Ph.D. Thesis by IRFAN ULLAH

MOLECULAR PROFILING OF BIOACTIVE CONSTITUENTS FROM MONOTHECA BUXIFOLIA (FALC.) FRUIT

Ph.D. Thesis By IRFAN ULLAH

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR, PESHAWAR, PAKISTAN

2016

MOLECULAR PROFILING OF BIOACTIVE CONSTITUENTS FROM MONOTHECA BUXIFOLIA (FALC.) FRUIT

IRFAN ULLAH

A THESIS SUBMITTED TO THE DEPARTMENT OF PHARMACY,

UNIVERSITY OF PESHAWAR

IN PARTIAL FULFILLMENT FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

PHARMACEUTICAL SCIENCES

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR, PESHAWAR, PAKISTAN

2016

This thesis, entitled, “MOLECULAR PROFILING OF BIOACTIVE CONSTITUENTS FROM MONOTHECA BUXIFOLIA (FALC.) FRUIT” submitted by Mr. Irfan Ullah is hereby approved and recommended as partial fulfilment for the award of Degree of Doctor of Philosophy in Pharmaceutical Sciences”.

Prof. Dr. Jamshaid Ali Khan ______

Research Supervisor, Department of Pharmacy, University of Peshawar.

Professor Dr. Muhammad Saeed ______

Chairman, Department of Pharmacy, University of Peshawar.

External Examiner ______

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR, PESHAWAR, PAKISTAN

2016

Dedication

Dedicated

My Dear Parents

Acknowledgements

I am thankful to Almighty Allah, Who made me sanctified with patience, understanding, countless blessing of health, and wellbeing to complete my Ph.D. project successfully, which is certainly a landmark in my life.

I would like to gratefully and sincerely thank Prof. Dr. Jamshaid Ali Khan for his guidance, understanding, patience, and most importantly, his friendship during my studies. His mentorship was paramount in providing a well-rounded experience consistent with my long-term career goals. He encouraged me to not only grow as an experimentalist but also as an instructor and an independent thinker. I am not sure many graduate students are given the opportunity to develop their own individuality and self-sufficiency by being allowed to work with such independence. For everything you’ve done for me, Sir, I thank you.

I am using this opportunity to express my gratitude to Meritorious Professor. Dr.

Zafar Iqbal (T.I), who supported me throughout the course of this Ph.D. project. I am sincerely grateful to him for sharing his fruitful and illuminating views on a number of issues related to the project.

I am also thankful to Prof. Dr. Muhammad Saeed, Chairman, Department of

Pharmacy, University of Peshawar, for the continuous support and encouragement throughout my research studies. I am also thankful to Prof. Dr. Fazal Subhan and

Prof. Dr. Muhammad Ismail for their aspiring guidance and providing the required facilities during the project work. I would also like to appreciate the cooperation of all the teaching and non-teaching staff of department.

Acknowledgements

It is my privilege to acknowledge the admirable facilitation, expert opinion and cooperation by Prof. Dr. Muhammad Iqbal Choudhary, Dr. Achyut Adhikari,

Prof. Dr. Khalid Mohammed Khan, Dr. Muhammad Ateeq, Dr. Ajmal Khan,

Mr. Shahid Ali Khan, Mr. Zafar Ali Shah, and Dr. Naveed Iqbal, International

Centre for Chemical and Biological Sciences (ICCBS), H.E.J Research Institute of

Chemistry, University of Karachi during this long course of research venture.

I am grateful to all my research seniors especially Dr. Abad Khan, Mr. Salar

Muhammad, Mr. Imran Ullah, Dr. Muhammad Imran, Dr. Muzaffar Abbas, Dr.

Waqar Ahmad Kaleem, Dr. Naveed Muhammad and Dr. Lateef Ahmad for enlightening me the first glance of research.

Indeed I would also like to thank all my fellow lab-mates especially, Mr. Safiullah,

Mr. Peer Abdul Hannan, Mr. Muhammad Shahid, Mr. Javaid Alam, Mr. Ismail,

Mr. Muhammad Hassan, Dr. Muhammad Shafiq, Ms. Mehreen Rehman, Mr.

Waheed Ur Rehman, Dr. Zia Ul Haq, Mr. Fawad Ahmed and Mr. Qasim Khan for the fruitful discussions, kind support, positive criticism and for all the fun we have had in the last four years.

Infact I am also thankful to the Department of Pharmacy, and University of Peshawar for providing me such a great opportunity of higher studies.

Last but not the least; I would like to thank my parents, brothers, sisters, my uncle

Mr. Muhammad Hayat (Advocate), my grandmother and all relatives for supporting me spiritually throughout my studies and my life in general, your support and prayers really made me too much confident.

Irfan Ullah Peshawar, 2016

Table of Contents Summary…..………………………………………………………………………….i-ii List of Abbreviations………..…………….……………………………………...... iii-vi 1. INTRODUCTION ...... 1 1.1. General Introduction ...... 1 1.1.1. History of plants as a source of drugs ...... 1 1.1.2. Medicinal importance of plants ...... 2 1.1.3. Modern day drugs from medicinal plants ...... 2 1.1.4. Some dietary supplements as preventive medicine ...... 4 1.1.5. Market value of natural products in the world ...... 5 1.1.6. Research on medicinal plants; a basic need of the day ...... 6 1.1.7. History of herbal products during 19th and 20th centuries ...... 8 1.1.8. Bioassay-guided isolation from natural products ...... 9 1.1.9. Medicinal plants as best sources of important drugs ...... 10 1.1.10. Secondary metabolites ...... 18 1.1.10.1. Major classes of plant specialized compounds…………………… .19 1.1.11. Status of medicinal plants in the world ...... 21 1.1.12. Medicinal plants status in Pakistan ...... 22 1.1.13. Family Sapotaceae ...... 24 1.1.14. Status of family Sapotaceae in medicinal plants ...... 25 1.1.15. Biological activities of family Sapotaceae ...... 25 1.1.16. The genus Monotheca ...... 31 1.2. Plant Introduction ...... 32 1.2.1. Monotheca buxifolia ...... 32 1.2.2. Synonyms ...... 32 1.2.3. Morphology...... 34 1.2.4. Fruit ...... 34 1.2.5. Distribution ...... 34 1.2.6. Ethnobotanical uses ...... 34 1.2.7. Previously reported activities of Monotheca buxifolia fruit ...... 35 1.3. Aim and Objectives ...... 36 2. MATERIAL AND METHODS ...... 37 2.1. General Experimental Requirements ...... 37

2.1.1. Experimental Conditions ...... 40 2.1.1.1 Physical Constants….……………………………………………… .40 2.1.2. Spectroscopy ...... 40 2.1.2.1 UV Spectra……………………………………………………… …..40 2.1.2.2 IR Spectra…………..…………………………………………… ….40 2.1.2.3 Nuclear Magnetic Resonance (NMR) ………….……………… ..…41 2.1.3. Spectrometry…………………………………………………………...42 2.1.3.1. Mass Spectra………………………………………………....… …42 2.1.3.2. Gas Chromatography-Mass Spectrometry (GC-MS)………..…… 42 2.2. Isolation and Purification of Compounds ...... 42 2.2.1. Column Chromatography (CC)...... 42 2.2.2. Thin-layer Chromatography (TLC) ...... 43 2.2.3. Visualizing Compounds Spot on Developed TLC...... 43 2.2.4. Ceric Sulphate Solution ...... 43 2.2.5. Dragendorff’s Solution ...... 43 2.2.6. Iodine solution ...... 44 2.2.7. Preparative HPLC (Recycling HPLC) ...... 44 2.3. Plant Materials ...... 44 2.3.1. Collection ...... 44 2.3.2. Extraction ...... 45 2.3.3. Fractionation ...... 45 2.4. Animals Used ...... 47 2.5. Isolation of Compounds ...... 47 2.5.1. Isolation of Compounds from Ethyl acetate Fraction ...... 47 2.6. Characterization of isolated compounds ...... 50 2.6.1. Buxifoline-A (1)...... 50 2.6.2. Buxifoline-B (2) ...... 51 2.6.3. Buxitriol (3) ...... 52 2.6.4. Buxilide (4) ...... 53 2.6.5. Buxiglucoside (5) ...... 54 2.6.6. Oleanolic acid (6) ...... 55 2.6.7. Glucosidic β-sitosterol (7)...... 56 2.6.8. 2-Hydroxy- epikatonic acid (8) ...... 57

2.6.9. Isoquercetin (9) ...... 58 2.7. Biological activities ...... 59 2.7.1. In-vitro biological activities ...... 59 2.7.1.1. Antibacterial activity ...... 59 2.7.1.2. Antifungal activity ...... 60 2.7.1.3. Leishmanicidal activity ...... 61 2.7.1.4. Cytotoxic activity (brine shrimp) ...... 61 2.7.1.5. Phytotoxic activity ...... 62 2.7.1.6. Insecticidal activity ...... 64 2.7.1.7. Urease inhibition assay ...... 64 2.7.1.8. Acetylcholinesterase inhibition ...... 65 2.7.1.9. Anticancer activity ...... 66 2.7.1.10. α-Chymotrypsin inhibition ...... 66 2.7.1.11. Cytotoxic activity (NIH 3T3 cell lines) ...... 67 2.7.1.12. Protein antiglycation ...... 68 2.7.1.13. Immune modulatory assay ...... 68 2.7.2. In-vivo Biological activities ...... 70 2.7.2.1. Acute toxicity ...... 70 2.7.2.2. Antipyretic activity against Brewer’s yeast induced pyrexia ...... 70 2.7.2.3. Antinociceptive activity ...... 71 2.7.2.4. Anti-inflammatory activity ...... 71 2.7.2.5. Prokinetic and laxative activity ...... 72 2.7.2.5.1. Charcoal meal transit test ...... 72 2.7.2.5.2. Laxative activity ...... 72 2.7.2.6. Hepatoprotective activity ...... 73 2.7.2.6.1. Isoniazid and Rifampicin induced toxicity ...... 73 2.7.2.6.2. Experimental design ...... 73 2.7.2.6.3. Blood Collection and Serum Preservation ...... 73 2.7.2.6.4. Biochemical assays ...... 74 2.7.2.6.5. Histology ...... 74 3. RESULTS AND DISCUSSION……………………………………………….…...75

3.1. Percent yield of the extract and subsequent solvents soluble fractions ...... 75 3.2. Structure elucidation of isolated compounds ...... 76

3.2.1. Buxifoline-A (1)...... 76 3.2.2. Buxifoline-B (2) ...... 79 3.2.3. Buxitriol (3) ...... 83 3.2.4. Buxilide (4) ...... 86 3.2.5. Buxiglucoside (5) ...... 89 3.2.6. Oleanolic acid (6) ...... 92 3.2.7. Glucosidic β-sitosterol (7) ...... 95 3.2.8. 2-Hydroxy-epikatonic acid (8) ...... 99 3.2.9. Isoquercetin (9) ...... 102 3.2.10. Composition of fixed oil ...... 106 3.3. Biological activities ...... 108 3.3.1. In-vitro activities ...... 108 3.1.1.1. Antibacterial activity…………………….………………...… .…108 3.1.1.2. Antifungal activity…………………….……….…………… …...113 3.1.1.3. Leishmanicidal activity………………………….……….…..…...117 3.1.1.4. Cytotoxic activity (Brine shrimp) ………………...…………..….118 3.1.1.5. Phytotoxic activity…………..………..…………….………….…122 3.1.1.6. Insecticidal activity…………………..…………….………….….125 3.1.1.7. Urease inhibitory assay………..……..…………….…………….128 3.1.1.8. Acetylcholineesterase inhibitory assay………..…….…….……..130 3.1.1.9. Anticancer activity (PC-3 cell lines) ……………….……………132 3.1.1.10. α-Chymotrypsin inhibitory assay………….………….………….134 3.1.1.11. Cytotoxic activity (NIH-3T3 cell lines) …………….…………...137 3.1.1.12. Protein antiglycation assay……………...…………….………….139 3.1.1.13. Immune modulatory assay ………………………....….…………..141 3.3.2. In-vivo activities ...... …………….……. 144 3.3.2.1. Acute toxicity ………………………………………….……144 3.3.2.2. Antipyretic activity………………………………………….……145 3.3.2.3. Analgesic activit……………..……………………………….…..148 3.3.2.4. Carrageenan induced paw edema………………………….….….149 3.3.2.5. Prokinetic and Laxative activity…...……………………….…….151 3.3.2.5.1. Effect of MBHE on charcoal meal intestinal transit.…….……151 3.3.2.5.2. Laxative activity …..………..…..………………………..……151 3.3.2.6. Hepatoprotective activity…………………………………...……155 3.3.2.6.1. Effect of hydroethanolic extract on biochemical parameter….155

3.3.2.6.2. Effect of hydroethanolic extract on liver histology………..….155 4. Conclusion……………………………………………………….……………161 5. References……………………………….………………...……….…….…...162

List of Tables

Table-1.1 Natural Products based drugs launched since 2005……...... 3 Table-1.2 Common herbs with their medicinal properties...... 5 Table-1.3 Different plants groups and their medicinal species...... 22 Table-1.4 Reported biological activities of family Sapotaceae...... 26 Table-1.5 Taxonomical classification of Monotheca buxifolia...... 32 Table-2.1 List of used chemicals and drugs...... 37 Table-2.2 Characterization of buxifoline-A...... 50 Table-2.3 Characterization of buxifoline-B...... 51 Table-2.4 Characterization of buxitriol...... 52 Table-2.5 Characterization of buxilide...... 53 Table-2.6 Characterization of buxiglucoside...... 54 Table-2.7 Characterization of oleanolic acid...... 55 Table-2.8: Characterization of glucosidic β-sitosterol...... 56 Table-2.9 Characterization of 2-hydroxy-epikatonic acid...... 57 Table-2.10 Characterization of isoquercetin...... 58 Table-2.11 Reference bacterial stains used...... 59 Table-2.12 Fungal strains taken for the study...... 60 Table-2.13 Composition of medium for phytotoxic activity...... 63 Table-2.14 Material used in immune modulatory assay...... 69 Table-3.1 Percent yield of the extract and subsequent solvents solublee fraction………………………………………………………..… 75 Table-3.2 13C- and 1H-NMR Chemical shift values of compound 1...... 78 Table-3.3 13C- and 1H-NMR Chemical shift values of compound 2...... 81 Table-3.4 13C- and 1H-NMR Chemical shift values of compound 3...... 85 Table-3.5 13C- and 1H-NMR Chemical shift values of compound 4...... 88 Table-3.6 13C- and 1H-NMR Chemical shift values of compound 5...... 91 Table-3.7 13C- and 1H-NMR Chemical shift values of compound 6...... 94 Table-3.8 13C- and 1H-NMR Chemical shift values of compound 7...... 98 Table-3.9 13C- and 1H-NMR Chemical shift values of compound 8...... 101 Table-3.10 13C- and 1H-NMR Chemical shift values of compound 9...... 105

List of Tables

Table-3.11 Compounds identified in MBHF through GC/MS...... 107 Table-3.12 Antibacterial activity of MBHE and subsequent fraction……..... 111 Table-3.13 Antifungal activity of MBHE and fraction...... 115 Table-3.14 Leishmanicidal activity of MBHE and fractions...... 118 Table-3.15 Cytotoxic potential of MBHE and fractions...... 120 Table-3.16 Phytotoxic activity of MBHE and fractions...... 123 Table-3.17 Insecticidal activity of the MBHE and fractions...... 126 Table-3.18 Urease inhibitory potential of the MBHE and fractions...... 129 Table-3.19 Acetylcholineesterase inhibition of MBHE and isolated compounds……………………………………………….....… 131 Table-3.20 Percent growth inhibition of the compounds against PC3 cell lines……………………………………………………………. 133 Table-3.21 α-Chymotrypsin inhibition assay of MBHE and fractions…...... 135 Table-3.22 α-Chymotrypsin inhibition assay of compounds...... 135 Table-3.23 Percent growth regulation potential against NIH 3T3 cell lines 138 Table-3.24 Protein antiglycation potential of MBHE and fractions……….…140 Table-3.25 Immune modulatory activity of compounds...... 142 Table-3.26 Evaluation of in-vivo acute toxicity of MBHE...... 144 Table-3.27 Antipyretic activity of MBHE against Brewer’s yeast induced pyrexia in mice…………..……………………………………….146 Table 3.28 Anti-inflammatory activity of Monotheca buxifolia (MBHE) against Carrageenan induced paw edema in mice…………….... 150 Table-3.29 Effect of atropine on the prokinetic effect of MBHE in mice...... 153 Table-3.30 Effect of atropine on the percent laxative effect of MBHE in mice………………………………………………………………154 Table-3.31 Hepatoprotective activity of MBHE by isoniazid and rifampicin induced hepatotoxicity in rats……..…………..…… 158 Table 3.32 Effect of MBHE on the severity of isoniazid and rifampicin induced hepatotoxicity after 21 days of treatment……...... ….….159

List of Figures

List of figures

Fig-1.1 Bio-assay guided isolated drugs from medicinal plants…….……… 17 Fig-1.2 Schematic pathways of synthesis of plants secondary metabolites from glycolysis, tricarboxylic acid (TCA) cycle and Calvin cycle.... 20 Fig-1.3 Status of medicinal plants in Pakistan...... 23 Fig-1.4 Isolated compounds from family Sapotaceae...... 30 Fig-1.5 Monotheca buxifolia specimen sheet at Department of Botany, University of Peshawar...... 33 Fig-2.1 Schematic diagram for extraction and fractionation………………. 46 Fig-2.2 Schematic diagram for isolation of pure compounds...... 49 Fig-3.1 Structure of buxifoline-A (1)...... 77 Fig-3.2 Key COSY and HMBC structure of buxifoline-A (1)...... 77 Fig-3.3 Structure of buxifoline-B (2)...... 80 Fig-3.4 Key COSY and HMBC structure of buxifoline-B (2)...... 80 Fig-3.5 Recycling HPLC Chromatogram showing purification of compound 1 and 2…………...... 82 Fig-3.6 Structure of buxitriol (3)...... 84 Fig-3.7 Key COSY and HMBC structure of buxitriol (3)...... 84 Fig-3.8 Structure of buxilide (4)...... 87 Fig-3.9 Key HMBC structure of buxilide (4)...... 87 Fig-3.10 Structure of buxiglucoside (5)...... 90 Fig-3.11 Key COSY and HMBC Structure of buxiglucoside (5)…...... 90 Fig-3.12 Structure elucidation of oleanolic acid (6)...... 93 Fig-3.13 Structure of glucosidic β-sitosterol (7)...... 97 Fig-3.14 Key COSY and HMBC structure of glucosidic β-sitosterol (7)…..... 97 Fig-3.15 Structure of 2-hydroxy-epikatonic acid (8)………………………… 100 Fig-3.16 Key COSY and HMBC structure of 2-hydroxy-epikatonic acid (8) 100 Fig-3.17 Structure of Isoquercetin (9)………………………………………... 104 Fig-3.18 Key COSY and HMBC structure of Isoquercetin (9)...... 104 Fig-3.19 GCMS chromatogram of the fixed oils of Monotheca buxifolia fruit………………………………………………………………... 106 Fig-3.20 Percent zones of inhibition of bacterial strains by MBHE and fractions...... 112

List of Figures

Fig-3.21 Antifungal activity of MBHE and fractions...... 116 Fig-3.22 Percent cytotoxic effect of MBHE and fractions...... 121 Fig-3.23 Phytotoxic effect MBHE and fractions...... 124 Fig-3.24 Insecticidal activity of the MBHE and fractions...... 127 Fig-3.25 Urease inhibitory potential of the MBHE, fractions and isolated 129 compounds...... Fig-3.26 Acetylcholineesterase inhibition of MBHE & isolated compounds 131 Fig-3.27 Percent growth inhibition of compounds against PC3 cell lines…... 133 Fig-3.28 α-Chymotrypsin inhibition assay of MBHE and fractions...... 136 Fig-3.29 α-Chymotrypsin inhibition assay of compounds...... 136 Fig-3.30 Percent growth regulation potential against NIH 3T3 cell lines...... 138 Fig-3.31 Protein antiglycation potential of MBHE and fractions...... 140 Fig-3.32 Immune modulatory activity of compounds...... 142 Fig-3.33 The effect of compound 7 on cell surface peptide loading of MHC-II molecules...... 143 Fig-3.34 Antipyretic activity of MBHE against Brewer’s yeast induced pyrexia in mice...... 147 Fig-3.35 Antinociceptive activity of MBHE in acetic acid induced abdominal constriction assay...... 148 Fig-3.36 Effect of atropine on the percent prokineteic effect of MBHE in mice………………………………………………………………... 153 Fig-3.37 Histopathological evaluation of isoniazid and rifampicin induced hepatotoxicity pre-treated with Monotheca buxifolia for 21 days…. 160

Summary

This Ph.D. thesis describes the phytochemical and pharmacological investigation of

Monotheca buxifolia fruit, used as laxative, digestive, purgative, vermicidal, antipyretic, and for the treatment of gastritis and urinary tract infection without scientific evidence(s).

