica seed. Results depict that EEVA has potent antioxidant activity with

IC 50 value of 95.10 % RSA of 89.70 against gallic acid (IC 50value: 23.43 ±0.43) and N- acetyl cysteine (IC 50 value: 111.44 ±0.7). Whereas HEVA was found to be inactive with % RSA of 40.03.

Table-12 Antioxidant bioassay of HEVA and EEVA

%RSA Sample IC 50 ±SEM (radical Scavenging activity) HEVA inactive 40.03 EEVA 95.10±2.01 mg/ml 89.70 Gallic acid 23.43±0.43 uM 93.93 N-Acetyl cysteine 111.44±0.7 uM 95.95

HEVA = Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica.

3.1.5.1 Radical Scavenging activity of EEVA:

Figure: 3 reveals the dose dependent effect of active antioxidant EEVA. 500 µg/ml of EEVA shows the maximal inhibition of 89.70 %

Percent RSA of EEVA at different concentrations 100 90 80 89.7 70 79.7 60

50 58.31 %RSA 40 40.23 30 20 22.121 10 12.156 0 15.62 31.25 62.5 125 250 500 CONCENTRATION OF EEVA (ΜG/ML)

Figure-3: Percentage radical scavenging activity of ethanol extract of

Vernonia anthelmintica seed

3.1.6 Antiglycation bioassay Table-13 shows the antiglycation activity of hexane and ethanol extract of seed of Vernonia anthelmintica. HEVC showed no inhibition while EEVC showed 45.60 % of inhibition as compared to standard agent Rutin (95%). Both the extracts are reported inactive as they showed less than 50 % activity at the concentration of 2 mg/ml.

Table-13 Antiglycation bioassay of HEVA and EEVA

Sample Concentration % inhibition IC 50 ±SEM (mg/ml) (µM) HEVC 2 -24.42 Inactive

EEVC 2 45.60 Inactive

Rutin 2 95 0.19 ± 0.01

HEVA = Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica.

3.1.7 Phytotoxic Bioassay

Table 14 and 15 shows the phytotoxic assay of hexane and ethanol extract of Vernonia anthelmintica. Results reveal the moderate phytotoxic activity of HEVA and EEVA as compared to standard drug paraquat at the concentration of 0.015 µg/ml.

Table-14 Phytotoxic bioassay of HEVA Name of plant Concentration No of Fronds % growth (µg/ml) sample control regulation Lemna minor 1000 4 20 80 100 10 50 10 16 20

HEVA = Hexane extract of Vernonia anthelmintica.

Table-15 Phytotoxic bioassay of EEVA Name of plant Concentration No of Fronds % growth (µg/ml) sample control regulation Lemna minor 1000 0 20 100 100 10 50 10 13 35

EEVA= Ethanol extract of Vernonia anthelmintica.

3.1.8 Insecticidal bioassay Table 16 and 17 show the insecticidal assay of hexane and ethanol extracts of seed of Vernonia anthelmintica. Both extracts show no insecticidal activity at the concentration of 1019.1 µg against standard insecticidal drug permethrin at the concentration of 239.5 µg/ml.

Table-16 Insecticidal bioassay of HEVA % mortality Name of insects Permethrine HEVA -ve control 239.5 µg 1019.1 µg Tribolium Castaneum 100 0 0 Callosbruchus 100 0 1%

HEVA = Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica.

Table-17 Insecticidal bioassay of EEVA % mortality Name of insects Permethrine -ve control EEVA 239.5 µg 1019.1 µg Tribolium Castaneum 100 0 0 Callosbruchus 100 0 0

HEVA = hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica.

3.1.9 Antileishmanial bioassay

Table 18 shows In-vitro antileishmanial activity of hexane and ethanol extracts of Vernonia antihelmintica. HEVA and EEVA show low antileismanial activity against standard drugs amphotericin B and Pentamide.

Table-18 Leishmanicidal bioassay of HEVA and EEVA

Leishmanicidal activity IC50 (µg/ml) ± S.D HEVA 79.06 ± 0.1 EEVA 73.09 ± 0.05 Amphotericin B 0.29 ± 0.05 Pentamide 5.09 ± 0.09

HEVA = hexane extract of Vernonia anthelmintica EEVA= Ethanol extract of Vernonia anthelmintica

3.2 In-Vivo Studies 3.2.1 Acute oral toxicity Table 19 shows the results of in vivo acute oral toxicity of HEVA, EEVA and

WDVA. The results of median lethal dose of in-vivo acute oral toxicity (LD50) for water decoction, hexane and ethanol seed extracts of Vernonia anthelmintica are given in table 18. The results clearly reveal that both extracts and water decoction of Vernonia anthelmintica has median lethal dose above 5000 mg/ kg on acute dosing as no mortality is observed even at the highest dose in the tested animals.

Table-19 Oral acute Toxicity of HEVA, EEVA and WDVA

GROUPS Dose (mg/Kg) No. of Mortality / total no. of animals PHASE 1: 10 100 1000

HEVA 0/3 0/3 0/3 EEVA 0/3 0/3 0/3 WDVA 0/3 0/3 0/3 PHASE 2: 1600 2900 5000 HEVA 0/3 0/3 0/3 EEVA 0/3 0/3 0/3 WDVA 0/3 0/3 0/3

HEVA = Hexane extract of Vernonia anthelmintica EEVA= Ethanol extract of Vernonia anthelmintica

3.2.2Sub-chronic studies 3.2.2.1: Effects on body weight Table-20 reveal the effects of HEVA, EEVA and WDVA on body weight of the tested animals on 60 days treatment with two different doses (150 mg/kg and 300 mg/kg). The results reveal the significant weight loss in all treatment groups as compared to control. Control group shows 1.3 % gain in weight as compared initial body weight. Whereas all other tested groups show significant percent weight loss as compared to their initial body weight.

Table-20 Effects of HEVA, EEVA and WDVA on body weight

Animal Dose Initial body Final body Weight % weight weight weight loss loss groups mg/kg (gms) Control - 1386±59.3 1405±55.5 -18.75±18.9 -1.3 HEVA 150 1466±91.8 1282±115.9 184±38.8* 12.55 HEVA 300 1549±69.2 1329±22.6 219±50.9** 14.1 EEVA 150 1548±132 1329±130.5 219±33.1** 14.1 EEVA 300 1569±97.18 1331±47.4 238±21.4** 15.1 WDVA 150 1546±128.2 1281±110.7 265±46.4** 17.1 WDVA 300 1660±56.5 1374±59.6 286±61.3** 17.2

n=10 Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant difference as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica.

3.2.2.2 Effects on hematological profile Table-21 shows the effects of different extracts of Vernonia anthelmintica (HEVA, EEVA, WDVA) on hematological parameters in rabbit model after 60 days dosing. The result revealed no significant change in hemoglobin, red blood cell count, hematocrit, MCV, MCH, MCHC, white blood cells and platelets in treatment groups as compared to control.

Table-21 Effects of Vernonia anthelmintica on hematological profile

Animal Dose Hb RBCs Hct MCV MCH MCHC WBC PLT groups mg/Kg (g/dl) (×103/cm) (%) (%) (pg/cell) (g/dl) (×103/cm) (×103/cm)

Control - 10.27±0.53 5.23±0.26 33.3±1.9 63.54±0.59 19.63±0.30 30.9±0.41 3.76±0.50 347.3±55.8

HEVA 150 11.16±0.10 5.55±0.03 35.9±0.18 64.59±0.28 20.08±0.10 31.0±0.16 3.68±0.50 405.7±20.6

HEVA 300 10.66±0.47 5.50±0.22 34.8±1.47 63.29±0.78 19.37±0.35 30.6±0.40 3.73±0.29 342.3±39.5

EEVA 150 10.47±0.23 5.05±0.15 33.8±0.70 67.23±1.37 20.80±0.45 30.9±0.19 3.88±0.42 384.8±43.49

EEVA 300 10.63±0.36 5.37±0.21 33.6±1.2 62.8±1.16 19.85±0.46 31.5±0.28 3.02±0.43 369.7±58.62

WDVA 150 10.4±0.22 5.16±0.18 33.7±0.7 65.57±1.12 20.20±0.33 30.8±0.34 3.77±0.60 453.7±25.2

WDVA 300 11.1±0.29 5.67±0.14 36.4±0.96 64.11±0.21 19.57±0.18 30.5±0.32 4.71±0.41 331.5±20.2

n=10. Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant difference as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica.

3.2.2.3: Effects on coagulation

Table -22 reveals the effects of different extract of Vernonia anthelmintica on coagulation. The results show non-significant alterations in TT (thrombin time), PT (prothrombin time) and aPTT (activated partial thromboplastin time) in all tested groups except WDVA that shows significant increase in these parameters as compared to control. Fibrinogen is found to be significantly decreased in all treatment groups as compared to control.

Table-22 Effects of Vernonia anthelmintica on coagulation

Animal Dose TT PT aPTT Fibrinogen groups mg/Kg (sec) (sec) (sec) (mg/dl) Control - 5.28±0.09 6.46±0.64 5.36±0.11 246.5±15.9

HEVA 150 5.13±0.03 11.4±0.25 5.45±0.12 154.1±11.4**

HEVA 300 5.16±0.02 12.5±3.03 5.23±0.04 138.0±14.02**

EEVA 150 5.91±0.7 10.7±1.62 7.73±0.92 168.8±13.9**

EEVA 300 5.33±0.09 10.6±1.41 10.5±1.83 191.7±6.01*

WDVA 150 5.17±0.02 10.8±1.25 7.70±3.00 187.4±5.36*

WDVA 300 11.45±2.33** 16.1±1.10** 11.7±3.86** 164.7±10.9**

n=10. Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant difference as compared to control’ HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= water decoction of Vernonia anthelmintica.

3.2.2.4 Effects on Glucose metabolism Table-23 shows the effects of different extracts of Vernonia anthelmintica (HEVA, EEVA, WDVA) on glucose metabolism in rabbit model after 60 days dosing. The result revealed no significant change in fasting blood sugar and HBA1c levels in treatment groups as compared to control.