In the present research work, Monotheca buxifolia hydroethanolic extract (MBHE), its sub-fractions and isolated compounds were screened for various biological activities, to provide a valid scientific rationale to its ethno-medicinal uses.

The MBHE and its fractions showed moderate antibacterial potential with maximum activity exhibited by n-hexane (69.56 and 60%) and ethyl acetate fractions (69.56 and

60%) against P. aeruginosa and E. coli respectively while other fractions showed mild antibacterial activity. Similarly the MBHE and its fractions showed a mild antifungal and no leishmanicidal potential.

Significant cytotoxicity against brine shrimp was observed for ethyl acetate and chloroform fractions with LD50 values of 14.74 and 31.22 µg/mL respectively. The

MBHE, aqueous and n-hexane fractions exhibited moderate cytotoxicity with LD50 values of 147.99, 117.06 and 279.95 µg/mL respectively. The phytotoxic potential observed against Lemna minor, was significant at dose of 1000 µg/mL and in order of chloroform > aqueous > n-butanol > ethyl acetate fractions. A mild insecticidal activity was showed by MBHE, chloroform, ethyl acetate and n-hexane fractions. The

MBHE and its fractions were found inactive in protein antiglycation assay.

In urease inhibition assay, ethyl acetate fraction showed a moderate inhibition (IC50 value of 151.3 µg/mL) while other fractions exhibited mild inhibition. Among the

Summary

isolated compounds, compound 9 showed significant inhibition (IC50 value of 51.6

µg/mL), while mild activity was observed for the rest of the compounds. In acetylcholineesterase and α-chymotrypsin inhibitory assays all the tested samples were found insignificant.

A significant anticancer activity was observed for compound 9 (55.55%) against PC3 cell lines, while compound 1 exhibited the least (5.34%) cytotoxic potential against

NIH 3T3 cell lines at 100 µM concentration. A moderate immune modulatory activity was also observed for compound 7.

The MBHE showed promising in-vivo anti-pyretic, analgesic, and anti-inflammatory activities at test dose of 150 mg/kg. MBHE also exhibited laxative activity in both charcoal meal transit and laxative (wet stool production) models. The laxative effect was partially antagonized by atropine. Furthermore, MBHE exhibited significant hepatoprotective activity against isoniazid and rifampicin induced hepatotoxicity in rats.

In phytochemical investigation, ethyl acetate fraction was subjected to various chromatographic techniques. Structures of the isolated compounds were elucidated using advanced spectroscopic and spectrometric techniques i.e. 1H-NMR, 13C-NMR,

COSY, HMBC, NOESY, HSQC, IR, UV, EI-MS, HR-EI-MS, FAB-MS,

26 HR-FAB-MS and [α] D. Among the isolated compounds, 4 were new, 1 was new natural product and 4 were previously reported but first time isolated from this plant.

The first time isolated from natural sources was buxifoline-A (1), new were buxifoline-B (2), buxitriol (3), buxilide (4) and buxiglucoside (5) while the other 4 compounds were oleanolic acid (6), glucosidic β-sitosterol (7), 2-hydroxy- epikatonic acid

(8) and iso-quercetin (9).

List of Abbreviations

3T3: 3-day transfer, inoculum 3×105 cells

ACh: Acetylcholine ACh-E: Acetylcholineesterase

AD: Alzheimer Disease

AIDS: Acquired Immune Deficiency Syndrome

APC: Antigen Presenting Cell

ARG: Argenine

ASP: Aspartate

AZT: Azathioprene

BB: Broad-Band

CAGR: Compound Annual Growth Rate CC: Column Chromatography CCh: Carbacholine CFU: Colony- Forming Unit

CHCl3: Chloroform CHS: Chalcone synthase

Co-A: Coenzyme A

COMP: Compound COPD: Chronic Obstructive Pulmonary Disease

COSY: Correlation Spectroscopy COX: Cyclo-oxygenase CV: Central Vein

CYP2E1: Cytochrome P2E1 Enzyme

DBSA: Di-bromo-salicyl-aldehyde

DCM: Dichloromethane

DELFIA: Dissociation-Enhanced Lanthanide Fluorescent Immunoassay

iii

List of Abbreviations

DEPT: Distortionless Enhancement by Polarization Transfer

DKK: Danish Krone

DMAPP: Dimethylallyl Diphosphate

DMSO: Dimethyl Sulfoxide DNA: Deoxyribonucleic Acid

EDTA: Ethylene Diamine Tetra Acetic acid

EI-MS: Electron Ionization Mass Spectrometry ELISA: Enzyme Linked Immunosorbent Assay

EtOAc: Ethyl acetate

FAB-MS: Fast Atom Bombardment Mass Spectrum

FALC: Falconer

FBS: Fetal Bovine Serum

FIG: Figure

GAP: Glyceraldehyde 3-Phosphate

GC: Gas Chromatography GCMS: Gas Chromatography Mass Spectrometry GEO Mean: Geometric Mean

GLN: Glutamine

GLU: Glucose

HLA: Human Leukocyte Antigen

HM: Herbal Medicine(s) HMBC: Heteronuclear Multiple Bond Coherence HPP: Health Promotion Programs HPPD: 4- Hydroxyl-Phenyl-Pyruvate Dehydrogenase

HPLC: High-Performance Liquid Chromatography

HREI-MS: High Resolution Electron Ionization Mass Spectrometry HRFAB-MS: High Resolution Fast Atom Bombardment Mass Spectrum

HSQC: Heteronuclear Single Quantum Coherence

List of Abbreviations

IC50: Concentration causing 50% Inhibition

ICCBS: International Centre for Chemical and Biological Sciences

IL-1: Interleukin 1

INH: Isoniazid

I.P.: Intra Peritonealy

IPP: Isopentenyl Diphosphate

IR: Infra Red

KI: Potassium Iodide

KBr: Potassium Bromide

LD50: Median Lethal Dose

MBHE: Monotheca buxifolia hydro-ethanolic (30:70) extract MTT: 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide

NaN3: Sodium azide

NCI: National Cancer Institute

NF: Necrotizing Factor

NIH: National Institute of Health

NMR: Nuclear Magnetic Resonance NNRTI: Non-Nucleoside Reverse Transcriptase Inhibitor

NPAA: Non-protein Amino Acids

ORN: Ornithine p: Probability PAL: Phenylalanine Ammonia Lyase

PBS: Phosphate Buffer Saline

PC3 Cells: Prostate Cancer Cells

PDC: Pyruvate Dehydrogenase complex

PI: Propidium Iodide

P.O.: Per Oral

List of Abbreviations

RIF: Rifampicin

RNA: Ribonucleic acid

RPM: Revolutions per Min

RUB: Russian Ruble

SALP: Serum Alkaline Phosphatase

SB: Serum Bilirubin

SDA: Sabouraud Dextrose Agar

SGOT: Serum Glutamic Oxaloacetic Transaminase

SGPT: Serum Glutamate Pyruvate Transaminase

TCA Cycle: Tri-Carboxylic Acid Cycle TLC: Thin Layer Chromatography TMS: Tri-Methyl Silane

TNF: Tumor Necrosis Factor

TP: Total Protein

TRP: the reductive pentose

TYR: tyrosine

NOESY: Nuclear Overhauser Effect Spectroscopy UTI: Urinary Tract Infections

UV: Ultra Violet

VLC: Vacuum Liquid Chromatography

WHO: World Health Organization £: British Pound €: Euro

µg: Micro gram

µL: Micro Litre

$: United States Dollar

Chapter: 1

INTRODUCTION

Chapter: 1 Introduction

1. INTRODUCTION

1.1. General introduction

Discovering excellent remedies for diseases that are efficacious, economical and have minimum adverse effects is the need of the hour, for discovering such products, herbal medicines can be the best choice, as plants produce a wide range of bioactive compounds, making them a rich source of different types of medicines1. Majority of drugs present today are due to extensive research on their isolation from plant sources2-3.

1.1.1. History of plants as a source of drugs

Human being has been gifted with medicinal plants fulfilling the basic needs of humanity. Since the beginning, plants are used not only for food, fuel, fragrances, spices, shelter and transport (boat), but also for medications. Fossils record show that human has been using plants as medicine since middle of paleolithic age somewhat

60,000 years ago2. Man is always in search of discovering products that suits best with the requirements of the advanced world, therefore he uses his knowledge, skills and experience to utilize natural products especially plants in a variety of ways for his existence. Archaic man started dividing plants into two broad categories i.e. plants used for treating ailments and for food purposes. Extensive research on plants made the man able to distinguish and use plants for particular purposes such as feeding silk worm with mulberry leaves4, making paper from bamboo (Bambuseae) marrow5-6 and using Berberis roots for treating oral and throat infections, wounds and injuries7-8.

Hence, a variety of plants are used widely for food while others are used for therapeutic purposes. The origin and development of such evidences prove the

Chapter: 1 Introduction knowledge, intelligence, skills and experience of human among all other creatures on the entire planet.

1.1.2. Medicinal importance of plants

Medicinal plants have got more attention from the belief that synthetic drugs have been identified to cause serious adverse effects9. Almost all of the drugs are basically derived from the nature, and it is still the largest source which is yet to be studied for discovering novel pharmacophores10. Higher plants are important sources of novel compounds that are either used directly as drugs, as a model for semi-synthetic or synthetic modifications and structural optimization as important pharmacological probes11. Plants are naturally gifted in compounds synthesis and synthesize compounds of complex stereochemistry, eventually inspires the natural product scientists, thus attracts the attention of such researchers to investigate more and more plants12. Recently a variety of compounds have been discovered from plant sources that serves man in treating many important diseases like cancers, malaria, hypertension, psychosis, tuberculosis, ulcers, diabetes, constipation and asthma13.

There are also many other examples to prove the significance of plants derived compounds (secondary metabolites) as feasible sources for advanced drugs development. The pharmacologically active compounds from plants along with the safe synthetic drugs can serve the humanity to combat the challenges of emerging and prevailing diseases.

1.1.3. Modern day drugs from medicinal plants

Majority of the present day drugs are directly or indirectly derived from medicinal plants. Extensive clinical research on plants made a gratifying achievements in many important fields such as anticancer (camptothecins and taxols)14, antimalarial (quinine

Chapter: 1 Introduction and artimether from species of Cinchona and Artimesia respectively)15-16 and leishmanicidal (f-narthexone, f-northexol from Asafoetida species)17. There is no doubt that natural products are the primary precursors of the majority of lead drugs. Present- ly, natural products, their derivatives and analogs represent 50% of all contempo- rary medicine in the clinical use18, including morphine19, atropine20, artimether16, codeine21, ephedrine22, quinine23, taxol14, vinblastine2, digoxin24 and vincristine25 etc.

Table-1.1: Natural Products based drugs launched since 200526.

Year Trade name Lead Source(s) Pharmacologic compound property 2005 Satives ® Dronabinol/ Natural-products Analgesic Cannabidol 2005 Flisint ® Fumagillin Natural-products Anti-Parasitic 2005 Finibax®/ Doribax Thienamycin Natural-products Antibacterial TM derivative 2005 Tygacel ® Tigecycline Natural-products Antibacterial (tetracycline) Semisynthetic derivative 2005 Prialt® Zinocotide Natural-products Pain 2005 Endeavor TM (Zo- Sirolimus Natural-products Cardiology tarolimus stent) Semisynthetic derivative 2006 Eraxis TM/ Ecalta TM Echinocandin Natural-products Antifungal (Anidulafungin) B Semisynthetic derivative 2006 ByettaTM (Ex- Exenatide-4 Natural-products Antidiabetic enatide) 2007 VyuanseTM Lisdex- Amphetamine Natural-products ADHD amphetamine Semisynthetic derivative 2007 AltabaxTM / Altar- Pleuromutilin Natural-products Antibacterial goTM (Retapamulin) Semisynthetic derivative 2007 ToriselTM Sirolimus Natural-products Anticancer (Temsirolimus) Semisynthetic derivative 2007 YondelisTM Trabectiden Natural-products Anticancer (Trabectiden) 2007 IxempraTM Epothilone B Natural-products Anticancer (Ixabepilone) Semisynthetic derivative

Chapter: 1 Introduction

1.1.4. Some dietary supplements as preventive medicine

There are number of epidemiological evidences about the use of fruits and vegetables in protecting from diseases including cancer27, cardiovascular disorders28, diabetes mellitus29, chronic infections30, constipation and neurologic disorders31-32.

Fruit and vegetables contain a variety of therapeutic constituents such as vitamins33, flavonoids34, crude proteins35, fibers36, alkaloids37, carotenoids38, tannins (polyphenols and proanthocyanins)39. All these substances are tremendously beneficial for health.

WHO strongly suggest the use of vegetables and fruits at least five times per day40.

Fruit of Punica granatum (Punicaceae) is used for the preventing cancer, cardiovascular-disease, diabetes mellitus, dental diseases, bacterial infections and skin damage due to ultraviolet radiation41. Quantitatively, fruit of P. granatum composed of flavonoids (app. 29.5 mg/g quercetin equivalent), phenolics (app. 330.9 mg/g gallic acid equivalent), tannins (30.6 mg/g catechin equivalentand) and anthocyanins (0.70 mg/g cyanidin--3--glucoside equivalent)42.

Similarly green leafy vegetables are primarily composed of crude protein in a range of

(app. 3.8 to 27.7 g/100 g), ascorbic acid (vitamen C 100 to 421.6 mg/100 g), and carbohydrates (2.9 to 47.9 g/100 g), furthermore the presence of saponins, inulins, alkaloids, and tannins have also been confirmed. Common diseases including headache, diarrhoea, high blood pressure, fevers, anaemia and female infertility are treated using more than 12 leafy vegetables43-44.

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Table-1.2: Common herbs with their medicinal properties45.

S.No Common name Botanic name Used for 1 Saw palmetto Sarenoa serrulata For enlarged prostate. 2 Garlic Allium sativum Reducing blood cholesterol level and decreasing risks of heart attack. 3 St. John’s wort Hypericum The treatment of depression with perforatum better patient compliance and fewer side effects 4 Ginger Zingiber officinale Alleviating vomiting and nausea

5 Black Cohosh Actaea racemosa Symptoms of menopause 6 Horse chestnut Aesculus Chronic venous insufficiency hippocastanum 7 Ginkgo Ginkgo biloba Improvement of mental performance in patient with AD

1.1.5. Market value of natural products in the world

The pharmacovigilance system of the World Health Organization (WHO) about natural products has opened many ways for the development of new marketable herbal medicines. More than 150 antibiotics were available in the market in 1983 that were derived from around 7,000 natural, along with 50 thousand semi-synthetically derived or synthetic compounds46-47. Natural products have highly impressed the

Western-world for its effectiveness in the treatment of a variety of ailments. In 1994 the annual sale of phytomedicines at retail level was more than 1.6 billion US$ while in 1998 the sale reached up to 4 billion US$. In European Union the sale of herbal medicine in 1996 were more than 7000 million US$48. In 1997 in Germany the total sale of plants based medicines were 1.8 billion US$ while in France, it was 1.1 billion

US$. In certain reports it has-been claimed that the sale of herbal-medicines (HM) have raised 43% during 1994-1998 in the market of UK and its worth was 50 million

Chapter: 1 Introduction

£, while this percentage raised up to 60% in 200346. In Denmark, the market value of herbal medicine with a moderate growth of 1% was DKK 50749. In Germany, the market value herbal products was €1.5 billion in 201450. The market value of herbal medicine in Russia with compound annual growth rate (CAGR) of 8% reached to a net worth of RUB 35.8 billion in 201451. In brazil, the herbal products, reached a total value sale of BRL1,508 million in 201452. In US among other herbal products, dietary supplements, dominated the market that account for three-quarters of the net sale.

Plants based traditional cold, cough, and allergy remedies retail value sales rose by

7% in 2014, aided by medicated confectionery products, to reach US$593 million53-54.

In the developed world including USA and Japan, there is high demand for nutraceuticals.

The proposed market value of nutraceutical in USA was more than $ 80-250 billion in 2010, with a similar market size in Europe and Japan55. Among the herbal medicine

India is enjoying a net share of US$1 billion while China is getting US$ 6 billion56. In

2008 the estimated global market for herbal medicine was US $ 83 billion, while the sale of herbal medicine was likely to get raise at an average of 6.4% annual growth rate, global targets for the herbal medicine market to reach US$107 billion by 201757-

58.

1.1.6. Research on medicinal plants; a basic need of the day

Medicinal plants have been used since long for therapeutic purposes and are still serving a huge population for their heath needs. Modern day drugs are derived from the natural products (plants) either directly or indirectly. In 2009, a total 250,000

-350,000 plant species have been identified, among them 35,000 are used globally for the treatment and/or management of various diseases2. It is also clear from the

Chapter: 1 Introduction available literature that 15% of medicinal-plants have been analysed phytochemically and only 6% are screened biologically59-60. Majority of these plants are used in unprocessed or semi-refined form or even in mixtures; hence, quality control and accurate clinical trials are required before their use in clinical practices61. The existing methodologies for the quality evaluation cannot fulfil the prime requirements of the safety and efficacy of herbal medicine. The basic reason might be composition of the purity (comparative to synthetic drugs) of herbal medicine as it is comprised of more than hundred compounds (substances). Yet, it befalls to be very complicated or practically impossible in majority of cases to recognize most of these constituents by currently available conventional methods62. Generally a few pharmacologically active components or marker are utilized to evaluate and authenticate the herbal medicines63.

Herbal medicines are composed of hundreds of compounds and majority of them are present in very low amounts. Similarly inconsistencies (variability) exist within the same product64. Chromatographic techniques are well established powerful tools for the separation of complex products (herbal medicines) into various relatively simple sub-systems65. Furthermore, hyphenated chromatographic approaches with several spectroscopic and/or spectrometric techniques such as high performance liquid chromatography-diode-array-detection (HPLC-DAD), gas-chromatography mass- spectrometry (GC – MS), capillary electrophoresis- diode array detection (CE-DAD) and high-performance liquid chormatography-mass spectrometry (HPLC-MS), could perform more accurately in expressions of the reduction of instrumental variation interference, selectivity, retention-time shift correction and measurement precision66- 67. The above techniques can be used to set references (standards) for the currently available herbal medicine(s) and can also lead to the isolation of new lead compound(s) that are more potent and desired65,68.

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The botanicals have begun to be accepted in allopathic system after the provision of valid scientific rationale. Just because of this revolution, WHO appreciated the merging of botanicals in health promotion-programs (HPP) of under develop or developing countries and points up the significance of scientific estimation of local herbal-remedies69.

1.1.7. History of herbal products during 19th and 20th centuries

Before 19th century, herbal medicines were used in crude form as tinctures (alcoholic extracts), infusions (herbal-teas), decoctions (boiled extracts of plant’s roots, leaves, flowers or stem bark), syrup (extract mixed in thick sugary solution) or used externally as ointments, balms, poultices and essential oils70. However, from late 19th century and onwards, researchers initiated the isolation, purification and identification of the bioactive constituents from the medicinal plants. This attempt led to the discovery of the highly important drugs that are even currently used extensively in modern medicine71-73. For instance, quinine isolated from Cinchona officinalis

(family Rubiaceae) plant and other species of the genus is a highly effective anti-malarial drug, morphine isolated from Papaver somniferum (family Pa- paveraceae) is a potent analgesic; taxol isolated from Taxus brevifolius (family Taxa- ceae) and vincristine from Catharanthus rosesus (family Apocynaceae) are effective anti-cancers. Similarly, serpentine isolated from Rauwolfia serpentia (family

Apocynaceae) is used for the management of hypertension70,73-74. Furthermore, many plant principles, other than biologically-active compounds, had served as lead molecules for designing, synthesis and development of new pharmacologically active drugs70. Similarly, some derivatives obtained after slight modification in compounds isolated from the medicinal plants have been proved to be less toxic and/or more effective71, such as, aspirin has been developed in 1953 by modifying the structure of

Chapter: 1 Introduction salicylic acid, which was isolated as active principle form number of plants, forlklorically used as analgesics71. Similarly, the anti-malarial drug artemisinin bearing low bioavailability and therefore, less effective, has been modified to many derivatives, including arteether, artesunate and artemether. In another example, the development of the popular hypoglycemic drug “metformin” was based on the utilization of extracts of Galega officinalis (Fabaceae) to treat diabetes mellitus. The blood sugar lowering activity of G. officinalis has been legitimated due to galegine, a guanidine type alkaloid. However, the galegine was identified to be toxic in human; therefore, various structural analogue of this compounds have been derived that are safe in clinical practices.

1.1.8. Bioassay-guided isolation from natural products

Natural products are rich sources of important pharmacologically active compounds.

A variety of pharmacologically active compounds been isolated from natural products using bioactivity guided sub-fractionation and isolation75. Such phytochemical profiling has been facilitated by advanced developments in bioassay methods. It is clear from evidences that, pharmacologically active compounds from plants are major secondary metabolites which becomes new drugs after passing through important phases such as identification, isolation, purification, characterization, modification, bioactivity screening and clinical trials76. Majority of such products are useful dietary-supplements, while others are useful commercial products such as latex, plants origin proteins and aromatic or volatile oils etc77-79. Structural modifications of the pharmacologically active compounds have proved to enhance the bioactivity profiles comparative to parent compounds80. A large number of semi-synthetic derivatives of such compounds have been approved for the clinical uses or undergoing sequential

Chapter: 1 Introduction clinical trials for the treatment of various ailments like cancers, diabetes, AIDS, hypertension, Alzheimer’s disease, pulmonary and other diseases81-82. Certain plants extracts are used as (traditional) medicines in many countries, bearing a specific identification for its existence in many areas of the world. In Asia, Kampo also known as Japanese-Chinese medicine, Traditional-Chinese medicine, Korean-Chinese medicine, Jamu (Indonesia) and Ayurvedic medicine (India), Hoemeopathy and Phyto- therapy in Europe, while in America the term “Alternative Medicines” is used for plants based drugs when used in combination with some other traditional medi- cines83-85. The term integrative medicine has been used for the combination of aforementioned traditional medicines with western medicine (conventional medicine) worldwide86.

1.1.9. Medicinal plants as best sources of important drugs

A variety of purification and isolation techniques have been developed to discover pharmacologically active moieties from medicinal plants that can serve as best remedies for different types of diseases. The methods include various chromatographic techniques, combinatorial chemistry, molecular modelling and synthetic chemistry, which have been used for obtaining drugs from natural products especially plants87-89. Although combinatorial chemistry, molecular modelling and other synthetic and semisynthetic chemistry are highlighted area for research, that are funded by pharmaceutical companies and organizations, natural products particularly plants are also an important sources of new pharmacologically active entities and lead drugs10,81 . Reports of a survey conducted in 2001-2002 claims that more than one quarter of overall selling drugs in the world were based on medicinal plants 81. Since

1981 to 2002 it has been noticed that more than 28% of new drugs have been derived

Chapter: 1 Introduction from plants70. Another survey conducted during the aforementioned period reported that more than 20% of lead drugs are the compounds that are derived from natural products70. Based on these reports it is clear that natural products account for more than 48% of lead drugs in that era of 30 years.

Furthermore, it is also clear that plants synthesize chemical compounds of complex chemistry with multiple stereo-centres, thus provide an idea for laboratory based total synthesis. Such compounds can also provide a base for its semi-synthetic derivatives as remodelled by structure modification90-93.

Certain natural products have many structural characteristics in common (chiral centres, complex ring systems, aromatic rings, degree of molecule saturation, and ratio of heteroatoms) that are considered to be very important in new drugs discovery efforts90-91,94-95. Compounds of different chemical classes, isolated from plants not only serves as new drugs, but also very useful for further structure modification followed by structure activity relationship studies. Sometime it is difficult to elucidate structures for complex compounds discovered from medicinal plants, in such cases compounds of known structures and bioactivities can provide some important directions. A variety of drugs have been discovered through molecule targeting techniques96. The advancements in high-throughput screening technique may help in specific activity directed towards these targets. A broad range of compounds have been isolated from folklorically used medicinal plants that have been proved to act on new molecular targets. Indirubin is an example that target and inhibit cyclin dependent kinases97-98, as another kamebakaurin, that has been proved to target and inhibit necrotizing factor (NF-nB)99-100. There are many known compounds that have been proved to act on new and novel molecular targets; such developments lead to

Chapter: 1 Introduction build attention in compounds that are isolated form plants. Similarly cucurbitacin-I

from NCI (National Cancer-Institute) is a diversity s.et of several identified compounds, that is shown to be vastly selective in the inhibition of JAK/STAT3 path-ways in cases of cancerous tumors in activated STAT3 condition101, other case in point is h-lapachone, that discriminatingly kills cancer cells and non-cytotoxic to normal cells by directly activating of check-point at some stage in the cell cycle102 and one of same type of compound is betulinic acid with discriminating anti-neuplastic potential that controls the cell-cycle by p38 activation103-105.

According to Balunas and Kindhorn (2005), four (new) drugs that originated from medicinal plants, have been approved for clinical use in U.S106. These drugs include

β-Arteether (Artemotil ®) a very effectual anti-malarial drug that has been imitated

from Artemis.inin, structurally it is a sesquiterpene-lactone and is obtained (isolated) from Artemisia species (family Asteraceae). This plant has been used in Chinese medicine for a variety of therapeutic purposes107-108. In Europe many other imitatives of artemisinin are under investigation in various stages of clinical trials as anti- inflammatory, anti-neoplastic and anti-malarial drugs109. Galanthamine or galantamine (Reminyl®) has been isolated through bioassay guided isolation from natural products in Russia in 1950s, that was first time isolated from Galanthus woronowii

(family Amaryllidaceae)110-111. Galantamine has been approved for the treatment of

Alzheimer’s disease. It inhibits acetylcholineesterase and slow down neurological degeneration process110-111.

Nitisinone (Orfadin®) a very recently discovered natural compound has been notified for controlling the tyrosinaemia, that proves the usefulness of lead structures derived from natural products112. Moreover, nitisinone, structurally a modified form of mes-

Chapter: 1 Introduction otrione, an herbicide derived from the natural product leptospermone, first time obtained from Callistemon citrinus (family Myrtaceae)113-114.

The aforementioned three tri-ketones inhibit a common type of enzyme,

4-hydroxyl-phenyl-pyruvate dehydrogenase (HPPD), when studied in maize and human 113-114. During studies in maize it inhibits the HPPD enzyme that shows herbicidal property by reducing plastoquinone and tocopherol bio-synthesis. While in human, inhibiting the enzyme, HPPD inhibits the catabolism of tyrosine and the accumulation of toxic metabolites in liver and kidneys114. Tiotropium (Spirival TM) is effective for the treatment of lungs disease i.e. chronic obstructive pulmonary disease

(COPD) is recently introduced to market for clinical use115-116. Tiotroprium is anticholinergic bronchodilator, derived from ipratropium that is derived from atropine, an alkaloid (isolated) obtained from Atropa-belladonna (family

Solanaceae)117-118. Tiotropium is a long acting drug comparatively to all other available medications for COPD117. Figure-1.1 shows some examples of drugs isolated from medicinal plant. Compounds (morphine-6-glucuronide, exatecan and vinflunine) are in clinical trials (Phase III) or under review for registration. They will modify the current regime for the management and treatment of various diseases.

Morphine-6-glucuronide a metabolic derivative of morphine, obtained from the

Papaver somniferum (family Papaveraceae), have less adverse effects than morphine and will be considered as best alternative of morphine in pain management119.

Vinflunine a modified form of vinblastine, obtained from Catharanthus roseus

(family Apocynaceae) is more effective anticancer than vinblastine120. Exatecan is a developed anticancer drug, similar to camptothecin obtained from Camptotheca acuminata (family Nyssaceae)81,121. The research on modification of the currently existing drugs isolated from plants can lead to the discovery of new chemical entities

Chapter: 1 Introduction with high therapeutic value(s). Calanolide-A is structurally dipyrano-coumarin obtained from Calophyllum lanigerum (family Clusiaceae), a rain forest tree of

Malaysia122-124. Research on Calanolide-A proved its effectiveness in the treatment of

HIV. It bears a very distinctive and unambiguous mode of action specifically, non-nucleoside reverse transcriptase(s) inhibitor (NNRTI) of HIV of type-I. It is also effective for combating strains resistant to Azathioprene (AZT)124-126. Calanolide-A is currently under investigation in Phase II clinical trials127.

Chapter: 1 Introduction

Arteether

Galanthamine

Nitisinone

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Tritropium

Calanolide-A

Exatecan

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Morphine -6-glucoronide

Vinflunine

Fig-1.1: Bio-assay guided isolated drugs from medicinal plants107,110,112,117.

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1.1.10. Secondary metabolites

Secondary metabolite(s) (SM) are organic compounds present in plants. They are not involved in the normal physiological functions and are produced in various metabolic processes, it has been reported that some SM are very specific to a particular set of species that came into same phylogenetic group. Some of the secondary metabolites may be extensively disseminated, that may be found in several taxa, while many other may-be present in a single species. Hence, SM are responsible for the majority of molecular variability in living organisms. Plants have metabolic diversity, due to which plants are gaining the attention of natural products scientists since long. It is often thought that the fixed mode of life is majorly responsible for metabolic diversity in plants. In contrast, animals have a less number of the specialized metabolites as their mood of life (mobility) let them to escape from predators. In comparison, plants depend majorly on chemical communication, whether to attract beneficial organisms

(pollinating insects), ward off pest, and/or signalling to other plants nearby. Generally the exact figure of SM produced by plants is not identified but is approximated to exceed 200,000 in number128.

It is also known that within a single plant, different tissues produce different types of

SM. Moreover, the environmental conditions and stage of development as well as physiological state also affect the specialized compound (SM) quantity in a given species. Currently many plant genomes have been sequenced completely, the number of sequenced genomes of plant is progressively increasing subsequent to the advancement in high-throughput sequencing, it has turn into more clear that the

(bio)synthetic properties of plants are quite higher than formerly thought, as imagined from the huge number of gene(s)-encoding proteins of unclear function but related to the different classes (types) of enzymes, which are identified to be concerned with

Chapter: 1 Introduction metabolic pathways. Yet, it has been approximated that up to 20% of all the genes of any plant species might be implicated in specific metabolism. Assuming that, there are 1.5 protein(s) per enzyme and 1 enzyme for each product, the model plant Ara- bidopsis thaliana (family Brassicaceae), with 26,500 genes can be approximated to generate 1750–3500 SM. Majority of plant species have even more larger genome

(e.g., rice has around 35,000 genes), and it may be concluded that they will be able to produce even further specialized compounds or SM129. There are several SM that are more common in many species, and every species even contributes a small fraction of the unique compounds to the plant kingdom. This marvellous diversity raises issues of their purpose such as how they are produced and which molecular mechanisms have endorsed this diversity to progress. By focusing on explicit examples, the issues of the development of SM biosynthesis and their locations of biosynthesis are addressed.

1.1.10.1. Major classes of plant specialized compounds

SM can be divided in various groups according to their chemical classes (nature).

Terpenoids is the most well-known class with more than 25,000 identified compounds. Alkaloids represent the second largest class with 21,000 compounds.

Phenolics’ (including henylpropanoids and flavonoids) is the third common class with more than 10,000 substances described. There are also many other classes with small- er numbers of compounds as they occur in a small number of species. SM are basically derived from precursors that are parts of the compounds of primary metabolism. A summary of the relations of the major classes of SM to central metabolism is presented in Figure: 1.2130.

Chapter: 1 Introduction

Fig-1.2: Schematic pathways of synthesis of plants secondary metabolites from glycolysis, tricarboxylic acid (TCA) cycle and Calvin cycle130.

Chapter: 1 Introduction

1.1.11. Status of medicinal plants in the world

Scientists are always in search of products that can fulfill or match the requirements of the advance world. In each and every part of the world people are often interested in getting the products that are recently discovered or even considered as newly introduced. Research and development section of every organization are struggling to rule on the market. Similarly, in medicine, majority of companies provide funds for numerous projects and spend huge revenue every year to introduce such remedies in the market that are more potent having no or low adverse effects and can combat diseases that are yet to be treated. Every year new drugs are introduced for management of various conditions such as cancers, hypertension, psychological disorders, diabetes, autoimmune diseases etc.

Studies discovering drugs for the management of diseases including cancers and infections are succeeded up to certain level. More detail studies are to be conducted to discover advance remedies that can combat certain condition like resistant strains of various pathological species.

Synthetic, semi synthetic derivatives or even pure compounds from natural products are discovered every year. Using traditional knowledge about medicinal plants, more than 119 plants based pharmaceuticals have been launched in the market and currently

89 medicinal plants based pharmaceuticals are used in the modern medicines131-132.

Throughout the world the aims of using medicinal plants as therapeutic agents are:

 Isolation of bioactive constituent from plants to be used directly as drugs, e.g.,

digitoxin, digoxin, morphine, reserpine, ephedrine, taxol, artimisinine,

vincristine, vinblastine, euginol;

Chapter: 1 Introduction

 Derivatization or remodeling of known or new compounds for semi synthesis

to introduce new pharmacologically active entities of high potency, e.g., arte-

mether, nabilone, metformin, oxycodon, teniposide, taxotere, galegine, vera-

pamil, amiodarone;

 To utilize such agents as pharmacological tools, e.g., lysergic acid

diethylamide, mescaline, yohimbine; and

 Specific part of a plant or whole plant as a herbal medicine, e.g., ginseng,

echinacea, cranberry, grapeseed, clove buds, feverfew, garlic, barley, ginkgo

biloba, saw palmetto133.

Table-1.3: Different plants groups and their medicinal species134.

S.No Name Number 1 Angiosperms A) Dicotyledones 3495 B) Monocotyledones 676 2 Gymnosperm 382 3 Pteridophytes 382 4 Bryophytes 39 5 Thalophytes 230

1.1.12. Medicinal plants status in Pakistan

Due to the presence of variety of climatic zones, Pakistan has a distinctive biodiversity. 1572 genera and more than 6000 species are present in Pakistan, among them 5521 medicinal plants species are found in hilly areas of the country135. 70% of the total species are present in the meticulous area of the country, while 30% are growing throughout the country136. Phyto-geograpically, Pakistan is divided into four main regions. Majority of medicinal-plants are found in Irano-Turanian region, some

Chapter: 1 Introduction

medicinal plants species are available in Himalayan and Sindian-region, while on In-

do-Pak boarder the least number of medicinal plants are found. This allocation is

shown in Figure-1.3.

In Pakistan, due to affordability and availability the demand of plants based medicines

is increasing day by day137. More than 84% of the total population across the country

is dependent on herbal-medicines138. Major part of that population is getting herbal

medicines from local practitioners called Tabibs or Hakims139. Knowledge about the

use of herbal remedies is transferred from generation to generation verbally and the

traditional practitioners are using herbal remedies without valid scientific

backgrounds140. The application of modern science and technology for the

determination of quality, efficacy and safety of such herbal product(s) is highly need-

ed. Biological screening of such plants using standard in-vitro and in-vivo proto-

cols, the phytochemical profiling is also required to supplement the pharma-

cological screening, and such phytochemical screening can aid in the discovery

of lead compounds.

Total plants species (6000)

Plant species with 30 % species are Plant species with known medicinal Phytogeographical multiregional and 70 unknown medicinal potentials (600 - regions % species are potential (5300 - 700) uniregional 5400)

Irano-Turanian Sindian region region Himalayan region Indo- Pak boarder

Fig-1.3: Status of medicinal plants in Pakistan136.

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In Pakistan majority of research on plants based drugs or medicinal plants are carried out at institutional or academia levels. Such research is either in the form of biological screening or phytochemical profiling. However, mostly pharmacological activities carried out are based on the folkloric use of such plants. Sometime preliminary screening (other than folkloric uses) may also be performed to investigate the plants for uses other than traditional uses. In Pakistan, medicinal plants are present in different areas and people of that area have centuries old knowledge about such plants. As a result of such research activities, majority of plants have been proved to be good medication for the management of a variety of ailments. Plants having no folkloric uses (previously not been used as medicinal plants) are totally ignored by the researchers working for screening medicinal plants. Investigating such plants can add important compounds to the list of pharmacologically active or potent compounds.

1.1.13. Family Sapotaceae

Sapotaceae is largely tropical family of flowering evergreen trees and shrubs, comprised of 53 genera and 1100 species141. In Pakistan it is represented by 6 genera and

7 species142. Many species produces edible fruits and/or having other economic uses.

Species noted for the edible fruits are Monotheca buxifolia143, Manilkara bi- dentata144, Chrysophyllum cainito (golden leaf or star-apple tree)145,

Pouteria sapota146, Vitellaria paradoxa (shea)147 and Sideroxylon australe (Australian native plum)148. Some trees of the genus Palaquium produces latex with a variety of uses149. Species like Argania spinosa produces edible oil and are traditionally harvested in Morocco150.

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1.1.14. Status of family Sapotaceae in medicinal plants

In medicinal plants, family sapotaceae is an ample source of bioactive compounds of different chemical classes such as alkaloids, glycosides, terpenoids, sterols, steroids, saponins, sapogenins, flavonoids, phenols, essential oils, tannins, carbohydrates, proteins and reducing sugars. Members of this family is significantly used in treatment of various diseases such as Maduca longifolia is used as analgesic, diuretic, tonic, rheumatism, chronic bronchitis, diabetes mellitus, Cushing’s disease, astringent, demulcent, anthelmentic, in pharyngitis, bronchitis, and bleeding gums151. A variety of compounds have been isolated from its members such as Cardiochrysine from leaves of Chrysophyllum perpulchrum152, Laburnine benzoate from Planchonella thyrsoidea and Planchonella anteridifera, Laburnine tiglate from Planchonella thyr- soidea153, toxifolin from various members of this family is used as anti- inflammatory, antineoplastic and antifungal agent154.

1.1.15. Biological activities of family Sapotaceae

This family is widely studied for a variety of biological activities including anticancer, antibacterial, leishmanicidal, antifungal and hematinic etc. Several members of this family including Mimusops elengi155, Bassia latifolia156, Chrysophyl- lum albidum157, Madhuca indica, Madhuca longifolia158, Autranella congolensis159,

Achras zapota160, and Pouteria campechiana161 have been evaluated for its hepatoprotective potential against various experimental models like acetaminophen, carbon tetrachloride, thioacetamide and D-galactosamine induced hepatotoxicity, a list of some reported biological activities are given in Table-1.4.

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Table-1.4: Reported biological activities of family Sapotaceae.

S.No Activity Source Ref. 1 Antimicrobial Tridesmostemon omphalocarpoides 162-163 2 Anti-trypanosomal Butyrospermum paradoxum 164 3 Antioxidant Monotheca buxifolia 165 4 Antidiarhoeal Vitellaria paradoxa 166 5 Antipyretic Madhuca indica 167 6 CNS depressant Mimusops elengi 168 7 Anti-Inflammatory Mimusops elengi 169 8 Anthelmintic Mimusops kummel 170 9 Analgesic Madhuca indica 167 10 Cytotoxicity Butyrospermum parkii 171 11 Antidiabetic Manilkara zapota 172 12 Anticancer Synsepalum dulcificum 173 13 Antiviral Tieghemella heckelii 174 14 Antihyperlipidemic Mimusops elengi 175-176 15 Antiulcer Mimusops elengi 177 16 Antiepileptic Madhuca longifolia 178 17 Nephroprotective Bassia malabarica 179 18 Hepatoprotective Chrysophyllum albidum, 157,180 Madhuca longifolia 19 Leishmanicidal Baillonella toxisperma, 181-182 Manilkara dissecta 20 Laxative Acharus sapota 183 21 Male sexual impotence Gambeya lacourtiana 184 22 Urolithiasis Mimusops elengi 185 23 Lumbago Baillonella toxisperma 184 24 Malaria Baillonella toxisperma 184 25 Anemia Baillonella toxisperma 184 26 Antispasmodic Pachystela brevipes 186 27 Rheumatic pain Diploknema butyracea 187 28 Fractured bone Madhuca indica 188 29 Asthma Madhuca indica 189 30 Anemia Chrysophyllum albidium 190

Chapter: 1 Introduction

31 Cough Madhuca indica 191 32 TB Mimusops elengi 192 33 Ulcer Manilkara hexandra 193 34 Dermatitis Vitellaria paradoxa 194 35 Sedative Bumelia sartorum 195 36 Anthelmintic Pouteria sapota 196 37 Cardioactive Chrysophyllum species 197 38 Anti-HIV Pouteria viridis 196 39 Respiratory infections Mimusops elengi 198 40 Spermatogenic Madhuca latifolia 199 41 Insulin secretion Madhuca longifolia 200 42 Diuretic Mimusops elengi 201 43 Neuroprotective Pouteria ramiflora 202 44 Pancreatic lipase inhibitor Chrysophyllum roxburghii 203 45 Anticaries Chrysophyllum roxburghii 203 46 Wound healing Madhuca longifolia 204 47 Trypsin inhibition Labramia bojeri 205 48 Anti-spasmodic Achras sapota 206 49 Miscarriage Capsicum annum 207 50 Antidepressant Manilkara subsericea 208

Chapter: 1 Introduction

Laburnine benzoate

Mol. Formula: C15H19NO2

Mol. wt.: 245.321

Source: Planchonella species

Isoretronecanol

Mol. Formula: C8H15NO

Mol. wt.: 141.213

Source: Planchonella species

Decanoyl

Mol. Formula: C40H68O2

Mol. wt.: 580.976

Source: Members of fam. Sapotaceae

Planchonelline

Mol. Formula: C12H19NO2S

Mol. wt.: 241.354

Source: Planchonella anteridifera

Chapter: 1 Introduction

Taxifolin

Mol. Formula: C15H12O7

Mol. wt.: 304

Source: Members of fam. Sapotaceae

Isotronecyl tigalate

Mol. Formula: C13H21NO2

Mol. wt.: 223.314

Source: Planchonella species

Isoretronecyl trans-B-methlthioacrylate

Mol. Formula: C12H19NO2S

Mol. wt.: 241.354

Source: Planchonella anteridifera

Bassic acid

Mol. Formula: C30H46O5

Mol. wt.: 486.690

Source: Madhuca longifolia

Chapter: 1 Introduction

Bassiasaponin A

Mol. Formula: C42H66O15

Mol. wt.: 810.974

Source: Bassia latifolia

Bassiasaponin B

Mol. Formula: C58H92O27

Mol. wt.: 1221.349

Source: Bassia latifolia

Fig-1.4: Isolated compounds from family Sapotaceae153,209.