Table-23 Effects of Vernonia anthelmintica on diabetes markers

Treatment Dose FBS HBA1c groups mg/Kg mg/dl % Control - 106.3±9.8 3.85±0.06

HEVA 150 110.4±4.9 3.61±0.10

HEVA 300 109.1±3.9 3.82±0.04

EEVA 150 105.1±4.7 3.66±0.07

EEVA 300 104.3±3.3 3.77±0.07

WDVA 150 107.6±6.3 3.77±0.04

WDVA 300 114.4±1.8 3.61±0.07

3.2.2.5 Effects on lipid profile Table 24 shows the effects of different extracts of Vernonia anthelmintica (HEVA, EEVA, WDVA) on lipid profile in rabbit model after 60 days dosing. The results reveal no significant change in cholesterol/HDL ratio, cholesterol, Triglycerides, HDL, LDL and VLDL in all treatment groups except EEVA that shows significantly higher cholesterol and HDL and significantly lower triglycerides and VLDL levels as compared to control.

Table -24 Effects of Vernonia anthelmintica on lipid profile

Animal Dose Chol/Hdl cholesterol TGs HDL LDL VLDL groups mg/kg ratio mg/dl Control - 1.89± 0.04 16.1± 0.7 39±2.1 8.7±0.49 6.6±1.05 7.8±0.42 HEVA 150 2.0± 0.02 23.5±1.6* 38±1.4 11.3±0.70 8.0±0.37 7.6±0.29 HEVA 300 2.0±0.01 25.7±2.6** 36.5±0.62 12.2±1.1 10.0±2.3 7.3±0.12 EEVA 150 1.9± 0.04 29.5±0.62** 31±0.84** 15.5±0.42** 11.0±0.84 6.1±0.25** EEVA 300 1.6±0.05** 22.1±1.02 28.5±1.0** 13.8±0.95** 7.0±0.80 5.7±0.20** WDVA 150 1.87±0.02 21.0±2.23 35.7±1.42 11.2±1.23 6.5±0.68 7.1±0.28 WDVA 300 1.91±0.09 13.5±1.21 36±2.28 7.0±0.53 4.25±0.81 7.2±0.45 n=10 Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant difference as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica.

3.2.2.6 Effects on liver function

Table 25 shows the effects of different extracts of Vernonia anthelmintica (HEVA, EEVA, WDVA) on liver function in rabbit model after 60 days dosing. The results reveal no significant change in total bilirubin, direct bilirubin, SGOT, SGPT, ALP, GGT as compared to control exceptional are EEVA and WDVA that have significant high levels of SGPT in the doses of 300 mg as compared to control.

Table -25 Effects of Vernonia Anthelmintica on liver function Animal Dose TBR DBR SGOT SGPT ALP GGT groups mg/kg mg/dl mg/dl U/L U/L U/L U/L Control - 0.41±0.1 0.21±0.08 95.5±8.07 55.1±5.25 45.3±10.4 9.8±1.2 HEVA 150 0.47±0.07 0.24±0.03 94.7±8.31 67.1±8.24 48.6±6.9 10.6±0.5 HEVA 300 0.29±0.07 0.17±0.04 82.7±8.94 91.5±8.8 43.9±10.0 10.5±1.3 EEVA 150 0.34±0.08 0.18±0.05 100±11.72 59.5±6.13 61.2±4.0 7.7±0.77 EEVA 300 0.31±0.06 0.21±0.05 98.8±8.90 98.3±12.8* 31.8±5.4 10.5±1.4 WDVA 150 0.29±0.06 0.18±0.05 95.6±10.50 84.3±8.79 51.4±2.4 7.7±1.40 WDVA 300 0.43±0.11 0.26±0.08 105±11.69 93.3±6.92* 73.0±11.1 11.9±1.0 n=10. Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant difference as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= water decoction of Vernonia anthelmintica.

3.2.2.7 Effects on renal function:

Table-26 shows the effects of different extracts of Vernonia anthelmintica (HEVA, EEVA, WDVA) on renal function in rabbit model after 60 days dosing. The results reveal no significant change in serum urea, uric acid, total protein and creatinine levels in any treatment group as compared to control.

Table-26 Effects of Vernonia anthelmintica on renal function Uric Total Animal Dose Urea Creatinine Acid Protein groups mg/Kg mg/dl mg/dl mg/dl mg/dl 1.22± Control - 54.9 ±7.12 0.91± 0.10 57.68 ±7.9 0.22 1.21± HEVA 150 65.7± 5.7 0.88± 0.12 76.5 ±7.6 0.19 1.50± HEVA 300 67.3 ± 6.7 1.45± 0.18 72.3 ±7.5 0.26 76.6 ± 1.01± EEVA 150 1.11± 0.22 65.0±7.8 2.83 0.18 68.3 ± 1.17± EEVA 300 1.06± 0.12 66.2±6.5 6.06 0.22 63.1 ± 1.13± WDVA 150 1.1± 0.21 75.3±5.3 6.03 0.15 72.8 ± 1.45± WDVA 300 1.27± 0.15 76.7±6.6 6.10 0.22 n=10. Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant difference as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica.

3.2.2.8 Effects on Electrolytes

Table-27 shows the effects of different extracts of Vernonia anthelmintica (HEVA, EEVA, WDVA) on serum electrolytes in rabbit model after 60 days dosing. The results reveal no significant change in sodium and calcium in treatment groups as compared to control except EEVA group that has significant high levels of sodium in the doses of 150 and 300 mg as compared to control.

Table-27 Effects of Vernonia anthelmintica on electrolytes Dose Sodium Calcium Animal groups mg/kg mg/dl Control - 125.1± 5.8 7.38±0.87 HEVA 150 129.8± 6.4 7.53±0.81 HEVA 300 132.9±4.8 8.15±0.67 EEVA 150 140.8±4.4 7.9±0.54 EEVA 300 146.4±4.2* 8.0±0.58 WDVA 150 146±2.06* 8.2±0.54 WDVA 300 134.7±8.5 9.0±0.65

n=10. Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant different as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica.

3.3 Pharmacological models 3.3.1 Anti-inflammatory activity Carrageenan induced rat paw model: Table 28 shows results of anti-inflammatory potential of three different extracts of Vernonia anthelmintica (HEVA, EEVA,AND WDVA) in two different doses (150mg/kg and 300 mg/kg) as compared to control and standard anti- inflammatory drug ibuprofen in the dose of 100 mg/kg. There was a significant decrease in paw edema of most of the treatment groups and standard group as compared to control at 1 h, 2 h, 3h and 4 h, 5h and 24 h.

In the present study, the effects of treatment and standard groups were assessed for volume of paw edema and percent inhibition as compared to control in carrageenan- induced paw edema rat model. The results reveal that standard drug ibuprofen in the dose of 100 mg/kg show highly significant decreased paw edema volume. This significant decrease is started from second hour and remains significant till 24 hours. The percent inhibition of edema volume as compare to control at time 1 h, 2 h, 3h, 4h, 5h and 24 h are shown in the table.

HEVA do not cause any significant decrease in edema volume at the dose of 150 mg/kg but 300 mg/kg dose of HEVA significantly decreased edema volume. This decrease is started at 2h and maximum at 3 h and remain significant till 24 h. The percent inhibition of edema in HEVA 300 group is found to be 58.2%, 70.9%

Table-28 Anti-inflammatory activity of Vernonia anthelmintica in rat paw Group Edema volume at time (ml) (dose/kg) % inhibition of inflammation at time (%) 1 h 2h 3h 4h 5h 24h Control 0.788±0.08 1.47±0.20 2.20±0.11 2.45±0.27 2.55±0.20 1.05±0.11

Ibuprofen 0.64±0.11* 0.35±0.04** 0.51±0.11** 0.66±0.11** 0.549±0.13** 0.35±0.07** (100) (18.7%) (82.3%) (76.8) (73.06%) (78.8) (71.60) HEVA 0.76±0.06 1.12±0.06 2.19±.11 2.40±0.26 2.52±0.34 0.89±0.15 (150) (3.5) (8.55) (0.4) (2%) (1.1) (15.2) HEVA 0.735±0.08 0.799±0.19* 0.64±0.11** 1.14±0.29** 1.13±0.28** 0.20±0.09** (300) (14.4) (58.2) (70.9) (53.4) (55.6) (80.9) EEVA 0.72±0.07 1.27±0.05 2.18±0.11 2.31±0.25 2.47±0.13 0.59±0.11** (150) (8.6) (21.3) (0.9) (5.71) (3.1) (43.8) EEVA 0.44±0.08 0.72±0.18* 1.10±0.11** 0.98±0.15** 1.14±0.15** 0.38±0.09** (300) (44.1) (58.2) (50) (60) (55.2) (68.8) WDVA 0.66±0.12 1.16±0.14 1.45±0.53 1.55±0.2 1.19±0.16** 0.44±0.03** (150) (16.2) (25.8) (34.0) (36.7) (53.3) (58.09) WDVA 0.56±0.13 0.72±0.16* 2.18±0.1 1.93±0.19 2.15±0.21 0.145±0.02** (300) (28.9) (42.1) (0.9) (21.22) (15.6) (86.1)

n=8 Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant difference as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica.

3.3.2 Analgesic activity 3.3.2.1 Hot plate method Table 29 shows results of analgesic potential of these different extracts of Vernonia anthelmintica (HEVA, EEVA,AND WDVA) in two different doses ( 150mg/kg and 300 mg/kg) as compared to control and standard analgesic drug aspirin in the dose of 300 mg/kg. Reaction time was measured in seconds, there was a significant increase in reaction time of all treatment groups and standard group as compared to control at 1 h, 2 h, 3h and 4 h.

Table-29 Analgesic activity of Vernonia anthelmintica by Hot plate method

Treatment Dose Basal Reaction time (s) Group mg/kg reaction Time(s) 1h 2h 3h 4h

Control - 4.25±0.18 3.99±0.07 3.75±0.05 3.84±0.07 3.90±0.15

Aspirin 300 4.24±0.08 7.7±0.70** 7.74±0.92** 7.39±0.60** 5.68±0.33**

HEVA 150 4.11±0.63 5.7±0.23* 6.01±0.21 6.32±0.26* 5.44±0.34**

HEVA 300 4.15±0.04 8.2±0.51** 8.49±0.79** 6.89±0.94** 6.7±0.35**

EEVA 150 4.12±0.04 5.19±0.21 7.1±0.30** 4.59±0.44 4.29±0.24

EEVA 300 4.11±0.09 6.97±0.50** 7.27±0.65** 6.33±0.25* 5.76±0.18**

WDVA 150 4.05±0.06 5.57±0.18 6.7±0.53** 5.81±0.27 5.38±0.23*

WDVA 300 4.19±0.06 7.09±0.24** 8.4±0.47** 7.64±0.34** 6.66±0.33**

n=8 Values are expressed as mean ±S.E.M *P ≤ 0.05 significant; **P ≤0.005 highly significant difference as compared to control HEVA= Hexane extract of Vernonia anthelmintica EEVA= Ethanol extract of Vernonia anthelmintica WDVA= Water decoction of Vernonia anthelmintica

3.3.2.2 Tail flick method Table-30 shows results of analgesic potential of these different extracts of Vernonia anthelmintica (HEVA, EEVA and WDVA) in two different doses (150mg/kg and 300 mg/kg) as compared to control and standard analgesic drug aspirin in the dose of 300 mg/kg. Reaction time was measured in seconds, there was a significant increase in reaction time of all treatment groups and standard group as compared to control at 1 h, 2 h, 3h and 4 h.

Table-30 Analgesic activity of Vernonia anthelmintica by tail flick method

Treatment Dose Basal Reaction time (s) Group mg/kg reaction Time(s) 1h 2h 3h 4h

Control - 1.24±0.16 1.2±0.03 1.15±0.05 1.098±0.64 1.00±0.03

Aspirin 300 1.27±0.15 2.7±0.11** 2.95±0.05** 3.44±0.22** 2.76±0.22**

HEVA 150 1.27±0.15 3.67±0.22** 3.67±0.40** 3.18±0.25** 2.47±0.14**

HEVA 300 1.38±0.23 5.27±0.47** 3.37±0.11** 2.55±0.26* 1.82±0.17

EEVA 150 1.28±0.13 3.45±0.38** 3.23±0.39** 2.86±0.38** 1.99±0.12*

EEVA 300 1.36±0.13 3.96±0.21** 5.16±0.04** 4.25±0.16** 2.95±0.19**

WDVA 150 1.27±0.12 2.54±0.33* 3.21±0.26** 3.09±0.02** 2.73±0.34**

WDVA 300 1.39±0.15 2.70±0.16** 3.31±0.27** 3.91±0.62** 1.95±0.27*

3.3.3 Hyperlipidemic model (HFHS)

3.3.3.1Antihyperlipidemic activity

Table-31 show the antihyperlipidemic effects of HEVA, EEVA and WDVA as compared to control, disease control and atorvastatin (standard antihyperlipidemic drug). In comparison to control, disease control show significant difference in terms of serum cholesterol, triglycerides, LDL and VLDL. Standard drug atorvastatin showed no significant difference as compared to normal control whereas HEVA, EEVA and WDVA show significant difference in HDL levels and the latter two are also significantly different in terms of total cholesterol as compared to control. In comparison to disease control all control and treatment groups are significantly different in terms of triglycerides, LDL and VLDL. In addition to these parameters EEVA also shows significant difference in terms of HDL. In comparison to standard anti-hyperlipidemic drug atorvastatin, disease control shows significant difference in terms of cholesterol, triglycerides, LDL and VLDL. HEVA, EEVA and WDVA also show significant higher levels of HDL and due to this reason EEVA and WDVA show significant higher total cholesterol levels as compared to standard drug.

Table-31 Effects of Vernonia anthelmintica on hyperlipidemia in HFHS rat model

Treatment Diet Dose Cholesterol triglyceride HDL LDL VLDL group mg/kg mg/dl Control Normal - 54.50±3.05 46.25 ±2.25 39±2.05 11±0.92 9.0±0.46 a** a** a** a**

Disease control HFHS - 102.50±9.19 83.25± 7.36 53± 2.4 48.5± 10.7 16.75±1.49 c** c** c** c* b** b** b* b* Atorvastatin HFHS 10 57.75± 2.41 34.50± 2.78 46.25±2.38 14±1.035 6.75±0.55 c** a** a** a** HEVA HFHS 300 80.25± 7.41 40.75±0.86 63.75±5.97 16.7± 1.49 8.0±0.26 c* a** c** a** a** b* EEVA HFHS 300 99.25±5.38 29.25± 1.91 79± 3.69 18.25±1.82 5.75±0.31 c** a** a** a** a** b** c* c** c* b** WDVA HFHS 300 97.25±1.23 46.25± 2.44 63.5± 3.36 29± 1.36 9.25±0.67 c** ** c** a** b** b* n=8 Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant ‘a’ different as compared to disease control. ‘b’ different as compared to standard. ‘c’ different as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica. HFHS= high fat high sugar.

3.3.3.2 Cardiac risk parameters: Table 32 and Fig-4, Fig-5and Fig-6 show the cardiac risk parameters including atherogenic index, Cholesterol /HDL ratio and LDL/HDL ratio of HEVA, EEVA and WDVA as compared to control, disease control and atorvastatin ( standard antihyperlipidemic drug). In comparison to control, disease control shows significant difference in terms of atherogenic index, cholesterol/HDL ratio and LDL/HDL. Standard drug atorvastatin and test groups show no significant difference as compared to normal control. In comparison to disease control all control and treatment groups are significantly different in terms of atherogenic index, cholesterol/HDL ratio and LDL/HDL. In comparison to standard antihyperlipidemic drug atorvastatin, disease control shows significant difference in terms of atherogenic index, cholesterol/HDL ratio and LDL/HDL

Table -32 Effects of seed extracts of Vernonia anthelmintica on cardiac risk parameters Treatment Diet Dose Atherogenic Cholesterol/HDL LDL/HDL Group mg/kg index ratio ratio Control Normal - 0.398± 0.023 1.39±0.02 0.28±0.01 a** a** a**

Disease control HFHS - 0.859±0.12 1.95±0.16 0.91±0.19 c**b* c**b* c**b*

Atorvastatin HFHS 300 0.256±0.33 1.25±0.03 0.30±0.01 a** a** a**

HEVA HFHS 300 0.16± 0.031 1.26±0.04 0.26±0.01 a** a** a**

EEVA HFHS 300 -0.06± 0.01 1.25±0.01 0.22±0.01 a**c** a** a**

WDVA HFHS 300 0.45±0.12 1.55±0.07 0.47±0.04 a* a* a**

n=8 Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant ‘a’ significantly different as compared to disease control. ‘b’ significantly different as compared to standard. ‘c’ significantly different as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica. HFHS= high fat high sugar.

atherogenic index

1.2

0.8 c** b** 0.859 0.6

0.4 a** a** 0.45 0.398

ATHEROGENIC INDEX ATHEROGENIC 0.2 a** a**c** a**c** -0.06 0.256 0.16 0 control disease control atorvastatin HEVA EEVA WDVA -0.2 TREATMENT GROUPS

Figure 4: Effects of extracts of Vernonia anthelmintica on atherogenic index in

HFHS rat model

cholestrol/ HDL ratio 2.5

2 c** b** 1.5 1.95 a** a** 1.55 1 1.39 a** a** a** 1.25 1.26 1.25

0.5 CHOLESTEROL/HD RATIO CHOLESTEROL/HD

0 control disease control atorvastatin HEVA EEVA WDVA TREATMENT GROUPS

Figure 5: Effects of extracts of Vernonia anthelmintica on Cholesterol /HDL ratio in

HFHS rat model

LDL/HDL Ratio 1.2

0.8 c**b** 0.91 0.6

0.4 LDL/HDL RATIO LDL/HDL a** 0.2 a** a** a** a** 0.44 0.28 0.3 0.26 0.22 0 control disease atorvastatin HEVA EEVA WDVA control TREATMENT GROUPS

Figure-6: Effects of extracts of Vernonia anthelmintica on LDL/HDL ratio in HFHS rat model

3.3.3.3 Anti-diabetic profile Table 33 and Fig-8 show the Fasting blood glucose levels of animals receiving HEVA, EEVA and WDVA as compared to control, disease control and atorvastatin (standard antihyperlipidemic drug). In comparison to control and standard drug, disease control shows significant difference in fasting blood glucose levels whereas in comparison to disease control all control and treatment groups show significantly lower blood glucose levels.

Table -33 Effects of Vernonia anthelmintica on fasting blood glucose in HFHS Rat model Treatment Group Diet Dose Blood glucose levels mg/kg mg/dl Control Normal - 124±2.97 a** Disease control HFHS - 182.1±8.16c**, b** Atorvastatin HFHS 300 126.6±7.7 a** HEVA HFHS 300 119.0±4.29 a** EEVA HFHS 300 116.3 ±10.99 a** WDVA HFHS 300 139.6±8.52 a**

n=8. Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant. a different as compared to disease control. b different as compared to standard c different as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica. HFHS= high fat high sugar.

250

200

c**b** 150 182.1 a** a** a** 100 a** a** 139.6 124 126.6 119 116.3

50 FASTING BLOODGLUCOSEMG/DL FASTING

0 control disease atorvastatin HEVA EEVA WDVA control TREATMENT GROUPS

Figure 8: Effects of extracts of Vernonia anthelmintica on fasting blood glucose levels in HFHS rat model

3.3.3.4 Hepatic profile

Table-34 show the Liver function tests of HFHS rat model receiving HEVA, EEVA and WDVA as compared to control, disease control and atorvastatin (standard antihyperlipidemic drug). There is no significant difference in levels of GGT and GPT in different groups. Standard drug shows significantly lower ALP levels as compared to disease control. GOT and TBR show different patterns of significant differences among different groups.