Chapter: 1 Introduction

1.1.16. The genus Monotheca

This genus contains large shrubs or small spiny trees, maximum 10 meters in height.

Branches are sericeous, sometime white pubescent. Strong spines, more often when sturdy with flowers and leaves. Leaves are spatulate elliptical, entire, glabrous above, coriaceous, with sericeous indumentums below, venation obscure and petiolate.

Inflorescence is sessile in clusters and axillary. Pedicellate bisexual flowers with 5 lobed calyx, lobes are imbricate, joined basally, and sometime broadly ovate, with sericeous indumentum. 5 lobed short tube corolla, sub-campanulate, lobes are ovate-oblong, contorted-imbricate, obtuse. 5 stamens with linear filaments, exceeding till the petals, anthers extrorse; 5 staminodes, alternating with that of stamens, with flattened base and filiform. Ovary globose, unilocular, hairy, placentation basal,

5-ovuled. Min stigma with cylindrical style. Fruit is fleshy berry, 1 seeded, globose.

Seed is globular with ruminate endosperm. Embryo is curved horizontally210.

Monotheca is mono-typic genus, widely distributed in the North-West of Pakistan, along boarder of Afghanistan, in Jubail mountain of Oman in Arabian Peninsula,

South-Ethiopia, North-Somalia and Dijibouti. Mostly occurs in the Juniperus forest, dry Olea and in ever-green scrub. Formerly genus Monotheca was included in

Myrsinaceae, but later on Radlkofer kept this genus in family Sapotaceae. Distinguish points of differentiation from others members of family Sapotaceae are larger stamens and no latex production except Palaquium species142.

Chapter: 1 Introduction

1.2. Plant Introduction

1.2.1. Monotheca buxifolia

Monotheca buxifolia is an edible fruit bearing wild tree which belongs to family

Sapotaceae165. Locally, fruit of this plant is known as Gurgura211. It was reported for the first time by Hugh Falconer (1808-65) a Scottish botanist in 1848. Therefore the complete suggested name for this plant is Monotheca buxifolia Falc212.

1.2.2. Synonyms

Edgeworthia buxifolia Falc.

Monotheca mascatensis A.DC.

Reptonia buxifolia A.DC.

Table-1.5: Taxonomical classification of Monotheca buxifolia212

Hierarchy Names Kingdom Planteae Division Magnoliophyta Class Magnoliopsida Order Ericales Family Sapotaceae Genus Monotheca Specie buxifolia Herbarium Department of Botany, University of Peshawar Sheet No. BOT. 20061(PUP)

Chapter: 1 Introduction

Fig-1.5: Monotheca buxifolia specimen sheet at Department of Botany, University of Peshawar.

Chapter: 1 Introduction

1.2.3. Morphology

Monotheca buxifolia is a large thorny shrub or small tree with axillary, terminal and short thorns. Leaves are alternate or often fascicled, 2.5-3.5 × 1-1.5 cm in dimension.

Glaucous lower surface while upper surface is glabrous rounded apex having somewhat cuneate base, recurved margin, 2 to 3 mm short petiole. Flowers up to 5 mm in diameter, pedicel is 1 mm long maximum. Small lobed calyx, which is 3 mm long and acute. Glabrous corolla lobes up to 2×1 mm. Stamens opposite to lobes and borne on the tube of corolla, petals are shorter than filaments; glabrous, subulate, anthers are versatile. Hairy ovary with tapering elongated style212.

1.2.4. Fruit

Fruit is berry with a variable diameter of 0.5 to 1 cm. It is dark black in color when fully ripen, bearing a single seed, seed to flesh ratio 1:0.3-0.5. Fruit is sweet in taste210.

1.2.5. Distribution

The plant is native to Pakistan and is widely distributed in the himalyan-alpine chain, mainly in South-West (Hindukush) area of the country. It is abundantly available in districts of Dir, Swat, Bunir and Darra Adam Khail143,211. It is also found in

Afghanistan, southern Iran. Among the arabian cape Monotheca buxifolia grow naturally in the northern mountains of Oman, Yemen and southern Saudi Arabia213.

1.2.6. Ethnobotanical uses

Leaves are used as fodder for cattle and also fed to cows and buffalos to restore milk taste214. Antimony made of its gum is used in the treatment of eye infections215.

Chapter: 1 Introduction

Fruit of Monotheca buxifolia bears digestive215, hematinic216, laxative162, purgative, vermicidal, and antipyretic properties217, it is also used in the management of urinary tract infection162.

1.2.7. Previously reported activities of Monotheca buxifolia fruit

Phytochemical screening218, in-vitro antioxidant potential, total phenolics and flavonoids165 contents of the fruit have been reported previously, but no data available regarding its chemical and biological profiles.

Aims and Objectives

1.3. Aim and Objectives

1. To explore the phytochemical constituents of Monotheca buxifolia fruit using

chromatographic, spectrometric and spectroscopic techniques;

2. To evaluate biological activities of the Monotheca buxifolia fruit to provide a

scientific rationale to its folk uses;

3. To ascertain certain uses, other than folk uses, of the Monotheca buxifolia fruit by

performing bioactivity screening of crude extract, fractions and isolated

compounds.

Chapter: 2

MATERIAL & METHODS

Chapter: 2 Material & Methods

2. MATERIAL AND METHODS

2.1. General Experimental Requirements

Drugs and chemicals showed in Table-2.1 were used in assorted experiments.

Distilled water was used as solvent for solubilization and dose preparation of

Monotheca buxifolia hydroethanolic extract (MBHE) for various pharmacological activities. MBHE was soluble at room temperature in distilled water. Normal saline was used as a negative control in in-vivo pharmacological activities. Commercial grade Solvents like ethanol, methanol, acetone, ethyl acetate, chloroform, dichloromethane (DCM), n-hexane and n-butanol were used. Solvents were distilled before use. Analytical grade solvents were used as mobile phase in recycling HPLC

(preparative HPLC).

Table- 2.1: List of used chemicals and drugs.

S. No Chemicals Source 1 Acetaminophen Sigma Chemical Co, 2 Acetic acid St Louis, MO, USA 3 Acetic Acid (Deuterated) 4 Acetylcholine 5 Acetylthiocholine 6 Adamantane ethanol 7 Amphotericin B 8 Atropine 9 Bismuth nitrate 10 Bovine Serum Albumin 11 Carbacholine 12 Charcoal 13 Chloroform (Deuterated) 14 Chymostatin 15 Dithiobis-nitrobenzoic acid 16 Dragendorff's reagent 17 Dulbecco’s Eagle’s medium 18 Etoposide

Chapter: 2 Material & Methods

19 Ferric chloride 20 Fetal bovine serum 21 Formalin 22 Imipenem 23 Isoniazid 24 Magnesium oxide 25 Methanol (Deuterated) 26 N-succinyl-phenylalanine-p-nitroanilide 27 Nutrient agar 28 Paracetamol 29 Phenol reagent 30 Potassium Bromide 31 Potassium dihydrogen phosphate 32 Potassium Iodide 33 Potassium nitrate 34 Potassium phosphate 35 Rifampicin 36 RPMI-1640 37 Sabouraud dextrose agar 38 Silymarin 39 Sephadex 40 Sodium molybdate 41 Sodium nitroprusside 42 Streptomycin 43 Thiourea 44 Tri Methyl Silane 45 Tris-HCl 46 Tween-20 47 Xyline 48 Boric acid Merck, Darmstadt, Germany 49 Calcium chloride 50 Calcium nitrate 51 Ceric sulphate 52 Copper sulphate 53 Dimethyl sulfoxide 54 Ethylenediaminetetraacetic acid 55 Eosin 56 Flash silica gel 57 Galantamine 58 Glucose 59 Hematoxyline

Chapter: 2 Material & Methods

60 Magnesium chloride 61 Magnesium sulphate 62 Manganese(II) chloride 63 Methanol 64 Monosodium phosphate 65 MTT 66 Paraffin 67 Penicillin 68 Permethrin 69 Potassium chloride 70 Propidium iodide 71 Preparative TLC plates 72 Sodium azide 73 Sodium bicarbonate 74 Sodium chloride 75 Sodium carbonate 76 Sodium phosphate 77 Silica gel 78 TLC plates 79 Biochemical assay kits for, SGPT Chema Diagnostica Italy 80 SGOT 81 Total Bilrubin 82 ALP 83 TP 84 Miconazole Tocris Bioscience Bristol 85 Pentamedine United Kingdom 86 Phosphate buffer saline Santa Cruz Biotech, USA 87 Carrageenan 88 Iodine crystals Angus Chemicals Germany 89 Diclofenac sodium Novartis Pakistan 90 Normal saline Otsuka Japan 91 Commercial salt mixture Instant Ocean, USA 92 Urease enzyme Sekisui Diagnostics, USA 93 Aspirin Reckitt Benckiser Pakistan 94 Silymarin Hisunny Chemicals, China 95 Atropine ShanDong Chemicals Chine 96 Thiourea Hebei Xinji Chemicals China 97 Brewer’s yeast Vahine Professional, France

Chapter: 2 Material & Methods

2.1.1. Experimental Conditions

Fruit processing, drying, crushing, extraction and fractionation were conducted at research laboratories of the Department of Pharmacy, University of Peshawar. While in-vivo experiments were carried out in Animal House and Bioassay Centre of the

Department of Pharmacy, University of Peshawar. Compounds isolation, spectroscopic, spectrometric studies and in-vitro biological activities were performed in the Hussain Ebrahim Jamal (HEJ) Research Institute of Chemistry, International

Centre for Chemical and Biological Sciences (ICCBS), University of Karachi, Pa- kistan.

2.1.1.1. Physical Constants

Melting points of the compounds were determined using Buchi-535 melting point

26 apparatus. Optical rotation ([α]D ) of the compounds was determined using digital polarimeter (JASCO –P 2000 polarimeter) equipped with Glan-Taylor Prism.

2.1.2. Spectroscopy

2.1.2.1. UV Spectra

UV spectra of the compounds were obtained using Thermo-scientific evaluation 300

UV/Vis spectrometer.

2.1.2.2. IR Spectra

The infra-red (IR) spectrum of the compounds was taken on potassium bromide (KBr) disc, using infrared spectrometer IR Bruker Vector 22.

Chapter: 2 Material & Methods

2.1.2.3. Nuclear Magnetic Resonance (NMR)

The Proton Nuclear Magnetic Resonance (1H-NMR) spectra of the isolated compounds were recorded using Bruker AV 300, AMX 400 HD, AMX 500 (with or without cryoprobe) and AV-600 (LC-NMR with or without cryoprobe), operated at

300, 400, 500 or 600 MHz. The homo-nuclear correlations of 1H-1H in compounds were obtained from correlation spectroscopy (COSY) experiments at 45o.

Heteronuclear single quantum coherence (HSQC) experiments were performed to get the short range hetero-nuclear couplings of 1H-13C. Heteronuclear multiple bond correlation (HMBC) experiments were performed to get long range hetero-nuclear connectivity. Nuclear Overhauser effect spectroscopy (NOESY) was also performed for the confirmation of elucidated structure. The aforementioned experiments were performed on AV 300, AMX-400 HD, AV-600 and AV-500 spectrometers. The values of the chemical shift were presented in ppm in reference to the solvent used

(MEOD, CD3OD, DMSO-d6, CDCl3 etc). The carbon-13 Nuclear Magnetic Res- onance (13C-NMR) spectra of the isolated compounds were obtained with the help of

AV 300, AMX 400 HD, AMX 500 (with or without cryoprobe) and AV-600 (LC-

NMR with or without cryoprobe), operated at 300, 400, 500 or 600 MHz. The signals for primary, secondary and tertiary carbons (CH, CH2 and CH3) in a compound were recognized from the spectrum of Distortion-less enhancement by polarization transfer (DEPT) at 90o and 135o. DEPT 90o spectrum was used to visualize CH signals only while DEPT 135° spectrum was used to visualize the three types of carbons appeared (CH3, CH2, and CH). The positive mode signals indicate CH and CH3, while the signals of CH2 appear in the negative mode. The broad-band (BB) decoupled 13C-NMR spectra were used to visualize all the multiplets of carbons including quaternary carbons. Tri-Methyl Silane (TMS) was used as internal standard.

Chapter: 2 Material & Methods

2.1.3. Spectrometry

2.1.3.1. Mass Spectrometry

For obtaining the low resolution electron ionization mass spectrum (EI-MS) of the compounds, Jeol-MS route JMS-600H attached with TSS-2000 JEOL was used. High resolution electron ionization mass spectrum (HR-EI-MS) was measured with the help of Thermo Finnigan MAT-95XP, attached with X-calibur. The fast atom bombardment mass spectrometry (FAB-MS) and high resolution fast atom bombardment mass spectrometry (HRFAB-MS) were obtained using Jeol JMS

HX-110 mass spectrometer that was attached with TGRAF-4200.

2.1.3.2. Gas Chromatography-Mass Spectrometry (GC-MS)

Shimadzu-GC having fused silica column (ZB-5MS), connected with mass spectrometer (JEOL JMS 600-H) was used for obtaining the quantitative and qualitative GC and GC-MS spectra of the essential oils.

2.2. Isolation and Purification of Compounds

A number of compounds were isolated from several sub-fractions of Monotheca buxifolia fruit hydro-ethanolic extract using chromatographic techniques.

2.2.1. Column Chromatography (CC)

Silica-gel mesh size 70-230 and silica-gel (flash silica) mesh size 230-400 were employed as stationary phase in column chromatography. Organic solvents including n-hexane, DCM, methanol, ethyl acetate, acetone and chloroform were used in single or combination in escalating order of polarity as mobile phase in column chromatography.

Chapter: 2 Material & Methods

2.2.2. Thin-layer Chromatography (TLC)

Two types of TLC plates were used i.e. for evaluating the purity of isolated compounds aluminum plates, pre-coated with silica gel (0.20 mm thickness, 20 × 20 cm) were used, while for purification purposes glass plates, pre-coated with silica gel

(0.5 mm thickness, 20 × 20cm) were used.

2.2.3. Visualizing Compounds Spot on Developed TLC

After developing the TLC, spots of the compounds were visualized on UV (254 nm and 364 nm) light for UV active compounds. Treating (developed TLC) with ceric sulphate followed by heating was used to visualize UV inactive compounds as well as for re-confirmation of UV active compounds.

While spraying Dragendorff’s solution on developed TLC plates was used to visualize and confirm spot of alkaloidal compounds.

2.2.4. Ceric Sulphate Solution

Ceric sulphate solution visualizes oxidizable substances on a TLC plate. Ceric- sulphate solution was prepared by solubilising ceric sulphate in H2SO4 (65%) aqueous solution. Certain compounds show different behaviour on treating with ceric sulphate solution followed by heating, such as appearance of a pink color confirms the presence of terpenoids. Oleanolic acid shows pink color wedge shape spot.

2.2.5. Dragendorff’s Solution

For preparation of the Dragendorff’s solution, potassium iodide (KI, 40%) solution was prepared by dissolving KI (8 g) in distilled water (20 mL) and bismuth nitrate solution was prepared separately by dissolving bismuth nitrate (0.85 g) in acetic acid

Chapter: 2 Material & Methods

(20%) and distilled water. Stock solution was prepared by mixing the aforementioned two solutions. Stock solution (5 mL) was diluted with 10mL acetic acid and 90 mL distilled water. Spraying with Dragendorff’s solution, appearance of light pink to orange or blackish brown indicates the presence of alkaloids.

2.2.6. Iodine solution

Few crystals of iodine were added to TLC tank and heated for five min at 40- 50oC.

Spots of compounds were visualized by placing the developed TLC tank (saturated with iodine fumes).

2.2.7. Preparative HPLC (Recycling HPLC)

Compounds were purified using recycling high performance liquid chromatography

(Japan Analytical Industry (JAI) LC-908W-C60). Size exclusion column

(JAICEL- GS320) and reverse phase column (JAIGEL-ODS-L-80) with pre-column

(JAIGE-OD-L-80-P) were used. Samples (10 mg/ml) were injected with a maximum

3 mL loop size. Compounds were detected with the help of UV (JAI UV Detector

310) and RI (JAI RI Detector RI-5) detectors with multiple wavelength and sensitivity were connected. The peaks were recorded with the help of Sekonic (SS 250F) recorder. Pumps used were made of Hitachi (L-7110).

2.3. Plant Materials

2.3.1. Collection

Fruit of Monotheca buxifolia (50 Kg) were collected from the northern areas of Pa- kistan in the month of August, authenticated by a taxonomist at the Department of

Chapter: 2 Material & Methods

Botany, University of Peshawar. A specimen has also been deposited in the herbarium of the University of Peshawar, reference number: Bot. 20061 (PUP).

2.3.2. Extraction

Seeds were separated and fleshy fruit pulp was dried under shade at ambient temperature. The dried pulp was crushed to powder using grinder (Moulinex-S200,

France). Powder (6.5 Kg) was macerated using sufficient hydro-ethanolic (30:70) solvent system for 15 days. After 15 days the soluble residue was filtered using

Whatman-1 filter paper. This procedure was repeated three times to obtain maximum soluble material. The filtrate was concentrated by vaporizing solvents under reduced pressure at 40 oC using a rotary evaporator (Buchi- R210, Switzerland) fitted with water-bath (Buchi-B491, Switzerland) and recirculating chiller (Buchi, Switzerland).

The semisolid mass was collected as crude extract “MBHE” (2.419 Kg).

2.3.3. Fractionation

MBHE (115 g) was kept for various in-vivo and in-vitro biological activities while the remaining (2.304 Kg) was mixed with 2.5 L distilled water and soaked overnight, extracted successively with n-hexane (3 × 5 L), chloroform (3 × 5 L), ethyl acetate (3

× 5 L), and n-butanol (3 × 5 L) to get hexane soluble (56 g), chloroform soluble (57.7 g), ethyl acetate soluble (34.9 g) and n-butanol soluble (54.5 g) fractions respectively, the remaining was considered as water soluble fraction i.e. aqueous fraction. The whole scheme is shown in Figure-2.1.

Chapter: 2 Material & Methods

Fig-2.1: Schematic diagram for extraction and fractionation.

Chapter: 2 Material & Methods

2.4. Animals Used

In a variety of in-vivo experiments, mice “BALB/c and NMRI” (both sexes) and male

Sprague Dawley rats were used. Animals were obtained from the Animal House and

Bioassay Centre of the Department of Pharmacy, University of Peshawar. They were kept in the animal house under standard laboratory conditions (25 ± 1 oC with 12 h light/dark cycle) and were fed with standard diet and free access to water ad-libitum until and unless specified. Guidelines of the Commission of Life Sciences, National

Research Council and Institute of Laboratory Animal Resources were strictly followed during the experiments219. The experimental protocols for in-vivo experiments were approved by the ethical committee of the Department of Pharmacy,

University of Peshawar, Pakistan (registration number: 04/EC-15/Pharm).