Table-34 Effects of Vernonia anthelmintica on liver function in HFHS model

Treatment Dose GPT GGT ALP GOT TBR Diet Group mg/Kg U/L U/L U/L U/L U/L Control Normal - 77.2± 4.08 1.17±0.29 288.4±9.36 134.5±5.8 0.25±0.03 a*, b** b* Disease control HFHS - 84.15± 4.23 1.57±0.20 352.0±28.5 166.4±8.6 0.15±0.03 b** c*, b** b** Atorvastatin HFHS 10 114.0± 13.6 2.31± 0.32 247.3±15.1 202.1±7.0 0.67±0.13 a** c** a**, c* HEVA HFHS 300 77.8± 4.95 1.95±0.36 296.4±17.1 149.7±6.9 0.87±0.10 b** a**,c* EEVA HFHS 300 71.9± 4.95 1.25±0.43 273.3±17.7 161.0±5.3 0.45±0.14 b** WDVA HFHS 300 71.2±72.34 2.58±0.50 252.4±32.7 190.3±4.0 0.35±0.01 c** n=8. Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant. a different as compared to disease control. b different as compared to standard. c different as compared to control. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica. HFHS= high fat high sugar.

3.3.4 Diabetic models 3.3.4.1 Normoglycemic rat model Table 35 shows the effects of different extracts of Vernonia anthelmintica (HEVA, EEVA, WDVA) on blood glucose levels at 0, 30, 60, 90 and 120 minutes in normoglycemic rat on acute dosing (300 mg/ kg). The results reveal that only standard drug glibenclamide significantly decreases the blood sugar level as compared to control at all tested corresponding times. Standard group also show significantly reduced blood glucose level when compared with its corresponding initial time. The tested groups HEVA and EEVA show significant increase in blood glucose level as compared to their corresponding initial levels. However WDVA show slightly different pattern as it shows significantly reduced blood glucose levels as compared to control at all tested times but do not decline to very low levels as seen in glibenclamide group.

Table-35 Effect of Vernonia anthelmintica on blood glucose level of normoglycemic rat

Treatment Dose Blood glucose levels (mg/dl) groups mg/kg 0 30 60 90 120 minutes Control - 68.2±2.1 88.5±0.6 70.5±1.7 89.0±0.4 93±1.2 Normal saline b** b** b** Glibenclamide 5 72.0±1.4 39±0.8 45.2±0.8 28.5±0.6 29±1.1 a**c** a**c* a**c** a**c**

HEVA 300 69.2±1.5 75.5±1.7 80.2±1.6 83.25±3.4 86.7±1.6 b**c** b**d** b** b**

EEVA 300 67.0±3.8 89.5±2.8 93.5±1.8 106±2.9 97.5±4.7 b** b**d** b**d** b**

WDVA 300 63.6±1.2 71.8±1.26 72.3±1.7 76.8±3.5 63.38±2.0 b**c** b** b**c** c**

n=8. Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant. a reduced from values at 0 minute in each corresponding group. b increased from values at 0 minute in each corresponding group. c reduced from control values of normoglycemic rats. d increased from control values of normoglycemic rats. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica. HFHS= high fat high sugar.

3.3.4.2 Glucose induced Hyperglycemic model:

Table 36 reveal the comparison of blood glucose levels of normal control, standard (glibenclamide 2.5 mg/kg) and treatment (HEVA, EEVA and WDVA 300 mg/kg) groups at 0,30,60,90 and 120 minutes following oral glucose challenge in rats on acute dosing.

Control group reveal sudden significant increase as compared to initial glucose levels as the result of glucose challenge at time 30 minutes. These levels gradually get normalized at time 60, 90 and 120 minutes. Thus at 30 minutes readings show significant increase followed by significant decrease in blood glucose levels at time 60,90 and 120 minutes

Standard and treatment groups are different from control as they resisted glucose challenge and does not show significant increase in blood glucose levels. 30 min reading of standard and all treatment groups is significantly decreased as compared to blood glucose levels of control group. However standard group is different from other treatment groups as the former shows significant decrease as compared to control values at all respective times as well as significantly reduced from their corresponding initial levels. HEVA, EEVA and WDVA showed slightly different pattern as the decline in blood glucose levels at time 60, 90 and 120 minutes are of lesser magnitude as compared to standard drug.

The most significant resistance to glucose challenge is observed in glibenclamide group followed by HEVA, EEVA and WDVA at time 30 minutes.

Table -36 Effect of Vernonia anthelmintica on blood glucose level of hyperglycemic rat

Blood glucose levels Treatment Dose (mg/dl) groups mg/kg 0 30 60 90 120 minutes Control 76.5±2.1 153.5±2.7 65.5±1.3 59.7±1.6 56.2±2.1 Normal saline a** a* a** a** Glibenclamide 5 68.2±2.5 58.0±1.0 55.0±1.3 51.7±0.8 57.0±3.5 a* b** a**b* a**b* a** HEVA 300 70.0±4.4 72.2±5.7 71.0±5.0 73.7±2.9 67.0±3.2 b** b** c* EEVA 300 71.0±2.0 81.5±2.9 70.5±0.5 66.2±1.7 64.5±2.5 a* b** a* WDVA 300 79.75±2.7 88.7±3.3 74.0±3.5 78.0±3.5 77.2±5.2 a* b** c* c** c** n=8. Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant. a different from values at time 0 in each corresponding group. b reduced from control values of hyperglycemic rats at corresponding time. c increased from control values of hyperglycemic rats at corresponding time. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica. HFHS= high fat high sugar.

3.3.4.3 Alloxan induced diabetic rat model:

Table 37 reveal the comparisons in fasting blood sugar of disease control (alloxan induced diabetic rats), treatment (HEVA, EEVA and WDVA 300 mg/kg) and standard (Glibenclamide 2.5mg/kg) groups at 30, 60, 90 and 120 minutes of acute dosing.

The results reveal that disease control group show significantly increased blood glucose levels at all tested times as compared to their corresponding initial levels.

Standard group show significantly reduced levels as compared to control as well as their corresponding initial readings at all tested times (30,60,90 and 120 minutes).HEVA, EEVA and WDVA all showed significant increase in blood glucose levels at time 30 minutes followed by significant decrease in HEVA and EEVA group at time 60, 90 and 120 minutes. However WDVA showed significant increase in blood glucose levels even at the 60, 90 and 120 minutes as compared to control as well as corresponding initial levels. Data suggest the significant hypoglycemic effect of standard, HEVA and EEVA as compared to disease control.

Table-37 Effect of Vernonia anthelmintica on blood glucose level of Alloxan induced diabetic rat Blood glucose levels (mg/dl) Treatment Dose 0 30 60 90 120 groups mg/kg minutes Disease Control - 267±7.7 300.5±9.3 341.75±6.8 343.7±12.7 318.6±10.9 b** b** b** b** Glibenclamide 5 287±7.4 262.2±14.4 220.1±11 187.2±8.6 163.5±14.6 a** c** a**c** a**c** a** c** HEVA 300 277±10.2 314.5±9.0 278.5±14.8 259±16.2 220.7±18.2 b** c** c** a** c**, EEVA 300 279±4.2 322.5±4.3 289.0±4.22 249.2±4.1 213±4.2 b** c** a**c** a** c** WDVA 300 280.5±10.1 344.5±13.8 356.2±14.6 361.2±16.7 344±8.3 b** d** b** b** b** n=8. Values are expressed as mean ±S.E.M. *P ≤ 0.05 significant; **P ≤0.005 highly significant. a reduced from values at 0 minute in each corresponding group. b increased from values at 0 minute in each corresponding group. c reduced from control values of diabetic rats. d increased from control values of diabetic rats. HEVA= Hexane extract of Vernonia anthelmintica. EEVA= Ethanol extract of Vernonia anthelmintica. WDVA= Water decoction of Vernonia anthelmintica. HFHS= high fat high sugar.

3.4 Histopathological Examination: Histological studies showing heart, kidney and liver of treated rabbits compared with normal controls.

3.4.1 Histopathology of Heart: Figure-9 shows the transverse section exhibiting myocardium of control animal with intact architecture. No findings of inflammation, fibrosis, and necrosis are seen. No signs of infarct are noticed. Figure 1

0, 11, 12, 13, 14 and 15 show the heart of animals treated with different extracts of Vernonia anthelmintica in two different doses for 60 days. The myocardium of treated animals show no histopathological variations as compared to control.

Figure-9: Cardiac tissue of control rabbit showing normal heart. (Magnification: 10x10)

Figure- 10: Normal cardiac tissue of rabbit treated with 150 mg/Kg of HEVA over 60 days period. (Magnification: 10x10)

Figure-11: Normal cardiac tissue of rabbit treated with 300 mg/kg of HEVA over 60 day period. (Magnification: 10x10)

Figure-12: Normal cardiac tissue of rabbit treated with 150 mg/kg of EEVA over 60 day period. (Magnification: 10x10)

Figure- 13: Normal cardiac tissue of rabbit treated with 300 mg/kg of EEVA over 60 day period. (Magnification: 10x10)

Figure-14: Normal cardiac tissue of rabbit treated with 150 mg/kg of WDVA over 60 day period. (Magnification: 10x10)

Figure-15: Normal myocardial tissue of rabbit treated with 300 mg/kg of WDVA over 60 day period. (Magnification: 10x10)

3.4.2 Histopathology of Kidney Figure-16 show Kidney sections of normal control with intact parenchymal system, renal parenchyma consist of multiple variable size glomeruli, no inflammation seen in interstitium. Figure-17, 18, 19, 20, 21 and 22 show the transverse section exhibiting renal tissues of animals treated with different extracts of Vernonia anthelmintica in two different doses for 60 days. The renal tissue of treated animals show no histopathological variations as compared to control.