2.5. Isolation of Compounds

2.5.1. Isolation of Compounds from Ethyl acetate Fraction

Based upon the biological activities, ethyl acetate fraction was selected for isolation of pure compounds. Ethyl acetate fraction (30 g) was subjected to vacuum liquid chromatography (VLC), using normal phase silica gel as stationary phase, eluted by using hexane, hexanes-ethyl acetate, ethyl acetate, ethyl acetate-methanol and methanol with increasing polarity. Matching the TLC results, 18 sub-fractions were obtained (IR. A→R).

Total 9 compounds were isolated and among them 1 was first time isolated from natural sources, 4 were new, while 4 were previously reported. Compound 1

(Buxifoline-A, first time isolated from natural sources), compound 2 (Buxifoline-B), compound 3 (Buxitriol), compound 4 (Buxilide) and compound 5 (Buxiglucoside) were isolated for the first time while the other 4 were compound 6 (Oleanolic acid),

Chapter: 2 Material & Methods compound 7 (Glucosidic β-sitosterol), compound 8 (2-Hydroxy- epikatonic acid) and compound 9 (Isoquercetin) were isolated for the first time from this plant (new source identified). Isolation scheme of the compounds is given in Figure-2.2.

Chapter: 2 Material & Methods

Fig-2.2: Schematic diagram for isolation of pure compounds.

Chapter: 2 Material & Methods

2.6. Characterization of isolated compounds

2.6.1. Buxifoline-A (1)

Compound 1 was isolated from the sub-fraction “B” of ethyl acetate fraction through column chromatography using ethyl acetate: hexane (1:9 → 2:8) as mobile phase and then purified by using recycling HPLC. Methanol: water (70:30) was used as mobile phase. Flow rate of the mobile phase was 4mL/min. Compound was detected using

UV detector. The retention time of the compound 1 was 36 min. White needle like crystals were obtained by evaporating solvents.

Table-2.2: Characterization of buxifoline-A.

S. No Parameters Observations 1 Physical state Needle like white crystals

2 Molecular formula C17H18N2O4 3 IUPAC name Dimethyl 4,4'-(methylenebis(azanediyl)) dibenzoate 26 4 [α] D 2.55 5 Melting point 334oC 6 UV activity UV active on TLC 7 Rf. Value 0.55 ethyl acetate: hexane (1:1) 8 Yield 67 mg 9 Solubility Methanol, Acetone (room temperature)

10 UV max 248, 232 and 210 nm 11 IR spectrum cm-1 3329 (NH), 1703 (C=O) and 1529 (aromatic) 12 1H-NMR (500 MHz) See Table-3.2 13 13C- NMR (300 MHz) See Table-3.2 14 HR-EIMS (m/z) 314.1228 (calcd. 314.1267)

Chapter: 2 Material & Methods

2.6.2. Buxifoline-B (2)

Compound 2 was also isolated from the sub-fraction “B” of ethyl acetate fraction through column chromatography using mobile phase ethyl acetate: hexane (1:9 →

2:8) and then purified by using recycling HPLC. Methanol: water (70:30) was used as mobile phase. Flow rate of the mobile phase was 4mL/min. Compound was detected using UV detector. The retention time of the compound 2 was 39 min. White amorphous powder was obtained by evaporating solvents.

Table-2.3: Characterization of buxifoline-B.

S.No Parameters Observations 1 Physical state White amorphous powder

2 Molecular formula C18H20N2O4 3 IUPAC name Ethyl 4-((((4- (methoxycarbonyl)phenyl)amino)methyl) amino)benzoate 26 4 [α] D 2.44 5 Melting point 455oC 6 UV activity UV active on TLC 7 Rf. Value 0.61 ethyl acetate: hexane (1:1) 8 Yield 12 mg 9 Solubility Methanol, Acetone (room temperature)

10 UV max 248, 243 and 209 nm 11 IR spectrum cm-1 3313 (NH), 1709 (C=O) and 1534 (aromatic) 12 1H-NMR (300 MHz) See Table-3.3 13 13C- NMR (500 MHz) See Table-3.3 14 HR-EIMS (m/z) 328.1383 (calcd. 328.1423)

Chapter: 2 Material & Methods

2.6.3. Buxitriol (3)

The sub-fraction “I” of ethyl acetate fraction was re-chromatographed using normal phase silica gel as stationary phase. The column was eluted using ethyl acetate:hexane

(6:4) as mobile phase.

Table-2.4: Characterization of buxitriol.

S.No Parameters Observations 1 Physical state White amorphous powder

2 Molecular formula C30H50O3 3 IUPAC name (2R,3R,4aR,6aR,6bS,8aS,11R,12aR,14aR,14bR)- 11-(hydroxymethyl)-4,4,6a,6b,8a,11,14b- heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11, 12,12a,14,14a,14b-icosahydropicene-2,3-diol 26 4 [α] D 2.44 5 Melting point 336oC 6 UV activity Active on UV 7 Rf. Value 0.32 hexane: DCM (6:4) 8 Yield 11 mg 9 Solubility Acetone, DCM (room temperature)

10 UV max 208, 244 nm 11 IR spectrum cm-1 3500 (Hydroxyl group) 12 1H-NMR (500 MHz) See Table-3.4 13 13C- NMR (600 MHz) See Table-3.4 14 HR-EIMS (m/z) 458.7162 (calcd. 458.3760)

Chapter: 2 Material & Methods

2.6.4. Buxilide (4)

Compound 4 was isolated from sub-fraction “M” of ethyl acetate fraction using normal phase preparative TLC. Mobile phase used were combination of 3 i.e. hexane: ethyl acetate: methanol (70:30:1).

Table-2.5: Characterization of buxilide.

S.No Parameters Observations 1 Physical state Off-white mild brownish crystals

2 Molecular formula C6H6O4 3 IUPAC name 4-hydroxy-5-(hydroxymethyl)-2H-pyran-2-one 26 4 [α] D 3.54 5 Melting point 328oC 6 UV activity Active on UV 7 Rf. Value 0.64 ethyl acetate: hexane (6:4) 8 Yield 16.2 mg 9 Solubility Methanol, Acetone (room temperature)

10 UV max 272 and 223 nm 11 IR spectrum cm-1 3388 (hydroxyl) and 1737 (lactone) 12 1H-NMR (300 MHz) See Table-3.5 13 13C- NMR (600 MHz) See Table-3.5 14 HR-EIMS (m/z) 142.0271 (calcd.= 142.0266)

Chapter: 2 Material & Methods

2.6.5. Buxiglucoside (5)

Compound 5 was isolated from the sub-fraction “M” of ethyl acetate fraction using normal phase preparative TLC. Mobile phase used were combination of 3 i.e. hexane: ethyl acetate: methanol (70:30:1).

Table-2.6: Characterization of buxiglucoside.

S.No Parameters Observations 1 Physical state Mild brownish sticky solid

2 Molecular formula C12H16O8 3 IUPAC name 5-(hydroxymethyl)-4-((2S,3R,4R,5R,6S) -3,4,5-trihydroxy-6-methyltetrahydro- 2H-pyran-2-yl)oxy)-2H-pyran-2-one 26 4 [α] D 5 5 Melting point 423oC 6 UV activity Active on UV 7 Rf. Value 0.48 ethyl acetate: hexane (6:4) 8 Yield 12.1 mg 9 Solubility Methanol, Acetone (room temperature)

10 UV max 270 and 221 nm 11 IR spectrum cm-1 3388 (hydroxyl) and 1737 (lactone) 12 1H-NMR (500 MHz) See Table-3.6 13 13C- NMR (500 MHz) See Table-3.6 (+ve) 14 HRFAB MS 289.0937 (calc. for C12H16O8 + H = 289.0923)

Chapter: 2 Material & Methods

2.6.6. Oleanolic acid (6)

Compound 6 was isolated from the subfraction “A” of ethyl acetate fraction of hydroethanolic (3:7) extract of Monotheca buxifolia fruit. Compound was purified through repeated column chromatography using normal phase silica gel as stationary phase. Mobile phase used were ethyl acetate: hexane (1:9).

Table-2.7: Characterization of oleanolic acid.

S.No Parameters Observations 1 Physical state While amorphous powder

2 Molecular formula C30H48O3 3 UV activity Inactive on UV 4 Rf. Value 0.43 ethyl acetate: hexane (2:8) 5 Yield 40 mg 6 Solubility DCM (room temperature) 7 1H-NMR (500 MHz) See Table-3.7 8 13C- NMR (500 MHz) See Table-3.7 9 HREI-MS 456.7003 (Calc. 456.6840)

Chapter: 2 Material & Methods

2.6.7. Glucosidic β-sitosterol (7)

Compound 7 was isolated from the sub-fraction “G” of ethyl acetate fraction. The compound was purified through repeated column chromatography. Normal phase silica was used as stationary phase. The column was eluted using ethyl acetate: hexane (1:1).

Table-2.8: Characterization of glucosidic β-sitosterol.

S.No Parameters Observations 1 Physical state While amorphous powder

2 Molecular formula C35H60O6 3 Yield 20.5 mg 4 Solubility DMSO (room temperature)

5 UV max 244 nm (DMSO-d6) 6 IR spectrum cm-1 3551 (hydroxyl), 1453(oleafinic double bond), 1049 (C-O) and 2948 (C-H) 7 1H-NMR (300 MHz) See Table-3.8 8 13C- NMR (400 MHz) See Table-3.8 9 HREI-MS (m/z) 576.4390

Chapter: 2 Material & Methods

2.6.8. 2-Hydroxy- epikatonic acid (8)

Compound 8 was isolated from the sub-fraction“I” of ethyl acetate fraction using column chromatography. Normal phase silica gel was used as stationary phase. The column was eluted using ethyl acetate: hexane (6.5:3.5).

Table-2.9: Characterization of 2-hydroxy-epikatonic acid.

S.No Parameters Observations 1 Physical state White sticky amorphous powder

2 Molecular formula C30H48O4 3 UV activity UV active on TLC 4 Rf. Value 0.41 hexane: DCM (6:4) 5 Yield 12mg 6 Solubility Methanol, DCM, chloroform (room temperature)

7 UV max 214, 223, 253, 260, 292 8 IR spectrum cm-1 3500 (hydroxyl) and 1736 (carboxyl) 9 1H-NMR (300 MHz) See Table-3.9 10 13C- NMR (600 MHz) See Table-3.9 11 HR-FAB (-ve) MS 471.6997 [M-H] (calcd. 472.3553)

Chapter: 2 Material & Methods

2.6.9. Isoquercetin (9)

Compound 9 was purified from the sub-fraction “M” of ethyl acetate fraction. The sub-fraction was re-chromatographed using normal phase silica gel as stationary phase. Compound 9 purification was made possible using solvent systems ethyl acetate: hexane (8:2).

Table-2.10: Characterization of isoquercetin.

S.No Parameters Observations 1 Physical state Yellow amorphous powder

2 Molecular formula C21H20O12 26 3 [α] D -76 4 Melting point 245oC 5 UV activity UV active on TLC 6 Yield 30.5 mg 7 Solubility Methanol, DMSO (room temperature)

8 UV max 213, 260 and 360 nm 9 IR spectrum cm-1 3415 (hydroxyl), 1650, 1433 (double bonds of aromatic rings) 10 1H-NMR (300 MHz) See Table-3.10 11 13C- NMR (400 MHz) See Table-3.10 12 HRFAB (-ve) MS 463.0012 [M-H]

Chapter: 2 Material & Methods

2.7. Biological activities

2.7.1. In-vitro biological activities

2.7.1.1. Antibacterial activity

Antibacterial potential of the MBHE and fractions was determined by agar well diffusion method220. In brief, nutrient agar (28 g) was dissolved in one litre (distilled and de-ionized) water and autoclaved at 121°C for 15 min, cooled to 45°C, transferred to 14 cm diameter petri-plates (8 mm thick layer) and covered aseptically, allowed to solidify at room temperature. The 24 hours old bacterial inoculum containing 104-106

CFU/mL (Mc-Farland turbidity equivalent to 0.5) was swabbed uniformly. Wells were made in the agar with sterilized borer (6 mm diameter). Test sample (100 µL) (3 mg/mL in DMSO) was added to each agar well and incubated at 37°C for 24 hours.

DMSO and imipenim were used as negative and positive control respectively. All the procedures were performed in triplicate and mean of zones of inhibition was calculated.

Percent inhibition was obtained by using formula220

zone of inhibition of sample % inhibition = X 100 zone of inhibition of + ve control

Table-2.11: Reference bacterial stains used.

S.No Bacterial Reference bacterial strains 1 Escherichia coli ATCC 25922 2 Bacillus subtilis ATCC 6633 3 Shigella. Flexeneri (clinical isolate) 4 Staphylococcus aureus ATCC 25923 5 Pseudomonas aeruginosa ATCC 27853 6 Salmonella typhi ATCC 19430

Chapter: 2 Material & Methods

2.7.1.2. Antifungal activity

Antifungal potential of MBHE and subsequent fractions was determined against five fungal strains by agar tube dilution method221. Briefly, test sample (24 mg) was dissolved in 1 mL sterile DMSO, served as stock solution. Sabouraud dextrose agar

(SDA) media was prepared, transferred to screw capped tubes and sterilized by autoclaving at 121oC for 15 min. Tubes were allowed to cool down to 50°C. Test sample

(66.6 µL, 400 µg/mL) was added to un-solidified media. Other media were supplemented with DMSO and standard antifungal drugs, served as control. Tubes were solidified at room temperature in slanting position. To each tube 4 mm diameter piece of a week old cultured fungus was added. Tubes were incubated at 28 ± 1°C for 7 days.

The tubes were examined for linear growth inhibition of fungi in millimetres. Percent inhibition was calculated with the reference to negative and positive controls.

Table-2.12: Fungal strains taken for the study.

S. No. Fungi Reference fungal strains 1 Candida albicans ATCC 2091 2 Aspergillus flavus ATCC 32611 3 Microsporum canis ATCC 11622 4 Fusarium solani ATCC 11712 5 Candida glabrata ATCC 90030

Chapter: 2 Material & Methods

2.7.1.3. Leishmanicidal activity

Leishmanicidal activity of MBHE and fractions was evaluated using standard protocol222. Simply, Leishmania major (DESTO) promastigotes were cultured at 22 to

25°C in RPMI-1640 (Sigma, USA). The media was added with 10% temperature inactivated (56°C for 30 min) fetal bovine serum (FBS). Promastigote culture in the log phase of growth was centrifuged (2000 rpm) for 10 min. Maintaining the same experimental conditions, they were washed (3 times) with sterile normal saline. Par-

6 asites were diluted to 10 -cells/mL by adding recently prepared medium. In a standard micro-titer plate of 96-wells, first row was supplemented with 180 µL of the recently prepared medium while other rows were added 100 µL. Medium was supplemented with samples (20 µL) and diluted serially. Parasite-culture (100 µL) was added to each well. DMSO was added to one row served as negative control, while standard drug was added to another row served as positive control. The loaded plates were incubated at 21-22°C for 72 hours. The living parasites were counted in

Neubauer’s chambers under microscope. Experiment was executed in triplicate and

IC50 was calculated with the help of a Windows based EZ-Fit 5.03 Perrella Scientific computer Software223.

2.7.1.4. Cytotoxic activity (brine shrimp)

Cytotoxicity assay was performed for MBHE and fractions using standard protocol224-225. Briefly, Artemia salina Leach (Brine shrimp) eggs were hatched in a shallow plastic dish (22 × 32 cm), filled with the artificial sea-water, which was made by dissolving the commercial salt mixture (Instant Ocean, Aquarium system,

Inc., USA) in redistilled water (pH 7.4). With help of a perforated partition the dish was divided into two compartments i.e. large compartment that was darkened and a

Chapter: 2 Material & Methods small compartment (remained open to ordinary light to attract larvae). Eggs (50 mg) were sprinkled to the large compartment. Stock solution of the test sample was made by dissolving 20 mg in 2 ml organic solvent. Samples (5, 50 and 500 µL) were added to separate vials in triplicate. The organic solvent was allowed to evaporate overnight.

Other vials were supplemented with organic solvent and reference cytotoxic drug

(etoposide) served as control. After two days nauplii were collected from the small compartment using Pasteur pipette. Seawater solution (1 mL) and 10 shrimps were added to each vial and final volume (5 mL) was made by adding seawater solution.

Loaded vials were incubated for 24 hours at 25 to 27°C. After 24 hours the numbers of living larvae were counted and results were obtained. LD50 was determined with the help of Finney Computer database226.

2.7.1.5. Phytotoxic activity

Phytotoxic activity were determined for the MBHE and fractions using established protocol227. The required medium was prepared by dissolving given components in distilled water (100 mL), potassium hydroxide was used for pH adjustment (5.5-6.5).

The medium was sterilized by autoclaving for 15 min at 121oC. Stock solution was prepared by dissolving test samples in methanol (20 mg/mL). In separate flasks samples were added in different concentration (10, 100 and 1000 µg/mL) and methanol was evaporated. Medium (20 mL) and Lemna minor (10 plants each containing 3 healthy fronds) plants were added to each flask. These flasks were kept in growth chamber for seven days. The phytotoxic effect of the samples was obtained by counting dead fronds.

Chapter: 2 Material & Methods

Following formula used for the calculation of growth regulation in percent227:

Number of fronds in test sample Percent Inhibition = 100 − X 100 Number of fronds in control

Table-2.13: Composition of medium for phytotoxic activity.

S.No Constituents Concentration (mg/mL) 1 Boric acid 2.86 2 Calcium nitrate 1180 3 Copper sulphate 0.22 4 Ethylene diamine tetra acetic acid 11.20 5 Ferric chloride 5.40 6 Magnesium sulphate 492 7 Manganese chloride 3.62 8 Potassium dihydrogen phosphate 680 9 Potassium nitrate 1515 10 Sodium molybdate 0.12 11 Zinc sulphate 0.22

Chapter: 2 Material & Methods

2.7.1.6. Insecticidal activity

The insecticidal potential of MBHE and fractions was determined against Tribolium castaneum, Rhyzopertha dominica and Callosobruchus analis using established protocol222. Briefly, test samples (20 mg) were dissolved in of methanol (3 mL). Test samples (1.019 mg/cm2) were loaded on 90 mm filter paper in petri plates separately.

The solvent was evaporated by keeping the plates overnight. Insects (10 active and equal size) were added to each plate. Plates were kept overnight in growth chamber at

27 oC and 50 % humidity. After 24 hours live insects were counted. Methanol and permethrin (239 μg/cm2) served as negative and positive controls respectively.

Results were obtained as percent mortality by using following formula222.

Number of living insects in test % Inhibition = 100 − X 100 Number of living insects in control

2.7.1.7. Urease inhibition assay

MBHE/fraction and isolated compounds were screened for urease inhibitory activity using protocol of Weatherburn228. Solution for the reaction was comprised of urease enzyme (Jack bean source 25 µL), urea (100 mM) and buffer solution (55 µL). The reaction solution along with test samples (5 µL, 1 mM) was incubated at 30oC for 15 min in 96-well plate. Indophenol’s procedure was adopted for measuring produced ammonia during the reaction. Briefly, 5µL of phenol reagent (1% w/v), alkali reagent

(70 µL) and sodium nitroprusside 0.005% (w/v) was added to each well. After 50 min of incubation, increase in absorbance was measured with the help of micro plate reader at 630 nm. Absorbance change (per min) was obtained with the help of Soft-

Max Pro software. Procedure was repeated for two more times. Thiourea was used as standard.

Chapter: 2 Material & Methods

Percent inhibition of urease enzyme was obtained by using formula228.

Optical density of test sample well % Inhibition = 100 − X 100 Optical density of control well

2.7.1.8. Acetylcholinesterase inhibition

MBHE and compounds were screened for acetylcholineesterase inhibitory potential.

Standard protocol was used with slight modification229. Briefly, the reaction mixture was prepared by mixing Tris-HCl (200 µL, 50 mM) at pH 8.0, BSA buffer (1%) and of the test sample (100 µL, 100 µg/mL). Dithiobis-nitrobenzoic acid (DTNB) (500

µL, 3 mM) was incubated for 2 min. at room temperature before the addition of substrate (acetylthiocholine iodide) (100 µL, 15 mM). After 4 min of the appearance of yellow color, the absorbance was measured at 405 nm. Galantamine (100 µg /mL) was used as standard.

The percent inhibition of acetylcholineesterase was obtained by using following formula:

A − B % AChe Inhibition = X 100 A

Where, A = Change in absorption without sample,

B = Change in absorption with test sample.

Chapter: 2 Material & Methods

2.7.1.9. Anticancer activity

Compounds were screened for anticancer potential. Standard MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric protocol was employed in 96–well (flat-bottom) micro plates230. Briefly, prostate cancer cells

(PC-3) were cultured in modified Dulbecco’s eagle’s medium by addition of Pen- icillin (100 IU/mL), Streptomycin (200 µg/mL) and fetal bovine serum (FBS) (5%) in

o three clean flasks. Flasks were incubated at 37 C in the presence of 5% CO2. The ex- ponentially growing-cells were harvested. Using haemocytometer, cell culture of

5 1×10 .cells/mL concentration was prepared. Cell culture (100 µL) was transferred to each well of micro plate. After 24 hours, medium was replaced by the freshly prepared medium (200 µL) with varying concentration of testing samples 1-100 µM.

Plates were incubated for 72 hours, MTT (50 L, 2 µg/mL) and DMSO (100 µL) were added to each well. Plates were further incubated for 4 hours.

Micro plate reader “ELISA” (Spectra Max Plus, Molecular Devices, USA) was used to observe the reduction of MTT, comparative to formazan in the cell culture; absorption was measured at 570 nm.

2.7.1.10. α-Chymotrypsin inhibition

MBHE, fractions and compounds were screened for α-chymotrypsin inhibitory potential using standard protocol231. Briefly, solution of α-chymotrypsin (12 units/mL) was prepared in Tris HCl (buffered pH 7.6) and pre-incubated with testing sample (final concentration prepared in 7% DMSO) at 30°C for 25 min.

N-succinyl-phenylalanine-p-nitroanilide (0.4 mM) was added to the reaction solution as substrate. During the reaction p-nitroaniline was released continuously. Absorption of p-nitroaniline was monitored with the help of micro-plate reader at 410 nm till the

Chapter: 2 Material & Methods appearence of significant change in coloration. Results were obtained using

Soft-Max-Pro software (Molecular Device, CA, USA).

Following formula was used calculating percent inhibition231:

Optical density of test sample well % Inhibition = 100 − X 100 Optical density of control well

2.7.1.11. Cytotoxic activity (NIH 3T3 cell lines)

Pure compounds were evaluated for cytotoxic activity against human B cell lymphocytes (15310-LN cell lines) using standard protocol232. Briefly, adamantane ethanol (AdEtOH) and test compounds (1, 4, 5, 6, 7 and 9) were administered to evaluate the cytotoxicity potential against NIH-3T3 cells in a flat-bottom plate of 96- wells using MTT assay.

The cytotoxicity observed against human B cells expressing HLA (Human leukocyte antigen) class II antigen, DRB1 (HLA-DRB1*0101 (15310-LN)) was determined by using the flow cytometry based propidium iodide (PI) staining. Simply, in the presence of test samples cultured cells were seeded at 7×104 cells/well. They were

o incubated at 37 C for 48 hours in the presence of CO2 (5%). After 48 hours the cells were centrifuged (1300 rpm for 5 min), rinsed with phosphate buffer saline (PBS) mixed with fetal bovine serum (5%) and stained with PI (0.5 mg/mL). The count of living (PI-non stained) and dead (PI-stained) cells were observed with the help of a flow cytomerter (FACS caliber-BD). The cytotoxicity was presented as percentage of dead cells from a histogram of PI-stained cells. Results were expressed as the percent growth inhibition and cell viability in percent.

Chapter: 2 Material & Methods

2.7.1.12. Protein antiglycation

Protein antiglycation assay was performed for MBHE and subsequent fraction by using the previous protocol233. Briefly, samples of bovine serum albumin (50 µL,

10 mg/mL), Magnesium oxide (50 µL, 14 mM) and phosphate buffer 0.1 M (pH 7.4) containing NaN3 (30 mM) in triplicate were incubated in presence or absence of testing sample (20 µL) for 9 days at 37C. Upon completion of incubation, glycation of protein was observed by monitoring the particular fluorescence (excitation, 330 nm and emission, 440 nm), comparative to blank, using microtitre plate spectrophotometer (Spectra-Max, Molecular Devices, CA, USA).

Rutin was used as standard (IC50 = 294 ± 1.50 µM).

2.7.1.13. Immune modulatory assay

To identify the compounds as MPLEs in ELISA (Enzyme linked immunosorbent assay) or DELFIA (Dissociation-Enhanced Lanthanide Fluorescent Immunoassay) based peptide loading experiment was done using standard protocol232. Briefly, first the plate (384 black well) was coated with desired anti-HLA-DR L243 antibody

(30uL/well) followed by incubation for 2 hours at 37oC. After the completion of incubation the plate was washed twice with wash buffer (50 uL/well) followed by taping on cloth. Furthermore, to prevent non specific interaction the plate was blocked via blocking solution (30-50 uL/well) followed by incubation at 37oC for 2 hours.

After incubation with blocking solution, the plate was washed four times followed by taping on cloth well. Meanwhile, proteins mixture was prepared during incubation

(blocking step) by mixing Biotineylated HA306-318 (80 ug/mL) with HLA-DR (100 nM). Similarly, in a separate 384 well white plate the desired adamantine compound at various concentrations was added followed by the addition of proteins mixture of

Chapter: 2 Material & Methods the same volume and was incubated at 37oC for 1hour. After the completion of incubation dibromosalicylaldehyde (DBSA, 1%, 40 uL/well) was added to each well having compound protein mixture followed by vigorous shaking. Meanwhile, transfer

20 µl from this diluted mixture to the 384 black well plates having coated anti-HLA-DR L243 antibody followed by incubation at 4oC for 1.5 hours. After the completion of incubation the plate was washed six times with washing buffer followed by the addition of Eu+3-labbled streptividine solutions (30 µL/well) and further incubated for 30 min. Similarly, the plate was washed eight times with washing buffer after incubation. Meanwhile, after washing enhancer’s solution was added (30 uL/well), bubbles were removed and fluorescence was recorded at 340nm excitation and 614 nm emission.

Table-2.14: Material used in immune modulatory assay232.

S.NO Name Ingredients

1 Carbonate Buffer 15 mM Na2CO3, 35 mM NaHCO3, 0.2 g/L NaN3 (pH 9.6). Use 1 μM synthetic peptide in carbonate buffer 2 Phosphate Buffered To prepare 1 L add 80 g sodium chloride Saline (10 × PBS) (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 L dH2O. Adjust pH to 7.4. 3 Wash Buffer 1X PBS containing 0.05% Tween-20 4 Blocking Buffer 10 mg/ml bovine serum albumin in wash buffer 5 Antibody Dilution Buffer 3% bovine serum albumin in wash buffer 6 Enhancement Solution DELFIA® (PerkinElmer Life Sciences # 1244-105) 7 Europium-labeled An- DELFIA® (PerkinElmer Life Sciences ti-mouse IgG for mouse #AD0124) primary antibodies or An- ti-rabbit IgG for rabbit primary antibodies.

Chapter: 2 Material & Methods

2.7.2. In-vivo Biological activities

MBHE was investigated for a variety of in-vivo biological activities.

2.7.2.1. Acute toxicity

In-vivo acute toxicity was evaluated for MBHE, using standard protocols234. In various concentrations (upto 2000 mg/kg), doses of MBHE were prepared in distilled water. Mice were distributed into various groups (n = 6). Solutions of MBHE were administered orally with predefined doses to their respective groups. Normal saline

10mL/kg was given orally to group 1 served as the negative-control group. After the stated treatment, animals were kept for observation for 24 hours. After 24 hours,

235 number of dead animals were counted in each group and LD50 was calculated .

2.7.2.2. Antipyretic activity against Brewer’s yeast induced pyrexia

The antipyretic potential of MBHE was screened against Brewer’s yeast induced pyrexia in BALB/c mice of either sex weighing 30-35 gm236. The animals were deprived of food for 5 hours before the start of experiment. Their normal rectal temperature was recorded using a digital thermometer, after which a 15% solution of

Brewer’s yeast was injected subcutaneously at a dose of 10 ml/kg to each animal.

The rise in body temperature was observed after 24 hours and animals showing at least 0.5oC rise of their body temperature were selected for the assay. The pyrexic animals were orally administered with MBHE (50, 100 and 150 mg/kg) and acetaminophen (150 mg/kg). The rectal temperature of animals was then recorded after 1 to 5 hours duration after treatment.

Chapter: 2 Material & Methods

2.7.2.3. Antinociceptive activity

The antinociceptive activity of MBHE was evaluated by acetic acid induced abdominal constriction assay in BALB/c mice of either sex weighing 21-24 gm237.

The animals were withdrawn from food 2 hours before experiment. MBHE was administered orally at doses of 50, 100, and 150 mg/kg body weight. Diclofenac sodium was administered at a dose of 50 mg/kg P.O. and served as positive control.

After 30 min of treatment, all animals were injected I.P. with 1% acetic acid. The number of writhes was counted after 5 min of acetic acid injection and was noted for

20 min.

2.7.2.4. Anti-inflammatory activity

The anti-inflammatory activity of MBHE was tested in carrageenan induced paw edema in BALB/c mice of either sex weighing 25-30 gm238. The animals were starved for 4 hours before the start of experiment. MBHE was administered orally at doses of

50, 100 and 150 mg/kg body weight. Aspirin was used as standard and was administered at a dose of 150 mg/kg P.O. After 30 min, all animals were challenged with a 50 μl of 1% solution of carrageenan, injected subcutaneously into the plantar surface of the left hind paw. The paw volume was measured using a digital ple- thysmometer at 1 to 5 hours after injecting carrageenan.

Chapter: 2 Material & Methods

2.7.2.5. Prokinetic and laxative activity

2.7.2.5.1. Charcoal meal transit test

MBHE was evaluated for its impact on GIT motility using standard protocol239. Brief- ly, BALB/C mice of either sex (25-30 gm) were selected. Animals were divided in several groups (n=6). Normal saline (10 mL/kg) was given orally to group-1 served as negative control, group-2 was given carbacholine (CCh.) I.P. (1 mg/kg) served as positive control. MBHE were given orally to group-3 and 4 at a dose of 100 and 300 mg/kg respectively. To understand the mechanism of action of MBHE as laxative; atropine (10mg/kg) was administered (I.P.) one hour before the administration of

MBHE (both doses) and CCh. to remaining groups i.e. 5-7.

After 15 min of the aforementioned treatments, aqueous suspension of charcoal

(0.3 ml) was given orally to each animal. After 30 min of charcoal administration animals were killed and dissected to expose the small intestine. Small intestine was removed and distance travelled by charcoal was measured.

Percent motility of G.I.T was calculated with the help of following formula239.

Distance covered by charcoal Percent motality = 100 − × 100 Tolal length of intestine

2.7.2.5.2. Laxative activity

MBHE was evaluated for laxative activity using standard protocol240. Briefly,

BALB/C mice of either sex were fasted for 12 hours before execution of the experiment. Animals were divided in several groups (6 animals in each group); group-1 was treated with the normal saline (10 mL/kg, P.O.), group-2 was given atropine 10 mg/kg I.P. while group 3 was administered carbacholine (CCh.) 1 mg/kg

Chapter: 2 Material & Methods

I.P. MBHE 100 and 300 mg/Kg was given orally to groups 4 and 5. After the stated treatment each animal was kept in a separate cage. They were deprived of food during experiment but having free access to water ad-libitum. To understand the mechanism of action of MBHE as laxative; atropine (10mg/kg) was administered (I.P.) one hour before the administration of MBHE and CCh. to remaining groups i.e. 6-8.

After 18 hours the total faeces (wet and dry) were counted for individual mouse and the percent rise was determined for wet faeces in contrast with the total faeces241.

2.7.2.6. Hepatoprotective activity

2.7.2.6.1. Isoniazid and Rifampicin induced toxicity

Suspension of isoniazid (INH) and rifampicin (RIF) were prepared separately. Rats were given suspension (50 mg/kg) for 21 days to induce hepatotoxicity242.

2.7.2.6.2. Experimental design

Animals were divided in 5 different groups (n = 6) i.e. group-1 fed with standard diet, to group-2 INH and RIF were given orally, group 3 INH, RIF and MBHE (150mg/kg) were given, group-4 fed with INH, RIF and MBHE (300mg/kg) and group-5 fed with

INH, RIF and silymarin (100 mg/kg).

2.7.2.6.3. Blood Collection and Serum Preservation

At the end of the experiment, animals were anaesthetized with ketamine (I.P.). Blood was collected and transferred immediately to centrifuge tubes (evacuated gel and clot activator, AST Diagnostics). Serum was separated by centrifugation at 3000 revolution per min (RPM) for 15 min (Centurion Scientific LTD, UK).

Chapter: 2 Material & Methods

2.7.2.6.4. Biochemical assays

The biochemical assays like Serum Glutamic Oxaloacetic Transaminase (SGOT),

Serum Glutamate Pyruvate Transaminase (SGPT), Serum Alkaline Phosphatase

(SALP), Total Protein (TP) and Serum Bilirubin (SB) were performed according to standard methods using an assay kit (Cheema diagnostica, Italy) through double beam

UV/Visible spectrometer (Lambda 25, Perkin Elmer, USA).

2.7.2.6.5. Histology

After 21 days of treatment, liver of each animal was removed and fixed immediately in 10% neutrally buffered formalin for 48 hours. The tissues were dehydrated in grad- ed ethanol solutions (50, 70, 80, 90, two changes each of 100%), cleared in two changes each of 100% xylene and were infiltrated and embedded in paraffin wax.

Tissue blocks were sectioned at 4 μm through a rotary microtome (SLEE Mainz CUT

5062, Germany), stained with Harris hematoxylin and eosin (H & E) for microscopic observation (Labomed Lx400 with digital camera iVu 3100, USA). Histopathological changes were scored as none (–), mild (+), moderate (++), or severe (+++) damage.

Chapter: 3

RESULTS & DISCUSSION

Chapter: 3 Results & Discussion

3. RESULTS AND DISCUSSION

3.1. Percent yield of the extract and subsequent solvents soluble fractions

Percent yield of the crude extract and subsequent solvents soluble, shown in

Table-3.1. The percent yield of hydroethanolic extract of Monotheca buxifolia fruit

(MBHE) was 4.838%, while in fractions the percent yield of aqueous fraction was maximum (4.435%) followed by chloroform fraction (0.115%), n-hexane (0.112%) and n-butanol fraction (0.108%). Yield of ethyl acetate fraction was the least one

(0.068%).

Percent yield provide information about the nature of the compounds in their respective solvents soluble fraction243. The essential oils and other non-polar compounds appear in the n-hexane fraction. While the polar majorly compounds appears in aqueous and n-butanol fraction. Compounds of intermediate polarity appear in the solvents like chloroform or ethyl acetate244.

Table-3.1: Percent yield of the extract and subsequent solvents soluble fractions.

S. No Name Quantity (g) Yield (%) 1 Crude extract 2304 4.838 2 n-Hexane fraction 56 0.112 3 Chloroform fraction 57.7 0.115 4 Ethyl acetate fraction 34.94 0.068 5 n-Butanol fraction 54.5 0.108 6 Aqueous fraction 1985.9 4.435

Chapter: 3 Results & Discussion

3.2. Structure elucidation of isolated compounds

3.2.1. Buxifoline-A (1)

Compound 1 was isolated as a crystalline material through repeated chromatographic techniques.

The EI-MS of compound 1 displayed the molecular ion peak M+ at m/z 314, while the

HREI-MS showed the M+ at m/z 314.1228, corresponding to molecular formula

C17H18N2O4 (calcd. 314.1267). The IR spectrum displayed sharp peaks at 3329 (NH),

1703 (C=O) and 1529 (aromatic) cm-1. Its UV spectrum showed maximum absorptions at 248, 232 and 210 nm.

The 1H-NMR spectrum showed two signals of four proton each at aromatic region, at

δ 7.10 (4H, d, J = 8.5 Hz, H-2, 2’, 6, 6’) and 7.30 (4 H, dd, J= 8.5 Hz, H-3, 3’, 5, 5’)

1 H-NMR also displayed two proton containing signal at δ 3.85 s, attributed to H2-9 and signal for oxygenated methyl protons at δ 3.70 (6H, s) assigned for H3-8 and

H3-8’.

The broad-band decoupled 13C-NMR spectrum showed a total of 10 carbon signals.

DEPT-90 and 135 spectra of compound 1 showed that there were one methyl, two methylene and four methine carbons and the remaining was found to be quaternary in nature. 2D-NMR spectra (HSQC, HMBC and COSY) were used to confirm the structure of compound (Figure-3.2).

Chapter: 3 Results & Discussion

Fig-3.1: Structure of buxifoline-A (1)

Fig-3.2: Key COSY and HMBC structure of buxifoline-A (1)

Chapter: 3 Results & Discussion

Table-3.2: 13C- and 1H-NMR Chemical shift values of compound 1

C. No. C H (J, Hz) 1 138.1 - 2 130.1 7.10 d (8.5) 3 120.4 7.30 d (8.5) 4 137.6 - 5 120.4 7.30 d (8.5) 6 130.1 7.10 d (8.5) 7 156.6 - 8 52.4 3.70 s 9 41.4 3.85 s 1’ 138.1 - 2’ 130.1 7.10 d (8.5) 3’ 120.4 7.30 d (8.5) 4’ 137.6 - 5’ 119.1 7.30 d (8.5) 6’ 130.1 7.10 d (8.5) 7’ 156.6 - 8’ 52.4 3.70 s

Chapter: 3 Results & Discussion

3.2.2. Buxifoline-B (2)

Compound 2 was isolated as white amorphous powder through repeated chromatographic techniques.

The EI-MS of compound 2 displayed the molecular ion peak M+ at m/z 328, while the

HREI-MS showed the M+ at m/z 328.1383, corresponding molecular formula

C18H20N2O4 (calcd. 328.1423). The IR spectrum displayed sharp peaks at 3313 (NH),

1709 (C=O) and 1534 (aromatic) cm-1. Its UV spectrum showed maximum absorptions at 248, 243 and 209 nm.

The 1H-NMR spectrum showed two signals of four proton each at aromatic region, at

δ 7.08 (4H, d, J = 8.5 Hz, H-2, 2’, 6, 6’) and 7.29 (4 H, dd, J= 8.5 Hz, H-3, 3’, 5, 5’).

1 H-NMR also displayed two proton containing signal at δ 3.85 s, attributed to H2-10 and signal for oxygenated methyl proton at δ 3.70 (3H, s) assigned for H3-8’, and signal for oxygenated methylene protons at δ 4.16 (2H, q, J = 7.0 Hz) and one signal for methyl protons at δ 1.28 (3H, t, J = 7.0 Hz)

The broad-band decoupled 13C-NMR spectrum showed a total of 18 carbon signals.

DEPT-90 and 135 spectra distinguish as two methyl, two methylene and eight methine carbons. The remaining was found to be quaternary in nature. The 2D-NMR spectra (HSQC, HMBC and COSY) were used to confirm the structure of compound

(Figure-3.4).

Chapter: 3 Results & Discussion

Fig-3.3: Structure of buxifoline-B (2)

Fig-3.4: Key COSY and HMBC structure of buxifoline-B (2)

Chapter: 3 Results & Discussion

Table-3.3: 13C- and 1H-NMR Chemical shift values of compound 2

C. No. C H (J, Hz) 1 138.2 - 2 130.1 7.08 d (8.5) 3 120.0 7.29d (8.5) 4 137.6 - 5 120.0 7.29 d (8.5) 6 130.1 7.08 d (8.5) 7 156.6 - 8 61.8 4.16 q (7.0) 9 14.9 1.28 t (7.0) 10 41.4 3.85 s 1’ 138.1 - 2’ 130.1 7.08 d (8.5) 3’ 120.0 7.29 d (8.5) 4’ 137.5 - 5’ 120.0 7.29 d (8.5) 6’ 130.1 7.08 d (8.5) 7’ 156.2 - 8’ 52.0 3.65 s

Chapter: 3 Results & Discussion

Fig-3.5: Recycling HPLC Chromatogram showing purification of compound 1 and 2.

Chapter: 3 Results & Discussion

3.2.3. Buxitriol (3)

Compound 3 was isolated from the ethyl acetate fraction of MBHE. Its molecular formula, C30H50O3, was determined from EI-MS spectrum, which showed molecular ion peak at m/z 458 and 13C-NMR values (BB, and DEPT). EI MS spectrum displayed fragments at m/z 224 and 234, associated with a retro-Diels–Alder fragmentation of the C ring of this pentacyclic triterpene, indicating that 3 is a member of an ole- an-12-ene system.

Absorption band at 3500 in the IR spectrum showed the presence of hydroxyl groups in compound 3.

1H- and 13C-NMR spectra of the compound is closely similar with compound 8 with only the difference of presence of hydroxyl group at C-29 position instead of carboxylic group in 3.

The 1H -NMR spectrum showed signals at δ 0.60, 0.81, 0.85, 0.89, 0.99, 1.0, 1.10

(each 3H, s), corresponding to seven methyl groups, two oxygenated methine hydrogens at δ 3.59 m and 2.90 d (J = 9.6 Hz) were assigned to H-2 and H-3 respectively, one olefinic methine proton resonated δ 5.28 t (J = 3.6 Hz) was attributed to H-12.