Figure-16: Renal tissue of control rabbit showing normal kidney. (Magnification: 25x10)

Figure-17: Normal renal tissue of rabbit treated with 150 mg/kg of HEVA over 60 day period. (Magnification: 25x10)

Slide-18: Normal renal tissue of rabbit treated with 300 mg/kg of HEVA over 60 day period. (Magnification: 10x10)

Figure- 19: Normal renal tissue of rabbit treated with 150 mg/kg of EEVA over 60 day period. (Magnification: 10x10)

Slide- 20: Normal renal tissue of rabbit treated with 300 mg/kg of EEVA over 60 day period. (Magnification: 10x10)

Figure-21: Normal renal tissue of rabbit treated with 150 mg/kg of WDVA over 60 day period. (Magnification: 10x10)

Figure-22: Normal renal tissue of rabbit treated with 300 mg/kg of WDVA over 60 day period. (Magnification: 10x10)

3.4.3 Histopathology of Liver: Following slides showed the hepatic transverse sections of control and treated animals.

Figure- 23 show normal liver tissue of control rabbit. It show the normal hepatocytes exhibiting intact architecture.

Figure- 24 show hepatic tissue of rabbit treated with 150 mg/kg of hexane extract of Vernonia anthelmintica over 60 day period. It reveal mild signs of inflammation within hepatocytes.

Figure- 25 reveal hepatic tissue of rabbit treated with 300 mg/kg of hexane extract of Vernonia anthelmintica over 60 day period. It show marked ballooning degeneration, mild microvascular steatosis, slight congestion of sinusoids and mild to moderate signs of inflammation are seen within hepatocytes.

Figure-26 revealed normal hepatic tissue of rabbit treated with 150 mg/kg of Ethanol extract of Vernonia anthelmintica over 60 day period. It showed no significant histopathological alterations as compared to normal control. Figure- 27 revealed hepatic tissue of rabbit treated with 300 mg/kg of water ethanol of Vernonia anthelmintica over 60 day period. It showed mild to moderate microvascular steatosis as well as inflammation within hepatocytes. However ballooning degeneration and fibrosis were not identified.

Figure-28 reveal normal hepatic tissue of rabbit treated with 150 mg/kg of water decoction of Vernonia anthelmintica over 60 day period. It shows no significant histopathological changes as compared to normal control hepatic tissue.

Figure-29 show hepatic tissue of rabbit treated with 300 mg/kg of WDVA over 60 day period. It shows slight congestion of sinusoids, mild steatosis with mild to moderate signs of inflammation within hepatocytes.

Figure-23: Normal liver tissue of control rabbit. (Magnification: 10x10)

Figure-24: Hepatic tissue of rabbit treated with 150 mg/kg of HEVA over 60 day period showing mild signs of inflammation. (Magnification: 10x10)

Figure-25: Hepatic tissue of rabbit treated with 300 mg/kg of HEVA over 60 day period showing mild to moderate signs of inflammation. (Magnification: 10x10)

Figure-26: Normal hepatic tissue of rabbit treated with 150 mg/kg of EEVA over 60 day period. (Magnification: 10x10)

Figure- 27: Hepatic tissue of rabbit treated with 300 mg/kg of WDVA over 60 day period showing mild to moderate signs of inflammation. (Magnification: 10x10)

Figure-28: Normal hepatic tissue of rabbit treated with 150 mg/kg of WDVA over 60 day period. (Magnification: 10x10)

Figure-29: Hepatic tissue of rabbit treated with 300 mg/kg of WDVA over 60 day period showing mild to moderate signs of inflammation. (Magnification: 10x10)

4. DISCUSSION

Herbal medicines have a prominent role in treatment and prophylaxis of varying ailments and usually contain potent pharmacological compounds. Apart from their known pharmacological activities in conventional medical systems its worth mentioning that these herbs are usually therapeutic at certain dose but shows toxicity on increasing the dose (Saad et al., 2006).

Previous reviews on safety of herbal medicine reported that most of the present herbs do not correspond to the plants described in the earlier literature as most of the herbs has undergone various primitive changes due to cultivating and environmental factors (Azaizeh et al., 2006). It is therefore recommended that every time any herb to be undertaken for pharmacological screening must go through acute toxicity, cytotoxicity and phytochemical assays to validate the exact data for the specific herb. To address the similar concerns present study was designed to evaluate the safety of different extracts of Vernonia anthelmintica seeds.

The phytochemical screening of HEVA in present study revealed the presence of alkaloids, cardiac glycosides, flavonoids, tannins, terpenoids and fixed oils. EEVA found to have similar phytochemical composition as HEVA except that carbohydrates were also present. WDVA differs drastically in comparison with hexane and ethanol extract as it contains only cardiac glycosides, flavonoids and terpenoids.

The initial step in the evaluation of toxic characteristic of certain herbal medicine is the determination of LD50. Results of acute toxicity serve as the basis of classification and labeling of drug, thus gives an idea about the possible mechanism of toxicity, helps in establishing the safe dose and analyzing the bioactive potential of drug (Ukwuani et al., 2012).

In present study in-vivo acute oral toxicity test clearly revealed the safety of water decoction, hexane and ethanol extract of Vernonia anthelmintica. LD50 estimated by Lorke’s method (1983) was found to be greater than 5000 mg/ kg body weight. All extracts were nontoxic and comes under the category 5 of global

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harmonization system that encircle all drugs with LD50 value >2000-5000mg/kg (Pratt, 2002).

The present results suggest that both hexane and ethanol extracts contains bioactive compounds that holds cytotoxic effects. The cytotoxic potential of drugs on the basis of brine shrimp lethality test was classified in various previous studies such that LC50 value above 1000 µg/ml is considered as nontoxic, LC50 value between 500- 1000 µg/ml is stratified as weakly cytotoxic, 100-500 µg/ml reveals moderate toxicity and 0-100 µg/ml is a predictor of strong cytotoxicity (Meyer et al., 1982a and Pimenta et al., 2003). Thus hexane and ethanol extracts of Vernonia anthelmintica shown to have moderate toxicity with the LC50 value of 216.1 and 104.1 µg/ml respectively at 24 hours.

Apart from the individual results of LD50 of acute oral toxicity and LC50 of brine shrimp assay this study also strengthen the correlation between LC50 of brine shrimp and LD50 of acute oral toxicity assay in accordance with Parra et al. (2001). According to Parra correlation data the brine shrimp LC50 <10 µg/ml holds LD50 in the range of 100-1000 mg/kg; LC50 <20 u/ml possesses LD50 in the range of 1000-2500 mg/kg and LC50 >25 µg/ml indicates LD50 in the range of 2500-8000 mg/kg with good correlation (r = 0.85; P < 0.05) (Parra et al., 2001). Thus in accordance with this correlation, the LC50 of both extracts of Vernonia anthelmintica in the current study correlates with LD50 of acute oral toxicity in mice.

Since the acute toxicity studies established its safety in acute exposure, therefore further assessment was conducted to establish the safe use of Vernonia anthelmintica extracts after dosing up to 60 days in rabbits. In the present study sub chronic toxicity profile of EEVA, HEVA and WDVA was evaluated using anthropometric, hematological, biochemical and histopathological parameters.

Oral treatment of HEVA, EEVA and WDVA over the period of 60 days in the doses of 150 mg/kg and 300 mg/kg were found to be associated with significant weight loss as compared to control animals in dose dependent manner. These results of the study on weight loss suggest that it might have this effect either due

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to appetite suppression or inhibition of nutrient utilization. Unfortunately the food consumption of experimental rats was not thoroughly observed that could provide beneficial insights to understand the weight losing potential of the tested drug.

However folk literature has shown Vernonia anthelmintica use in various weight reducing recipes. One approach to weight loss was reported to keep blood sugar levels stable. Increasing blood glucose levels too high may cause weight gain by secreting insulin and this overplay of insulin may results in low levels of glucose resulting in increased appetite. Thus preventing blood sugar peaks is a good approach to decrease appetite and hence weight (Ebbeling et al., 2012, Juanola- Falgarona et al., 2014).

Present results of Vernonia anthelmintica on glycemic profile (sub chronic and acute effects) supports this hypothesis of weight loss as it is capable of reducing postprandial glucose levels that in contrast with glibenclamide were not found to fall too low to be hypoglycemic. Thus the glucose modulating effect of HEVA, EEVA and WDVA might accounts for its weight losing capability. However more studies are needed to justify its weight losing potential by carefully observing intake and output of animals on long term basis.

Effects of drug on physiological and pathological status in human and animals can be easily determined by its effect on hematopoietic system as it is considered as one of the most sensitive target for assessing toxicity (Adeneye et al., 2006). Moreover hematologic profile gives preliminary and vital information regarding the functionality of bone marrow and intravascular effects like anemia and hemolysis (Voigt and Swist, 2011).

Evaluation of hematologic parameters in the present study revealed no significant difference in any of the parameter between control and treated groups on repeated dosing of HEVA, EEVA and WDVA for 60 days. Thus a normal pattern of hematologic profile further strengthens the nontoxic nature of Vernonia anthelmintica on blood.

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Coagulation parameters in the present study revealed no significant alterations in TT, PT and aPTT in treatment groups as compared to control except WDVA that showed highly significant increase in TT and highly significant increase in PT and aPTT. However all treatment groups except WDVA 300 has shown increase in PT and aPTT but, has not achieved significance. Fibrinogen concentration in all treatment groups was found to be significantly decreased with varying intensities as compared to control group, with highest decline in HEVA followed by WDVA and EEVA. This decrease in coagulation times might be due to fall in fibrinogen inhibitory effects on coagulation factors.

Sub-chonic effects of 150 and 300 mg doses of HEVA, EEVA and WDVA on the liver function were also evaluated. The results revealed no significant alteration in liver enzymes (ALT, AST, ALP and GGT) and bilirubin (total and direct) in any treatment group as compared to control after 60 days of oral treatment. However exception to these results there was significant elevation in ALT at 300 mg/kg in animals received EEVA and WDVA. The results of liver parameters are in accordance with the histopathological findings of EEVA and WDVA since there was mild inflammation but histological findings were not severe enough to cause significant alteration in most of the liver enzymes evaluated. The liver is the main vital organ responsible for detoxification and metabolism of most drugs. Changes in liver function parameters are strong predictors of liver damage and hence toxicity of drugs (Woodman, 1996). Thus the potential of EEVA and WDVA in elevating ALT should be further studied since all other liver enzymes were normal.

Daily oral administration of HEVA, EEVA and WDVA for 60 days resulted in non-significant alteration in renal function parameters (Urea, uric acid, creatinine and total protein) in all treated groups as compared to control. Electrolytes levels (sodium and calcium) were also same in all treatment groups and control except for EEVA and WDVA that showed significant increase in sodium level in the doses of 300 mg/kg and 150 mg/kg. These mild elevations may be due to increased dietary intake or decreased clearance of sodium.

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The safety of drug on renal function was further justified by the histopathological findings that showed no difference in the echo texture and histo-morphological features between control and treatment groups. Thus all extracts of Vernonia anthelmintica were found to be safe and nontoxic to renal function in tested doses.

Sub chronic treatment of all extracts was also evaluated for its effects on lipid and glucose metabolism. The results revealed that HEVA showed significant increase in total cholesterol levels but this elevation is attributable to the increase of HDL more than LDL. Whereas TGs, HDL, LDL and VLDL were altered insignificantly at 150 and 300 mg/kg.

However EEVA on sub-chronic dosing showed dose independent increase in HDL and total cholesterol and decrease in TGs and VLDL at 150 and 300mg/kg doses. However EEVA in the dose of 300 mg/kg also reduce the cholesterol/ HDL ratio as compared to control. WDVA showed non-significant alterations in all lipid parameters.

The results indicated the favorable profile of all extracts of Vernonia anthelmintica on lipid parameters. EEVA and HEVA were more potent as compared to WDVA in lowering LDL and increasing HDL and hence can be used for the development of effective antihyperlipidemic drug.

Fasting blood sugar and HBA1c (glycated hemoglobin) level were also assessed after sub-chronic dosing to establish the role of Vernonia anthelmintica on glucose metabolism. The results revealed non-significant changes in FBS and HBA1c levels in all treatment groups as compared to control. Since erythrocytes are easily permeable to glucose, hence glycated hemoglobin is formed by the attachment of glucose to hemoglobin. This glycosylated hemoglobin provides an excellent assessment tool for glycemic control. Increase in HBA1c (a form of glycosylated hemoglobin) along with elevated fasting blood sugar is a marker for poor glycemic control (Jameson and De Groot, 2010).

Present study revealed that all extract of Vernonia anthelmintica did not cause any hypoglycemic effect in normal animals in spite of the fact that this herbal seeds

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have known antidiabetic potential. Thus it can be concluded that Vernonia anthelmintica might have potential to decrease blood sugar levels in hyperglycemic conditions but they would not cause hypoglycemia in normoglycemic subjects in contrast to various oral hypoglycemic drugs.

Present study revealed no significant antibacterial potential of hexane and ethanol extracts of Vernonia anthelmintica on tested pathogens in the concentration of 50 µg/ml. These results were in accordance with previous studies that reported it inactive against tested bacterial strains (Ratnam et al., 2014, Syed, 2012). However it is possible that further characterization, isolation and increase in the tested concentration may exhibit antibacterial potential as reported in few previous studies (Alam and Qureshi, 2010). Hence further studies should be conducted to elucidate its antibacterial potential.

Cancer differs in types and forms in human population but do share some similar properties i.e. inability to respond to inhibitory signals of cell growth. Apoptosis is inhibited and angiogenesis is unopposed within the cancer cells, hence resulted in prolong survival of cancer cells (Kumar et al., 2013). Herbal compounds have shown to display their anticancer potential due to anti proliferative and apoptosis induction properties (Greenwell and Rahman, 2015). In modern medicine, plants still hold a strong potential to be developed as anticancer agents. Effective anticancer agents such as vinblastine and camptothecin were conventionally obtained from different parts of catharanthus roseus and camptotheca acuminate respectively (Volkov and Grodnitskaya, 1994; Liu and Wang, 2004).

Previous studies on Vernonia anthelmintica have revealed its anti-cancer potential on various cancer cell lines. Chloroform fraction of Vernonia anthelmintica was reported to possess dose dependent cytotoxic effects against A549 (non-small cell lung carcinoma), MCF-7 (breast cancer), PC-3 (prostate cancer ) and WRL-68 (normal hepatic cell) cell lines with the IC50 value of 31.42 ± 5.4, 8.1 ± 0.9, , 22.61 ± 1.7 and 54.93 ± 8.3 μg/mL respectively (Arya et al., 2012a).

In the present study ethanol and hexane seed extracts of Vernonia anthelmintica were assayed against 3T3 (mouse fibroblast), Hela (human epithelial cell cancer),

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PC-3 (prostate cancer), MDA-MB 231 and MCF-7 (breast cancer) cell lines. The result of the study revealed significant activity of ethanol extract against 3T3 cell line with the IC50 value of 38.6 ± 4.9 at 50 µg/ml. Though non-significant but EEVA showed % inhibition of 36.5, 36.5, 29.1 and 38.59 against Hela, PC-3, MDA-MB 231 and MCF-7 cell lines respectively at 50 µg/ml. whereas HEVA showed % inhibition of 21.8, 11.3, 49.2, 21.7 and 27.2 against 3T3, Hela, PC-3, MDA-MB 231 and MCF-7 cell lines at same concentration. Plants in comparison with chemical agents are unique as they contain high concentrations of flavonoids like flavones, anthocyanins, flavonols, chalcones and all these can be found in a single part of plant e.g. seeds (Wen et al., 2014).

Previous studies indicated the anticancer activity of flavonoids against human hepatic, breast and cervical carcinomas (Wen et al., 2014). Some of the mechanism proposed for anticancer activity of flavonoids is their ability to scavenge free radicals (Cao et al., 2013), inhibition of phosphorylation of proteins required for the survival of cancer cells like STAT (signal transducer and activator of transcription) proteins, inhibition of NF-κB expression vital for cancer cell survival, inhibition of angiogenesis and proliferation (Kumar et al., 2013).

Present study also revealed the presence of flavonoids in both ethanol and hexane extracts and EEVA has found to have strong free radical scavenging activity which may be the possible mechanism of anticancer potential of EEVA against 3T3 cell line noted in this study. However non-significant but EEVA and HEVA has shown considerable inhibition against various cancer cell lines. Further characterization and isolation of flavonoids from Vernonia anthelmintica seeds may show strong anticancer potential in future studies.

The advantage of finding anticancer drug from plant derived compounds is their selectivity for cancer cells i.e. these remedies are toxic to cancer cells but safe for normal cells (Greenwell and Rahman, 2015). Present study is in accordance with this hypothesis of selectivity as EEVA and HEVA both showed cytotoxic

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potential in brine shrimp lethality test but found to be quite safe in in-vivo acute and sub-chronic toxicity studies.

Present study revealed that both hexane and ethanol extracts of Vernonia anthelmintica possess moderate phytotoxic activity against lemna minor in dose dependent manner. EEVA showed 100%, 50% and 35 % growth regulation at the concentration of 1000,100 and 10 µg respectively, whereas HEVA showed 80%, 50% and 20% growth regulation at the similar concentrations respectively.

Weed control is the foremost task in crop production. The major reason behind the environmental pollution and herbicide resistance is overuse of synthetic herbicides. Natural plant based herbicides offer an attractive solution to this problem as they are biodegradable and environmental friendly (Islam and Kato- Noguchi, 2014). The secondary metabolites of plants have been claimed to act as chemical agents against herbal pathogens, insects, pests and weeds and many natural compounds can be developed as herbicides, fungicide and insecticides (Duke et al., 2000).

Present study revealed the potential of Vernonia anthelmintica in controlling small seeded weeds and regulating the growth of crops. However further studies are required to ascertain the basic mechanism and components involved in phytotoxic potential of seeds. Present study had also undertaken insecticidal assay of hexane and ethanol extracts of Vernonia anthelmintica and the results showed that both extracts are inactive against insects Tribolium castaneum and Callobruchus at 1019.1 µg.

Leishmaniasis is a set of tropical disease that is caused by varying protozoal parasites belonging to the genus Leishmania. Modern medicine has been provided with more effective pharmaceuticals for the treatment of protozoal infections due to scientific evaluation of herbal medicine used in folk remedies. Quinone, alkaloids and terpenes are few metabolites effective against protozoal infections. It is worth mentioning that earlier antiprotozoal drugs such as emetine, quinine and artemisinin were all derived from different species of plants. This fact

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prompted to discover new bioactive antiprotozoal compounds of plant origin (Phillipson and Wright, 1991).

Previous data is lacking for the effect of Vernonia anthelmintica on leishmaniasis. Hence present study has undertaken the in-vitro antileishmanial assay of hexane and ethanol extracts of Vernonia anthelmintica and the results showed low antileishmanial activity with IC50 value 79.06±0.1 and 73.09±0.05 µg/ml. This antileishmanial activity of the extracts may be attributable due to the presence of alkaloids, flavonoids and terpenoids. Further isolation and characterisation of bioactive metabolites may lead to the development of more effective antileishmanial drugs.

Diabetes mellitus is a one of the most common chronic metabolic disease that is described by high levels of glucose in blood. The underlying cause of this abnormality is either impaired secretion or resistance of insulin (Association, 2006). Diabetes is associated with various chronic complication of varying severities e.g. hyperlipidemia, atherosclerosis, neuropathy, nephropathy (Sheetz and King, 2002). Glucose metabolism is a complex process that involves various metabolic pathways. Sustained hyperglycaemia therefore results in diverse cellular change leading to varying complications. Oxidants, glycation products and hyper-osmolarity are the pathophysiological derivatives of hyperglycaemia that exerts direct effects on tissues (Sheetz and King, 2002).

Established therapies for diabetes embrace insulin and oral hypoglycaemic agent’s e.g. sulphonylureas, α-Glucosidase inhibitors, thiazolidinedione etc. however these agents are known to be effective hypoglycaemic agents but none of them is devoid of side effects (Moller, 2001). Hence the search of herbal products have an advantage over synthetic medicine in the way that bioactive substances presents in plants possess antioxidant, hypoglycaemic and hypolipidemic potential (Marles and Farnsworth, 1995).