The 13C NMR spectrum of 3 indicated the presence of 30 signals, corresponding to seven quaternary, six methine, ten methylene and seven methyl carbon atoms on the basis of the DEPT experiment. Four signals at δ 139.8, 126.1, 84.4 and 69.4 were respectively assigned to the C-13, C-12, C-3 and C-2.

Structure of the compound was further confirmed by 2D-NMR spectra such as COSY,

HSQC, HMBC and NOESY (Figure-3.7).

Chapter: 3 Results & Discussion

Fig-3.6: Structure of buxitriol (3)

Fig-3.7: Key COSY and HMBC structure of buxitriol (3)

Chapter: 3 Results & Discussion

Table-3.4: 13C- and 1H-NMR Chemical shift values of compound 3

C. No. C H (J, Hz) 1 48.2 1.60, 1.92 m 2 69.4 3.59 m 3 84.4 2.90 d (9.6) 4 38.1 - 5 56.6 0.83 6 19.5 1.40, 1.52 7 34.2 1.34, 1.54 8 40.5 - 9 54.4 2.20 10 38.1 - 11 23.8 1.62, 1.94 12 126.1 5.28 13 139.8 - 14 41.6 - 15 26.9 1.61, 1.91 16 33.6 1.0, 1.29 17 33.0 1.32, 1.50 18 40.4 1.35 19 41.5 1.61, 1.69 20 36.0 - 21 29.1 1.09, 1.91 22 38.2 0.95, 1.35 23 29.3 1.0 24 14.4 0.60 25 17.8 0.81 26 17.6 0.85 27 24.0 1.10 28 17.2 0.89 29 63.0 3.51, 3.53 30 17.5 0.99

Chapter: 3 Results & Discussion

3.2.4. Buxilide (4)

Compound 4 was isolated from the ethyl acetate fraction of MBHE. Its molecular formula, C6H6O4, was determined from HREI MS spectrum, which showed molecular

13 ion peak at m/z 142.0271 (calc. for C6H6O4 = 142.0266) and C-NMR values (BB, and DEPT). EI MS spectrum displayed molecular ion peak at m/z 142.0 and fragments at m/z 113 and 69. Absorption band at 3388 and 1737 in the IR spectrum showed the presence of hydroxyl and lactone groups in compound 4. UV spectrum displayed absorptions at 272 and 223 nm.

1H- NMR spectrum showed two downfield signals at δ 6.49 s and 7.94 s which were attributed to H-3 and H-6 respectively and one two proton signal at δ 4.39 s for H2-7.

13C-NMR spectrum (BB and DEPT) displayed resonances for six carbons which include one methylene, two methine and three quaternary carbons. Structure of compound was further confirmed by using 2D-NMR spectra such as COSY, HSQC,

HMBC and NOESY (Figure-3.9).

Chapter: 3 Results & Discussion

Fig-3.8: Structure of buxilide (4)

Fig-3.9: Key HMBC structure of buxilide (4)

Chapter: 3 Results & Discussion

Table-3.5: 13C- and 1H-NMR Chemical shift values of compound 4

C. No. C H (J, Hz) 2 170.4 - 3 110.7 6.49 s 4 176.9 - 5 147.4 - 6 141.0 7.94 s 7 61.2 4.39 s

Chapter: 3 Results & Discussion

3.2.5. Buxiglucoside (5)

Compound 5 was isolated from the ethyl acetate fraction of MBHE by repeated column chromatography. Its molecular formula, C12H16O8, was determined from

HRFAB (+ve) MS spectrum, which showed molecular ion peak at m/z 289.0937 (calc.

13 for C12H16O8 + H = 289.0923) and C-NMR values (BB, and DEPT). EI MS spectrum displayed peaks at m/z 142.0, 113 and 69. Absorption band at 3388 and

1737 in the IR spectrum showed the presence of hydroxyl and lactone groups in compound 5. UV spectrum displayed absorptions at 270 and 221 nm.

1H- NMR spectrum showed two downfield signals at δ 6.49 s and 7.94 s which were attributed to H-3 and H-6 respectively and one two proton signal at δ 4.39 s for H2-7.

Presence of rhamnose sugar in molecule indicated by presence of signals at δH/ δC

[4.26 br s/101.5, CH-1’; 4.60 br s/72.5, H-2’; 4.24 overlap/72.8, H-3’; 4.23 overlap/74.5, H-4’; 4.28 overlap/69.4, H-5’ and 1.41 d (J = 6.0 Hz)/18.2, H-6’.

Position of the rhamnose was confirmed by using HMBC correlation which displayed cross peaks between H-1’ and C-4, structure of compound was further confirmed by using 2D-NMR spectra such as COSY, HSQC, HMBC and NOESY. Key HMBC correlation in compound 5 is shown in Figure-3.11.

Chapter: 3 Results & Discussion

Fig-3.10: Structure of buxiglucoside (5)

Fig-3.11: Key COSY and HMBC Structure of buxiglucoside (5)

Chapter: 3 Results & Discussion

Table-3.6: 13C- and 1H-NMR Chemical shift values of compound 5

C. No. C H (J, Hz) 2 170.4 - 3 110.7 6.49 s 4 176.9 - 5 147.4 - 6 141.0 7.94 s 7 61.2 4.39 s 1’ 101.5 4.26 br s 2’ 72.5 4.60 br s 3’ 72.8 4.24 overlapped 4’ 74.5 4.23 overlapped 5’ 69.4 4.28 overlapped 6’ 18.2 1.41 d (6.0)

Chapter: 3 Results & Discussion

3.2.6. Oleanolic acid (6)

Compound 6 was isolated from the ethyl acetate fraction through normal phase column chromatography. The EI-MS showed molecular ion peak at m/z at 456, while the HREI-MS was in agreement with molecular formula C30H48O3 at m/z 456.7003

(Calc. 456.6840). The molecular formula indicated seven degrees of un-saturation.

Six degrees of un-saturation indicated the presence of six double bonds, while remaining one corresponded to the presence of a double bond in compound 6.

The 1H-NMR (MeOD, 300 MHz) spectrum of compound 6 showed an olefinic H-12 one proton integration as a broad triplet at δ 5.22. The OH-substituted methine (CH-3) appeared as a double doublet at δ 3.17 (1H, dd, J3, 2 = 5.1 Hz, J3, 2β = 10.8 Hz). The

H-18 showed a signal at δ 2.21 (1H, dd, J18, 19 = 11.1 Hz, J18, 19β = 16.1 Hz). Seven methyl groups appeared in the 1H-NMR spectrum of compound 6. The germinal

CH3-23 and CH3-24 appeared at δ 0.98 and 0.95 (3H, s) respectively. Similarly

CH3-25, CH3-26 and CH3-27 appeared as singlets at δ 0.98, 0.94 and 1.15, respectively. The CH3 -29 and CH3-30 also appeared as a singlet at δ 0.80 and 0.77, respectively.

Chapter: 3 Results & Discussion

Fig-3.12: Structure elucidation of oleanolic acid (6)

Chapter: 3 Results & Discussion

Table-3.7: 13C- and 1H-NMR Chemical shift values of compound 6

C. No. δC δH (J, Hz) 1 38.1 1.57, 1.32 2 27.9 1.73, 1.46

3 79.7 3.16 (1H, dd, J3,2 = 4.8 Hz / J3,2β = 2.0 Hz) 4 39.8 –– 5 56.8 1.39 (1H, m) 6 19.5 1.54, 1.27 (2H, m) 7 33.9 1.57, 1.36 (2H, m) 8 42.7 –– 9 47.3 1.43 (1H, m) 10 38.1 –– 11 23.9 2.04, 1.79 (2H, m)

12 123.6 5.24 (1H, t, J12,11 = 6.5 Hz) 13 145.2 –– 14 42.8 –– 15 28.9 1.34, 1.03 (2H, m) 16 24.5 1.71, 1.18 (2H, m) 17 47.7 ––

18 42.9 2.16 (1H, dd, J3,2 = 4.8 Hz, J3,2β = 2.0 Hz). 19 46.3 1.51, 1.45 (2H, m) 20 30.7 –– 21 34.9 1.61, 1.35 (2H, m) 22 33.0 1.72, 1.48 (2H, m) 23 23.9 0.94 (3H, s) 24 23.9 0.93 (3H, s) 25 15.9 0.97 (3H, s) 26 16.3 0.90 (3H, s) 27 26.4 1.15 (3H, s) 28 24.5 –– 29 24.1 0.77 (3H, s) 30 181.8 0.80 (3H, s)

Chapter: 3 Results & Discussion

3.2.7. Glucosidic β-sitosterol (7)

The EI-MS shows molecular ion peak M+ at m/z = 576.1, The HREI-MS was in agreement with molecular formula C35H60O6 at m/z 576.4390.The IR (KBr) shows absorption signals for characteristic hydroxyl groups at 3551 cm-1. The IR also shows an absorption peak for Olefinic double bond at 1453 cm-1. The C-O is appeared at

1049 cm-1 while the C-H bond appeared due to stretching vibration at 2948 cm-1. The

UV (DMSO-d6) shows absorption at 244nm which indicates the presence of a double bond in compound.

The H-NMR (DMSO-d6) shows a br,s at δ 5.11 due to H-6 of olefinic proton. The anomeric proton of glucose moiety appeared as a broad doublet at δ 4.87 (1H, d, J 1’,

2’ = 4.8Hz). The anomeric proton of glucose moiety also appeared as a broad singlet at δ 5.01 (1H, S). The H-3 methine OH substituted proton appeared as one proton integration multiplet at δ 3.62 (1H, m). The compound 7 contained 6 methyl groups.

The CH3-19 appeared as a singlet at δ 1.23. The other methyl CH3-18 appeared as a singlet at δ 1.73 (3H, S), CH3-21 (3H, d, J21, 20 = 13.4 Hz). The CH3-29 appeared as a triplet at δ 0.811(3H, t, J 29, 25 = 11.1 Hz). While the CH3-27 appeared at δ 1.00

(3H, dd, J27, 26 = 6.4 Hz). The CH3-28 also appeared as a doublet at δ 0.91 (3H, d,

J28,26 =6.3 Hz).

The (broad band decoupled) 13C-NMR shows a total of 7 carbon atoms. The quaternary olefinic carbon C-5 of compound 7 appeared at δ 138.9. The C-6 olefinic methane carbon atom appeared at 117.152 while the olefinic methane C-22 and C-23 appeared at δ 137.9 and 128.9. The anomeric carbon of glucose appeared in the BB decoupled 13C-NMR at δ 100.8.

Chapter: 3 Results & Discussion

The oleafinic double bond shows HMBC correlation with quaternary carbon C-5 and

C-4. The CH3-19 shows HMBC correlation with quaternary carbon C-4, glucose substituted C-3 and quaternary carbon C-10. The anomeric carbon shows connectivity with OH-substituted C-3, so form HMBC it was confirmed that the glucose was attached to C-3 position through oxygen linkage (Figure-3.14).

Chapter: 3 Results & Discussion

Fig-3.13: Structure of glucosidic β-sitosterol (7)

Fig-3.14: Key COSY and HMBC structure of glucosidic β-sitosterol (7)

Chapter: 3 Results & Discussion

Table-3.8: 13C- and 1H-NMR Chemical shift values of compound 7

C. No. δH (J, Hz) Multiplicity

1 1.50, 1.32 (2H, m) CH2

2 1.42, 1.48 (2H, m) CH2 3 3.62 (1H, m) CH

4 2.20,2.28 (2H, m) CH2 5 –– 138.9 6 5.11 (1H, br s) 117.2

7 2.26 1.65 (2H, m) CH2 8 1.40 (1H, m) CH 9 1.41 (1H, m) CH 10 –– C

11 1.28, 1.51 (2H, m) CH2

12 1.45, 1.41 (2H, m) CH2 13 –– C 14 1.40 (1H, m) CH

15 1.83,1.80 (2H, m) CH2

16 2.20, 1.73 (2H, m) CH2 17 1.51 (1H, m) CH 18 1.73 (CH3, s) CH3 19 1.23 (CH , s) CH 3 3 20 2.24 (1H, m) CH

21 0.67 (CH3-21, d, J21,20 = 5.4 Hz CH3 22 4.9 (1H, s) 137.9 23 5.10 (1H, m) 128.9 24 2.28 (I H, m) CH

25 1.13 (2H, m) CH2 26 1.80 (1H, m) CH 27 1.00 (CH3, d, J27,26 = 3.3 Hz) CH3 28 0.91 (CH , d, J = 3.3 Hz) CH 3 28,26 3 29 0.81 (CH3, m, overlapped) CH3

1’’ 4.87 (1H, d, J 1’’, 2’’ = 4.8Hz). 100.8 2’’ –– 76.7 3’’ –– 73.7 4’’ –– 76.3 5’’ –– 70.0 6’’ –– 76.7

Chapter: 3 Results & Discussion

3.2.8. 2-Hydroxy-epikatonic acid (8)

2-Hydroxy-epikatonic acid (8) was isolated from the ethyl acetate fraction of MBHE.

(-ve) Its molecular formula, C30H48O4, was determined from HRFAB-MS spectrum, which showed pseudo molecular ion peak at m/z 471.3549.

(Calc. 471.3553) and 13C-NMR values (BB, and DEPT). EI MS spectrum displayed fragments at m/z 224 and 248, associated with a retro-Diels-Alder fragmentation of the C ring of this pentacyclic triterpene, indicating that 8 is a member of an ole- an-12-ene system8.

Absorption bands at 3500 and 1736 cm-1 in the IR spectrum showed the presence of hydroxyl and carboxyl groups in compound 8.

1H- and 13C-NMR spectra of the compound is closely similar with reported compound

Epikatonic acid245, with only the difference of substitution of hydroxyl group on C-2 position.

The 1H -NMR spectrum showed signals at δ 0.60, 0.81, 0.85, 0.89, 0.99, 1.0, 1.10

The 13C NMR spectrum of 8 indicated the presence of 30 signals, corresponding to eight quaternary, six methine, nine methylene and seven methyl carbon atoms on the basis of the DEPT experiment. Five signals at δ 181.8, 139.7, 126.1, 84.6 and 69.5 were respectively assigned to the C-29, C-13, C-12, C-3 and C-2.

Structure of the compound was further confirmed by 2D-NMR spectra such as COSY,

HSQC, HMBC and NOESY (Figure-3.16).

Chapter: 3 Results & Discussion

Fig-3.15: Structure of 2-hydroxy-epikatonic acid (8)

Fig-3.16: Key COSY and HMBC structure of 2-hydroxy-epikatonic acid (8)

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Chapter: 3 Results & Discussion

Table-3.9: 13C- and 1H-NMR Chemical shift values of compound 8

C. No. C H (J, Hz) 1 48.3 1.60, 1.92 m 2 69.5 3.59 m 3 84.6 2.90 d (9.6) 4 39.2 - 5 56.6 0.83 6 19.5 1.40, 1.52 7 34.2 1.34, 1.54 8 40.8 - 9 54.3 2.20 10 40.5 - 11 24.4 1.62, 1.94 12 126.6 5.28 t (3.6) 13 139.7 - 14 43.3 - 15 25.3 1.61, 1.91 16 31.8 1.0, 1.29 17 33.0 1.32, 1.50 18 40.4 1.35 19 38.1 1.61, 1.69 20 43.3 - 21 29.1 1.09, 1.91 22 40.0 0.95, 1.35 23 29.3 1.0 24 17.2 0.60 25 17.5 0.81 26 17.6 0.85 27 24.1 1.10 28 17.6 0.89 29 181.8 - 30 17.8 0.99

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Chapter: 3 Results & Discussion

3.2.9. Isoquercetin (9)

Compound 9 was isolated from the ethyl acetate fraction using repeated column chromatography. The FAB (-ve) of compound 9 showed pseudo-molecular ion peak

[M-H] at m/z at 463. The HRFAB-MS (-ve) indicated the molecular formula at

C21H20O12 at m/z 463.0012. The IR (KBr) shows absorption characteristic for flavonoids appeared at 3415 (OH), 1650, 1433 Cm-1 (due to double bonds of aromatic rings). The C-H stretching appeared at 2918 while C-O appeared at 1056 Cm-1. The

UV (MeOH) shows absorption signal at 213, 260 and 360 nm indicated the conjugated system in compound.

The 1H-NMR (MeOD, 300 MHz) showed a meta coupled doublet appeared at δ 7.84

(1H, d, J 2’, 6’ = 2.1 Hz, H-2’). Similarly the H-6’ showed a meta coupling with H-2’ appeared at δ 7.59 (1H, d, J 6’2’ = 2.1 Hz). The H-3’ appeared at δ 6.87 (1H, d, J 3’2’ =

8.4 Hz) which showed an artho coupling with H-2’ of aromatic ring B. The H-8 of ring A appeared at δ 6.87 (1H, d, J8, 6=1.8 Hz). Similarly the H-6 of ring A appeared at δ 6.20 (1H, d, J 8, 6 = 2.1 Hz). From the J value it shows that both are meta coupled to each other. The glucose moiety with ring C of compound 9 shows the anomeric proton appeared at δ 5.17 (1H, d, J 1’’, 2’’=7.5 Hz).Hz The H-2’’ of glucose appeared at δ 3.84 (1H, d, J2’’, 1’’=2.4/J2’’, 3’’ = 1.8 Hz).The H-3’’ appeared at δ 3.78 (1H, brs). The H-4’’ appeared at δ 3.67 (1H, dd, J4’’) The H-5‘’ exhibited at 3.5 (1H,m)

The H-6’’ methylene proton appeared at δ 3.54 (2H,m)

The 13 C-NMR (BB and DEPT) showed a total of 21 carbon atoms. Among which 10 are quaternary and 10 are methine (CH) carbon atom which the dept 135 shows the presence of one methylene. The OH substituted carbon atom of aromatic ring A

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Chapter: 3 Results & Discussion appeared at δ 166.2 and 163.0 for C-5 and C-7. The OH bearing carbon atom of ring

B appeared at δ 149.0 and 145.8 for C-4 and C-5.

The quaternary carbon atom C-9 and C-10 appeared at δ 158.8 and 105.6. The ring C quaternary carbon atoms appeared at δ 158.4 and 135.7 for C-2 and C-3. The C-6 and

C-8 methine (CH) carbon atoms of compound exhibited at δ 99.9 and 94.7. The methines of aromatic ring B at C-2’ and C-6’ appeared at δ 122.9 and 116.1. The compound 9 also contains a glucose moiety the anomeric carbon atom C-1 appeared at δ 105.4. The OH- substituted carbon atoms C-2’’, C-3’’, C-4’’ appeared at δ 75.1,

73.2 and 70.0 respectively. The methine C-5’’ of compound 9 appeared at δ 77.1. The methylene C-6’’ of glucose moiety of compound 9 appeared at δ 61.9. The α/β unsaturated carbon atoms of ring C appeared at δ 179.5

The H-2’ shows HMBC connectivity with C-1’, C-4’ and C-5’ of aromatic ring B. the

H-2’ also connected to C-2 of pyran ring C though HMBC correlation. The H-6’ shows correlation with C-2’ and C-5’ of aromatic ring B, while it also shows connection to C-2 of ring C. The H-3’ shows connectivity to C-1’, C-4’, C-5’ and

C-6’ of ring B. the H-8 of ring A gives its connection through HMBC correlation with

C-4 carbonyl, C-9 and C-10 quaternary carbon and C-7 OH substituted carbon and

C-6 methine carbon atom. The position of H-6 confirmed through HMBC correlation by its connectivity to the adjacent carbon atoms (C-8, C-10, C-5 and C-7). The anomeric proton of glucose moiety shows connectivity to C-2’’ and C-3carbon atom.

So from HMBC it was confirmed that the glucose moiety attached to C-3 of ring C which shows HMBC correlation between anomeric protons of glucose to C-3 at 136.0

(Figure-3.18).

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Chapter: 3 Results & Discussion

Fig-3.17: Structure of Isoquercetin (9)

Fig-3.18: Key COSY and HMBC structure of Isoquercetin (9)

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Chapter: 3 Results & Discussion

Table-3.10: 13C- and 1H-NMR Chemical shift values of compound 9.

C.No δ C δH (J, Hz) 1 –– –– 2 158.4 –– 3 138.7 –– 4 178.9 ––

5 166.2 6.20 (1H, d, J 6, 8 = 2.1 Hz) 6 99.9 ––

7 163.0 6.87 (1H, d, J8, 6=1.8 Hz) 8 94.7 –– 9 158.8 –– 10 105.6 ––

1' 122.5 7.84 (1H, d, J 2’, 6’, 2.1)

2' 122.9 6.87 (1H, d, J 3’2’ = 8.4 Hz) 3' 117.8 –– 4' 149.0 ––

5' 145.8 7.59 (1H, d, J 6’2’ = 2.1 Hz)

'6 116.1 5.17 (1H, d, J1’’, 2’’ =7.5 Hz).