Present study has undertaken thee different diabetic models to assess the acute antidiabetic effect of Vernonia anthelmintica extracts. The results in

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normoglycemic model revealed that all thee extracts i.e. hexane, ethanol extracts and water decoction did not decrease the blood glucose levels and showed the same pattern of blood glucose levels as that of control when measured at 30, 60, 90 and 120 minutes. However standard drug glibenclamide significantly reduced the blood glucose levels as compared to control as well as baseline levels. This activity clearly revealed that Vernonia anthelmintica does not decrease blood sugar levels in normoglycemic individuals and might act either as alpha- glucosidase inhibitor that acts by decreasing absorption of sugar in GIT.

Oral glucose tolerance test is a tool to diagnose diabetes, insulin resistance or any other disturbance in carbohydrate metabolism (Li et al., 2014). Present study revealed the effects of prior dosing of Vernonia anthelmintica extracts in oral glucose tolerance test in rat model (OGTT). The results revealed that all HEVA, EEVA, WDVA and standard drug glibenclamide significantly lowered levels of blood glucose as compared to hyperglycaemic control at time 30 minutes. It is also worth mentioning that extracts of Vernonia anthelmintica have not declined it to very low levels as compared to control and standard group at 60, 90 and 120 minutes. The results revealed that Vernonia anthelmintica resisted the glucose induced increase in blood sugar levels in tested animals comparable to glibenclamide. The hypoglycaemic effects observed in this study might be unrelated to its pancreatic mechanism however delayed glucose absorption in intestine or enhanced uptake in muscle and liver may be responsible (Qureshi et al., 2009).

The third animal model tested for acute antidiabetic action of Vernonia anthelmintica is alloxan induced diabetic model. The result of this study revealed that HEVA, EEVA and glibenclamide significantly decreased the blood glucose levels at most of the time intervals tested and most profound at 120 minutes. Whereas WDVA showed no antidiabetic activity in alloxan induced hyperglycaemic model. These results give a clear indication that Vernoniaanthelmintica has glucose modulating effect rather than simply decreasing it. It has a strong antidiabetic potential without hypoglycaemic side effect.

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Increase intake of sugar and high fat diet is associated with insulin resistance and if remained untreated resulted in diabetes. Lipogenic effects of high sugars involves increased triglyceride synthesis in liver after reaching to hepatocytes though GLUT-5 i.e. insulin-independent glucose transporter (Armato et al., 2015). Hypertriglyceridemia might be the reason of insulin resistance characterized by impaired carbohydrate metabolism, high LDL and VLDL and low HDL levels (Hsieh et al., 2013).

Present study revealed the same spectra of disorders in HFHS (high fat high sugar) model. All these disturbances in carbohydrate and lipid metabolism were observed to be improved by treating with extracts of Vernonia anthelmintica. The results revealed significantly increased levels of Fasting blood glucose in disease control as compared to normal control. The treatment with atorvastatin, EEVA, HEVA and WDVA for 30 days has resulted in significant decrease in fasting blood sugar. This decrease in FBS is most profound in EEVA followed by HEVA, atorvastatin and WDVA.

Present study also revealed the In-vitro antiglycation activity of hexane and ethanol extract of Vernonia anthelmintica seeds. HEVA showed no inhibition while EEVA showed 45.60 % of inhibition as compared to standard agent Rutin (95%). Though insignificant but ethanol extract showed 45% inhibition in glycation assay. Thus the antihyperglycemic activity of ethanol extract may also be attributable to antiglycation effects of extract. Generation of AGE (advanced glycation end products) may lead to increased ROS (reactive oxygen species) production leading to varying complications of diabetes due to oxidative stress. Further isolation and characterization of polyphenols, flavonoids and alkaloids present in seeds may improve the antiglycation profile in future studies (Kayama et al., 2015).

Thus present results for antihyperglycemic potential of Vernonia anthelmintica provide the information that would fill the gaps left in the previous studies. The results revealed that the antihyperglycemic effects of Vernonia anthelmintica seeds are not straight forward rather involves various mechanisms i.e. increased

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glucose utilization and insulin secretion by overexpressing GLUT-2 and GLUT- 4 receptors by non-cytotoxic proliferation of β-TC6 in cell line studies, (Arya et al., 2012c) α- glucosidase inhibition (Ani and Naidu, 2008), Inhibition of protein tyrosine phosphatase-1B (PTP-1B) i.e. an enzyme involved in negative regulation of insulin signalling pathways (Tsou et al., 2012) and antiglycation potential of selected extracts.

Present study clearly indicates that Vernonia anthelmintica has strong antidiabetic potential in hyperglycaemic condition without producing hypoglycaemia. These effect of Vernonia anthelmintica extracts may be attributable to the presence of bioactive secondary metabolites such as flavonoids, alkaloids, terpenoids and other polyphenols including gallic acid, caffeic acid, protocatechuic acid, ellagic acid, quercetin, ferulic acid, and kaempferol (Ani and Naidu, 2008).

Atherosclerosis progressing to cardiovascular disease (CVD) is the principal cause of morbidity and mortality worldwide. Atherosclerosis referred to complex set of complication that involves many cellular components along with varying circulating mediators that leads to an inflammatory state. Lesion of atherosclerosis starts with the deposition of lipoproteins, macrophages derived from monocytes and lymphocytes along with arterial wall. Even with the advancement in cardiology medicine, atherosclerosis remains a prominent medical complication (Burnett, 2004).

Current pharmacotherapy of hyperlipidaemia has various side effects whereas herbal drugs are supposed to be effective in lowering lipids and hence CVD. Herbal medicine are cheap and easily available and have comparatively low risks (Kaur and Meena, 2013)

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In present study, high fat high sugar hyperlipidemic rat model was used for the assessment of antihyperlipidemic effect of Vernonia anthelmintica extracts as it is very close to metabolic syndrome caused by high dietary consumption of fat and sugar presumably due to unhealthy diet practices in our population (Munshi et al., 2014).

Lipid profile, liver function tests and fasting blood glucose levels were estimated at the end of study period. The results revealed that in rats LDL levels are far less as compared to HDL levels in all control and treated groups except for disease control. Although these results are contrary to human LDL levels which are greater than HDL levels but it is in accordance with various previous studies that also revealed same pattern of lipid levels in rats (Munshi et al., 2014; Lehmann et al., 1993).

High levels of low density lipoproteins (LDL) have long been recognized as a primary risk factor for the development of cardiovascular disease due to atherosclerosis and hence hold a principal target for the treatment and prevention of disease. In the present study disease control group showed significantly higher levels of LDL cholesterol, but 30 days dosing of standard drug atorvastatin, HEVA, EEVA and WDVA significantly reduced LDL levels. The LDL lowering effect is more profound in atorvastatin followed by EEVA, HEVA and WDVA. Increase in the levels of triglycerides has been reported as an independent risk contributor to CVD (Cullen, 2000). It is also reported that it might be a possibility that in spite of being acting as sole atherogenic agents, high levels of triglycerides may serve in increment of triglyceride rich remnant lipoproteins that may lead to atherosclerosis (Havel, 1990). In the present study elevated levels of triglycerides in disease model were significantly reduced by Vernonia anthelmintica extracts and standard drug. This decrease was of higher magnitude in EEVA followed by atorvastatin and HEVA whereas WDVA revealed non-significant reduction in triglycerides.

Several studies have indicated the role of flavonoids in cholesterol synthesis and shown to have both stimulatory and inhibitory effects depending on the type and

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dose of flavonoid (Gebhardt, 2001). Moving more towards mechanistic approach flavonoids are known to produce their cholesterol lowering effects by inhibition of cholesterol synthesis, modulation of HMG-CoA reductase activity, increase biliary secretion of cholesterol, decrease production of Apo-B100 and increase LDL receptor expression (Pal et al., 2003;Park et al., 2001). Few studies also revealed the role of flavonoid in inhibition of lipases and serum triglycerides levels in animal models (Kawaguchi et al., 1997). Hence flavonoids in most of the studies revealed that its use is associated with low total cholesterol, high HDL cholesterol and high HDL/LDL ratio (Vinson and Dabbagh, 1998).

Previous studies have shown the cholesterol lowering effects of alkaloids derived from plants. The main mechanism involved in protection of alkaloids against atherosclerosis is its cholesterol lowering effects. Alkaloids have shown increased LDL receptor expression. Alkaloids also control atherosclerosis due to its antioxidant, anti-inflammatory actions as well as its involvement in inhibition of vascular cell proliferation and endothelial dysfunction. Alkaloids for example berberine has shown to have total cholesterol, LDL, and triglyceride lowering potential as well as capable of increasing HDL (Pirillo and Catapano, 2015).

Present study has revealed the presence of flavonoids and alkaloids in HEVA and EEVA that might be responsible for its anti hyperlipidemic effects and as WDVA lacks alkaloids as revealed in phytochemical evaluation hence its antihyperlipidemic potential is of lesser magnitude as compared to EEVA and HEVA that contains both flavonoids and alkaloids.

As total cholesterol is the sum of all cholesterols that’s why EEVA, HEVA and WDVA does not show significant reduction in its levels as these test drugs increases HDL-C in the course of reducing LDL-C. Hence increase in cholesterol in disease control depicts the increment in LDL and triglyceride fraction whereas increase in total cholesterol in HEVA, EEVA and WDVA represent favourable increase in HDL.

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Present study estimated the cardiac risk by calculating atherogenic index, cholesterol/ HDL ratio and LDL/HDL ratio with the help of measured lipid and cholesterol value. Disease control revealed significantly higher atherogenic index, cholesterol/ HDL ratio and LDL/HDL ratio as compared to normal control and treated groups. On treatment with standard and test extracts the cardiac risk is reduced. This reduction in cardiac risk is most profound in EEVA, HEVA and atorvastatin and even lower as compared to normal control and significantly lower as compared to disease control.

In the course of treating hyperlipidaemia most of the antihyperlipidemic drugs causes liver injury of hepatocellular pattern with very rare incidence of cholestatic damage. The most common mechanism proposed for these damages are alteration in cytochrome P450 system, disruption in bile acid functions, immune-mediated apoptosis and inflammatory responses to the drug or its metabolite and oxidative damage (Bertolami, 2005). Present study was thus designed to estimate the effects of Vernonia anthelmintica extracts on liver while treating hyperlipidaemia in HFHS model.

AST (aspartate transaminase) and ALT (alanine transaminase) are predominantly concentrated in liver. However AST is also present in skeletal muscles, heart, kidneys, blood and brain cells whereas ALT is also present in kidneys and skeletal muscles. Thus ALT is more specific for indicating hepatic damage. Elevation of both transaminases indicates the hepatocellular damage (Rej, 1989). ALP is an enzyme that is responsible for the transportation of metabolites though cell membranes. Elevation of ALP is the sign of liver and bone diseases as at these sites ALP is present predominantly (Dufour et al., 2000). ALP of hepatic origin is present on the surface of epithelium of bile duct. Cholestasis increases the production and release of ALP as well as bile salt accumulation also leads to its increase (Moss, 1997). Drug induced liver damage may appear as cholestatic pattern with preferential increase in ALP and conjugated bilirubin and negligible increase in amino transferases (Velayudham and Farrell, 2003). GGT (gamma glutamyl transpeptidase) is an important enzyme present in biliary epithelial cells,

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renal tubules, pancreas and intestine. Same mechanism of alteration as described for alkaline phosphatase is applicable here. Though it lacks specificity but it is very sensitive for liver disease and its alteration in context with changes in ALP is highly diagnostic for liver damage (Giannini et al., 2005).

In the present study disease control of HFHS model revealed increased ALP levels which may be an indication of cholestatic pattern. Whereas ALT, AST, GGT and total bilirubin showed no significant alterations. Hence disease model showed no hepatocellular damage. The HMG-CoA reductase inhibitors are the class of drugs used for the treatment of hyperlipidemia and prevention from coronary artery disease. Atorvastatin is reported to be associated with hepatic injury (Liu et al., 2010a).

Present study has evaluated its effects on liver in HFHS model of hyperlipidemia. The results revealed that atorvastatin has increased aminotransferases as compared to control and disease control. ALT and GGT were increased as compared to control but this change did not achieve significance whereas AST showed significant increase as compared to control. Total bilirubin levels were also increased as compared to control and disease control. However ALP levels were found to be decreased as compared to disease control. Thus atorvastatin showed mild potential of causing hepatocellular damage but improved the cholestatic damage. EEVA and HEVA showed most favorable effects on liver enzymes with no marked alterations in liver enzymes as compared to control. It is worth mentioning that HEVA and EEVA showed significantly lower levels of AST as compared to atorvastatin. Total bilirubin levels showed no marked elevation except for HEVA that revealed significantly higher bilirubin levels as compared to control and disease control but as the ALP levels showed no significant increase so it would not indicate cholestatic damage.

The results of the present study revealed the safety of Vernonia anthelmintica extracts in the course of treating hyperlipidemia in rats. This safety may be attributed to antioxidant activity of these extracts.

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In the present study, the effects of control, treatment and standard groups were assessed for volume of paw edema and percent inhibition as compared to control in carrageenan-induced paw edema rat model. The result revealed the strong anti- inflammatory activity of Ibuprofen in the dose of 100 mg/kg with the maximum inhibition (82.3%) at 2 hour which remained decrease till 24 hour. HEVA in the dose of 150mg/kg did not show any significant anti-inflammatory activity but 300 mg/kg dose revealed strong anti-inflammatory potential started at 2 hour, maximum at 3 hour (70.9%) and remain significant till 24 hour of treatment. EEVA in the dose of 150 mg/kg showed significant decreased paw edema only at 24 hour that would be more appropriately considered as reversal of inflammation. However 300 mg/kg dose of EEVA revealed strong anti-inflammatory potential that was started at 1sthour, achieve significance at 2 hour, became maximum at 4 hour and remained significant till 24 hours. WDVA showed a little aberrant result as 150 mg/kg dose produces its anti-inflammatory effect at 5 hour and 300 mg/ kg dose only showed significant decrease in paw volume at 2nd hour of treatment.

Carrageenan model was chosen because of its sensitivity towards orally administered anti-inflammatory agents against acute inflammatory conditions (Di Rosa et al., 1971)

The results of present study clearly depicts that HEVA and EEVA in higher dose possess a strong anti-inflammatory potential that is comparable to standard anti- inflammatory drugs. Whereas WDVA showed milder potential against acute inflammation. Towards the mechanistic approach HEVA and EEVA in the doses of 300 mg/kg might showed this potential due to potent effects on second phase of inflammation that is caused by increased production of prostaglandins, free radicals and cyclooxygenase pathway products and weak effects on first phase that is merely due to release of mediators (histamine, serotonin and kinins) and accounts for first hour of inflammatory changes after carrageenan injection (Vinegar et al., 1969).

Present study also revealed the strong antioxidant potential of EEVA and hence strong potential to scavenge free radicals and it is the added advantage of ethanol

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extract in producing sustained anti-inflammatory effect. WDVA on contrary showed a different pattern as 150 mg/kg dose showed more pronounced effects on second phase of inflammation whereas 300 mg/kg dose showed activity in first phase only. These results are in accordance with a previous study which showed that petroleum ether and alcohol extracts of Vernonia anthelmintica on oral intake suppressed the edema after 2 hour and sustained till 5 hour having strong prostaglandin inhibition effect (Ashok et al., 2010b).

The present study suggested that all extracts of Vernonia anthelminticum showed significant analgesic activity in thermal induced pain models. Tail flick method and hot plate method induced thermal noxious stimuli are pain models that involve central mechanism of pain. HEVA, EEVA and WDVA all observed to have anti nociceptive potential at tested doses with more pronounced effects at 300 mg/kg dose as compared to 150 mg/ kg.

The present study revealed that HEVA, EEVA and WDVA showed dose dependent significant analgesic potential in Hot plate pain model. The effects are more pronounced at first hour and high doses (300 mg/ kg) whereas low doses showed minimized effects as compared to high dose in all tested groups. The results of HEVA in tail flick method revealed that though significant increase in reaction time as compared to basal reaction time was observed in 150 mg/kg dose but most pronounced effect was seen at the dose of 300 mg/kg at first hour of experiment. EEVA and WDVA showed most pronounced anti nociceptive effect at 300 mg/ kg at second hour of experiment but both low and high doses showed significant analgesic potential at tested times.

Thus results revealed that with the exception of few observations all groups revealed significant analgesic activity in dose dependent manner and both models showed same pattern of pain inhibition. Anti-nociceptive effects of high doses in HEVA, EEVA and WDVA groups are comparable to aspirin 300 mg/kg that was used as standard analgesic drug in this experiment. The mechanism involved in attenuation of pain might be either inhibition of pain transmission to CNS or by enhancing the inhibitory pathway from brain. (Katzung et al., 2004). This effect

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might be due to inhibition of pain receptor sensitization at the inflammation site by prostaglandins (Barar 2006). The extracts might achieved this analgesic activity by blocking phospholipase A2 or cyclooxygenases (Bhaskar and Balakrishnan, 2015).

The present study is in accordance with the previous observations that revealed analgesic effects of petroleum ether and alcohol extracts of Vernonia anthelmintica in the doses of 100 and 200 mg/kg (Purnima et al., 2009b).

Oxygen is supposed to be a necessary element required by all living cells for their survival. In normal physiology and metabolism, certain percentage of oxygen becomes univalently reduced to generate free radicals such as nitric oxides, hydroxyl, superoxides and hydrogen peroxides. These free radicals termed as ROS (reactive oxygen species) exert oxidative stress to human cells and their extent is so much so that each cell faces thousands of oxidative hits per second (Mondal et al., 2006).

Medicinal plants and their purified extracts have shown to exhibit antioxidant potential. This antioxidant capacity of plants is due to presence of varying secondary metabolites namely flavones, flavonoid, isoflavones, coumarins, anthocyanins, lignans, catechins and isocatechins (Aqil et al., 2006). Drugs with antioxidant principal are required and being in use for the prevention and cure of various complex diseases of chronic nature like stroke, diabetes, atherosclerosis, alzheimer’s and cancer (Devasagayam et al., 2004).

General mechanism of antioxidant to combat with free radicals is to break up the propagation of their chains by simply providing an electron or hydrogen atom and thus receiving their excessive energy (Lachman et al., 1986). It is documented need to find natural antioxidants in order to replace synthetic antioxidants because later cannot be used freely because of their potential side effects like carcinogenicity (Kumaran and Karunakaran, 2007). The natural antioxidants work by scavenging free radicals, complexing metals with oxidant potential, exhausting single oxygen and as reducing agents (Ebadi, 2006).

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Present study has undertaken DPPH assay in order to estimate the free radical scavenging capacity of Vernonia anthelmintica extracts. The results revealed that HEVA showed non-significant radical scavenging activity (% RSA 40.03). Whereas EEVA has shown to have strong dose dependent DPPH radical scavenging activity with an IC50 value 95.10± 2.01 and maximal inhibition of 89.70 %. Present work further strengthen the claims made for chloroform fraction of Vernonia anthelmintica as an antioxidant in a previous study (Arya et al., 2012a).

Thus adding up with the previous findings present study demonstrated the role of Vernonia anthelmintica as a natural source of antioxidant metabolites which could play a favourable role in scavenging free radicals and hence can be used in the prevention and cure of diseases caused by oxidative stress. The pharmacological activities such as lipid lowering, antihyperglycemic, anti-inflammatory, analgesic observed in the present study are also partly attributable to antioxidant potential of Vernonia anthelmintica as the capability of scavenging free radical may hinder tissue damage and inflammation by attenuating several signalling pathways and hence provides protection to the living cells.

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5. CONCLUSION

Conclusion:

Vernonia anthelmintica is known for its wide traditional use in the subcontinent. The present study revealed significant evidence to establish its safety for use in manufacturing of pharmaceutical as well as herbal remedy for the treatment of diabetes, dyslipidaemia, inflammation, cancer and nociception. Furthermore Vernonia anthelmintica found to possess strong anti-oxidant potential and can be used in combating various chronic diseases attributable to the hazardous effects of free radicals. Phytotoxic and antileishmanial potential of Vernonia anthelmintica are discovered in this study and need further workup to establish its role in this aspect. Weight reducing capability reported in present investigation should be further studied in order to design anti-obesity preparations from active metabolites of seeds.

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