1’’ 105.4 3.84 (1H, d, J2’’, 1’’ , J2’’, 3’’ = 2.4, 1.8 Hz 2’’ 75.1 3.78 (1H, brs

3’’ 73.2 3.67 (1H, dd, J4’’,3’Hz/J4’’, 5’’) 4’’ 70.0 3.5 (1H,m) 5’’ 77.1 3.54 (2H,m) 6’’ 61.9 ––

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Chapter: 3 Results & Discussion

3.2.10. Composition of fixed oil

Fig-3.19: GCMS chromatogram of the fixed oils of Monotheca buxifolia fruit.

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Chapter: 3 Results & Discussion

The GC/MS analysis of n-hexane fraction resulted in identification of 13 compounds given in Figure-3.19 and Table-3.11. They were identified by matching their mass fragmentation pattern. Compounds detected were saturated, unsaturated fatty acids, sterols and triterpenoids. Relative concentrations were obtained on the basis of the relative area of the peaks in chromatogram.

Table-3.11: Compounds identified in MBHF through GC/MS

S. No. Compound Name Molecular formu- Mol. Wt. % Abundance la

1 Hexadecanoic acid, C17H34O2 270 1.9 methyl ester

2 Ethyl 9-hexadecanoate C18H34O2 282 3.5

3 Hexadecanoic acid, C18H36O2 284 0.40 ethyl ester

4 Cis-1,2-Cyclodode C12H24O2 200 0.46 canediol

5 (E)-9-Octadecanoic C20H38O2 310 0.31 acid ethyl ester

6 Octadecanoic acid C18H36O2 284 0.1

7 Pthalic acid, di(2- C24H38O4 390 0.17 propylpentyl) ester

8 Chondrillasterol C29H48O 412 3

9 α- Amyrin C30H50O 426 29

10 Lupeol C30H50O 426 17

11 12-oleanen-3yl- ace- C32H52O2 468 20.9 tate

12 Lup-20(29)-en-3-ol C32H52O2 468 3

13 α- Neogammacer-22 C32H52O2 468 0.3 (29)-en-3-ol, acetate

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Chapter: 3 Results & Discussion

3.3. Biological activities

3.3.1. In-vitro activities

3.3.1.1. Antibacterial activity

Antibacterial potential of MBHE and fractions was evaluated against various bacterial strains. Results are presented in Table-3.12 and Figure-3.20. It is clear from the results that all the samples were active (mild to moderate) against tested bacterial strains except B. subtilits. Ethyl acetate fraction was most active followed by n-hexane fraction while aqueous fraction was least active comparatively. Percent inhibition was obtained by comparing the zones of inhibition of samples with the zone of inhibition of standard antibacterial drug (imipenem, 10µg/disc). E. coli was most susceptible to ethyl acetate and n-hexane fraction with inhibitory zone 15 mm each and percent affect 60%, followed by MBHE, n-butanol and aqueous fraction with zones of inhibition 12, 11 and 8 mm respectively and the percent inhibitory effect of these fractions were 48, 44, and 32% respectively. Chloroform fraction was least active against E. coli with a zone of inhibition of 6 mm and percent inhibition 24%.

The maximum inhibitory potential against S. flexeneri was shown by n-butanol fraction followed by n-hexane and ethyl acetate fractions with zones of inhibitions of

14, 13 and 11 mm respectively and their percent inhibitory activity was 50, 46.43 and

22.92%. Mild inhibitory activity of against S. flexeneri was observed in case of

MBHE and aqueous fraction with 7 mm zone of inhibition and 25% inhibitory potential. S. flexeneri was completely resistant to the antibacterial effect of chloroform fraction. The highest antibacterial potential against S. aureus was exhibited by n-hexane and chloroform fraction with zones of inhibition 15 mm and their percent inhibitory effect was 31.25% respectively. S. aureus was mildly inhibited by n-butanol, MBHE and aqueous fraction with zones of inhibition of 11, 9

108

Chapter: 3 Results & Discussion and 8 mm respectively and 22.91, 18.75 and 16.66% inhibitory effect. S. aureus was resistant to the antibacterial effect of ethyl acetate fraction. The maximum antibacterial effect against P. aeruginosa was shown by n-hexane and ethyl acetate fractions with inhibitory zones of 16 mm each and percent inhibition 69.56% respectively. Comparatively chloroform and n-butanol fractions were having better antibacterial effect against P. aeruginosa with zones of inhibition of 13 mm each and

56.52 percent inhibition respectively. In case of aqueous fraction and MBHE a very low inhibitory activity was observed against P. aeruginosa with zones of inhibition 6 and 5 mm and percent inhibitory effect 26.08 and 21.74% respectively. The growth of

S. typhi was appreciably inhibited by n-hexane fraction the zone of inhibition was 13 mm with 46.43% inhibition. MBHE and chloroform showed 10 mm zone of inhibition each with 35.71% effect respectively. Ethyl acetate fraction showed a mild inhibitory effect against S. typhi with 5 mm zone of inhibition and percent effect 17.85%.

S. typhi was completely resistant to the antibacterial effect of n-butanol and aqueous fraction. It is also clear from the results that B. subtilis was completely resistant to the antibacterial effect of MBHE and fractions.

It can be concluded that MBHE and subsequent fractions are the loaded source of anti-bacterial agents. A variety of antibacterial are present in the market, these drugs are quite efficacious in diseases caused by various pathogens except resistant species.

Majority of them are synthetic or semi synthetic derivatives246. Studies on medicinal plants are of quite interest to discover antibacterial drugs that are safe, potent and efficacious against current resistant species. A variety of plants have been screened for their antibacterial activity247. The aforementioned protocol is significant in the identification of antibacterial plants and their extracts for antibacterial potential248. In the current experiment both MBHE and fractions were screened against few bacterial

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Chapter: 3 Results & Discussion strains, all the tested samples were effective while, B. subtilis was resistant to the antibacterial action of the tested samples. M. buxifolia fruit or extract/fractions can be used in the management of diseases caused by the above mentioned bacterial strains except B. subtilis. As the fruit is used forlklorically in the treatment of various infections such as urinary tract infections (UTI) and gastritis therefore, this anti-bacterial bioassay provides a valid scientific rationale to the traditional use of this fruit162.

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Table-3.12: Antibacterial activity of MBHE and subsequent fractions

Standard MBHE n-Hexane Chloroform Ethyl acetate n-Butanol Aqueous

Name

Bacteria inhibition

(%) (%) (%) (%) (%) (%) (mm) (mm) (mm) (mm) (mm) (mm) (mm)

Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition

Zone of inhibitionZone of inhibitionZone of Zone of inhibitionZone of inhibitionZone of inhibitionZone of inhibitionZone

E. coli 25 11 44 15 60 6 24 15 60 12 48 08 32 S. flexeneri 28 7 25 13 46.43 -- -- 11 22.92 14 50 7 25 S. aureus 48 9 18.75 15 31.25 15 31.25 -- -- 11 22.91 8 16.66 P. aeruginosa 23 5 21.74 16 69.56 13 56.52 16 69.56 13 56.52 06 26.08 S. typhi 28 10 35.71 13 46.43 10 35.71 05 17.85 ------B. subtilis 50 ------

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E. coli S. flexeneri 80 60

60 40

20 20

Percent inhibition Percent inhibition

0 0

MBHE MBHE Aqueous Aqueous n-Hexane n-Butanol n-Hexane n-Butanol ChloroformEthyl acetate ChloroformEthyl acetate

S. aureus P. aeruginosa 40 80

30 60

20 40

10 20

Percent inhibition Percent inhibition

0 0

MBHE MBHE Aqueous Aqueous n-Hexane n-Butanol n-Hexane n-Butanol Chloroform Ethyl acetate ChloroformEthyl acetate

S. typhi B. subtilis

Percent inhibition Percent inhibition

0 0

MBHE MBHE Aqueous Aqueous n-Hexane n-Butanol n-Hexane n-Butanol ChloroformEthyl acetate ChloroformEthyl acetate

Fig-3.20: Percent zones of inhibition of bacterial strains by MBHE and fractions.

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3.3.1.2. Antifungal activity

The results of antifungal activity of MBHE and subsequent fractions are shown in the

Table-3.13 and Figure-3.21. It is clear from the results that MBHE showed 20% inhibition against Fusarium solani, while other strains were resistant to its antifungal effect. n-Hexane fraction exhibited 25% inhibition against Microsporum canis, while no fungicidal activity was noticed against other tested strains. Chloroform extract showed 25% inhibition against Fusarium solani; other strains were resistant to its antifungal property. Ethyl acetate fraction showed a maximum inhibitory activity against Fusarium solani followed by Microsporum canis with percent inhibition of 50 and 10% respectively, while no antifungal activity was observed against other strains. n-Butanol fraction showed maximum antifungal effect against Microsporum canis followed by Fusarium solani with inhibition of 30 and 20% respectively; growth of other strains were not affected by n-butanol fraction. Aqueous fraction was only active against Microsporum canis with 30% inhibitory potential. Amphotericin B and miconazole were used as standard drugs. The MBHE and fractions exhibited 20% antifungal potential against Fusarium solani, while no antifungal potential was observed against other strains.

Study on the above mentioned fungal strains is significant in human point of view, as these strains are the root causes of various types of infections in human249. A variety of diseases caused by Microsporum canis such as tinea corporis250, tinea faciei251, tinea capitis250, dermatophytosis252 and various mycetomas in human and certain pets. Fusarium solani causes invasive mycosis such as onychomycosis in both immunocompromised and immunosuppressed patients, cutaneous hyalo hyphomycosis, systemic infections with high mortality rate, while in immunocompetent patients causes mycotic keratitis when the fungi get penetrates

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Fruit of M. buxifolia is used in traditional medicine for the treatment of various ailments, yet it is clear from the results of the above mentioned assay that n-hexane, ethyl acetate, n-butanol and aqueous fractions of M. buxifolia fruit can be used against diseases caused by Microsporum canis, while MBHE, chloroform, ethyl acetate and n-butanol fractions can used in the treatment of diseases caused by Fusarium solani.

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Table-3.13: Antifungal activity of MBHE and fraction

Name of fungi Standard MBHE n-Hexane Chloroform Ethyl acetate n-Butanol Aqueous Candida albicans Miconazole 110.8 µg/ml 0 0 0 0 0 0 Aspergillus flavus Amphotericin B 20.20 µg/ml 0 0 0 0 0 0 Microsporum canis Miconazole 98.4 µg/ml 0 25 0 10 30 30 Fusarium solani Miconazole 73.25 µg/ml 20 0 25 50 20 0 Candida glabrata Miconazole 110.8 µg/ml 0 0 0 0 0 0

Values are given in percent inhibition.

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MBHE n - Hexane 25 30

20 20 15

10 10 5

Percentinhibition Percentinhibition

0 0

A. flavus M. canis F. solani A. flavus M. canis F. solani C. albicans C. glabrata C. albicans C. glabrata

Chloroform Ethyl acetate 30 60

20 40

10 20

Percentinhibition

0 0

A. flavus M. canis F. solani A. flavus M. canis F. solani C. albicans C. glabrata C. albicans C. glabrata

n - Butanol Aqueous 40 40

30 30

20 20

10 10

Percentinhibition Percentinhibition

0 0

A. flavus M. canis F. solani A. flavus M. canis F. solani C. albicans C. glabrata C. albicans C. glabrata

Fig-3.21: Antifungal activity of MBHE and fractions.

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3.3.1.3. Leishmanicidal activity

The MBHE and subsequent solvent fractions were tested against Leishmania major.

The results are presented in Table-3.14. The tested samples didn’t show any significant leishmanicidal activity. Their IC50 were more than 100 and no appreciable inhibition was observed.

Leishmaniasis, a common disease in the sub-tropical and tropical regions of the

255 world, is an infection of protozoa.l parasite of genus Leishmania . Currently leishmaniasis is considered as a serious disease due to lack of availability of specific treatment; still some semi synthetic and synthetic drugs are implicated to treat leish- maniasis256. The currently available drugs are either expensive, ineffective or possess potential adverse effects257. Sometimes drugs i.e. emetic tartar (antimony potassium tartrate), megulamine antimoniate, stibamine and sodium stibogluconate causes severe adverse effects on the patients258. Drugs such as amphotericin B and pentamidine are also used in the management of leishmaniasis, lacking the desired efficacy259-260.

Study on antileishmanial drugs is a highlighted area of research. In the current sit- uation, it is highly needed to discover safe, effective, economical drugs for the inti- mate treatment of this painful and common disease17.

Keeping in view the importance of the above mentioned disease, the MBHE and following fractions were screened for their antileishmanial potential. Tested samples were inactive in the assay.

Table-3.14: Leishmanicidal activity of MBHE and fractions

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Test organism Extracts/ fractions IC50 (µg/ml) MBHE >100 n-Hexane >100 Chloroform >100 Leishmania major Ethyl acetate >100 (DESTO) n-Butanol >100 Aqueous >100 Amphotericin B 0.29 ± 0.05 Pentamidine 5.09 ± 0.09

3.3.1.4. Cytotoxic activity (Brine shrimp)

The cytotoxic bioassay (brine shrimp lethality assay) was performed for MBHE and fractions. Samples were tested in three different concentrations (10, 100, and 1000

µg/mL). Results are shown in Table-3.15 and Figure-3.22. MBHE showed a maximum cytotoxicity (70%) at high concentration (1000 µg/mL) while mild activity (40 and 30%) was observed at lower concentrations (100 and 10 µg/mL) respectively. The n-hexane fraction showed a moderate activity (56.66%) at high concentration (1000

µg/mL). A mild cytotoxic effect (43.33 and 36.66%) was observed at lower concentrations (100 and 10 µg/mL) respectively. Chloroform fraction showed a highest cytotoxic behaviour (73.33%) at high concentration (1000 µg/mL); while at lower concentrations (100 and 10 µg/mL) a moderate response (50 and 46.66%) was noticed.

Ethyl acetate fraction showed an appreciable activity (63.33%) at high dose (1000

µg/mL) while, having a moderate potential (53.33 and 50%) at lower doses (100 and

10 µg/mL) respectively. n-Butanol showed a mild activity of 16.66% at high concentration (1000 µg/mL) while, at lower concentration insignificant cytotoxic response was observed. The aqueous fraction at highest dose (1000 µg/mL) showed a significant cytotoxicity (66.66%) while, mild to moderate response (40 and 30%) was ob-

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Chapter: 3 Results & Discussion served at lower concentrations (100 and 10 µg/mL) respectively. The maximum cytotoxic activity was exhibited by ethyl acetate fraction followed by chloroform fraction, aqueous fraction, MBHE and n-hexane fraction with LD50 of 14.74, 31.22,

117.07, 147.99 and 279.95 µg/mL respectively. The LD50 of standard drug

(etoposide) was 7.4432 µg/mL. Insignificant LD50 was observer in case of n-butanol.

Generally the cytotoxicity shown by plant material is well intended to be the existence of active anticancer components261. Previously brine shrimp has been used as zoological specimen for evaluating plants for their cytotoxicity262. However this assay is relatively insufficient to elucidate the mechanism of action of cytotoxiciy, still it is considered to be very helpful to evaluate the bioactivity of plants extracts, fractions and isolated pure compounds263.

Ethyl acetate fraction (LD50 = 14.74 µg/mL) was eluted using chromatographic techniques. A number of compounds of different chemical classes were isolated; these compounds were investigated for their anticancer effect against prostate cancer (PC3) cell lines. Compound 9 (refer to Table-3.20) showed a good anticancer activity

(55.56%).

The potential activity of ethyl acetate fraction in the above mentioned assay can be correlated with the presence of compound 9.

Table-3.15: Cytotoxic potential of MBHE and fractions

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S. No Dose No. of No. of No. of LD50 Std. LD50 µg/mL shrimps survived dead µg/mL drug µg/mL shrimps shrimps MBHE 1 1000 30 9 21 2 100 30 18 12 147.99 Etoposide 7.4432 3 10 30 21 09 n-Hexane 1 1000 30 13 17 2 100 30 17 13 279.95 Etoposide 7.4432 3 10 30 19 11 Chloroform 1 1000 30 8 22 2 100 30 15 15 31.22 Etoposide 7.4432 3 10 30 16 14 Ethyl acetate 1 1000 30 11 19 2 100 30 14 16 14.74 Etoposide 7.4432 3 10 30 15 15 n-Butanol 1 1000 30 25 05 2 100 30 29 01 -- Etoposide 7.4432 3 10 30 30 00 Aqueous 1 1000 30 10 20 2 100 30 18 12 117.07 Etoposide 7.4432 3 10 30 21 09

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10 µg/mL 60

Percent effect Percent

MBHE Aqueous n-Hexane n-Butnaol Chlorofom Ethyl acetate

100 µg/mL 60

Percent effect Percent

MBHE Aqueous n-Hexane n-Butnaol Chlorofom Ethyl acetate

1000 µg/mL 80

Percent effect Percent 20

MBHE Aqueous n-Hexane n-Butnaol Chlorofom Ethyl acetate

Fig-3.22: Percent cytotoxic effect of MBHE and fractions.

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3.3.1.5. Phytotoxic activity

The phytotoxic potential of MBHE and subsequent fractions were studied against

Lemna minor in order to evaluate its promoter of inhibitor effect on plant growth264.

Results are shown in Table-3.16 and Figure-3.23. Samples were used in different concentrations (10, 100 and 1000 µg/mL). The paraquat was used as standard phytotoxic drug (0.015 µg/mL). The MBHE showed a mild to moderate phytotoxic activity 10, 25 and 35% at concentrations of 10, 100 and 1000 µg/mL respectively.

The n-hexane fraction also showed a mild to moderate activity of 15, 20 and 25% phytotoxicity at 10 and 100 and 1000 µg/mL respectively. The chloroform fraction showed a maximum activity of 85% at 1000 µg/mL, while at lower concentration (10 and 100 µg/mL) a mild phytotoxic activity (15 and 20%) was observed respectively.

The ethyl acetate fraction was significantly active at high concentration (1000 µg/mL) showing a percent phytotoxicity of 75%, while a mild to moderate response, 15 and

35% was observed at lower concentrations 10 and 100 µg/mL respectively. The n-butanol fraction at high concentration (1000 µg/mL) showed a maximum phytotoxic effect (80%), while showed a mild activity (10 and 25%) at lower concentrations (10 and 100 µg/mL) respectively. The aqueous fraction was also showed a maximum phytotoxic effect (80%) at high concentration (1000 µg/mL), while a mild phytotoxic activity (10 and 30%) at lower concentrations 10 and 100

µg/mL respectively.

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Table-3.16: Phytotoxic activity of MBHE and fractions

Sample No. of Conc. of No. of No. of Percent fronds sample fronds fronds growth (3/plant) (µg/mL) survived died regulation 20 10 18 02 10 MBHE 20 100 15 05 25 20 1000 13 07 35 20 10 17 03 15 n-Hexane 20 100 16 04 20 20 1000 15 05 25 20 10 17 03 15 Chloroform 20 100 16 04 20 20 1000 03 17 85 20 10 17 03 15 Ethyl ace- 20 100 13 07 35 tate 20 1000 05 15 75 20 10 18 02 10 n-Butanol 20 100 15 05 25 20 1000 04 16 80 20 10 18 02 10 Aqueous 20 100 14 06 30 20 1000 04 16 80

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10 µg/mL 20

Percent regulation

MBHE Aqueous n-Hexane n-Butanol ChloroformEthyl acetate

100 µg/mL 40

Percent regulation

MBHE Aqueous n-Hexane n-Butanol ChloroformEthyl acetate

1000 µg/mL 100

Percent regulation

MBHE Aqueous n-Hexane n-Butanol ChloroformEthyl acetate

Fig-3.23: Phytotoxic effect MBHE and fractions.

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Chapter: 3 Results & Discussion

3.3.1.6. Insecticidal activity

MBHE and fractions were evaluated for contact insecticidal activity against

Rhyzopertha dominica, Tribolium castaneum, and Callosobruchus analis. Results are shown in Table-3.17 and Figure-3.24. Permethrin (235.9 µg/cm2) and distilled water were used as positive and negative control respectively. Samples were applied in a concentration of 1019.10 µg/cm2. The maximum mortality (40%) was shown by chloroform fraction against Tribolium castaneum. A moderate insecticidal activity

(20%) was observed in case of n-hexane and MBHE against Rhyzopertha dominica and Callosobruchus analis respectively. A mild mortality (10%) was observed in case of MBHE to Tribolium castaneum, ethyl acetate fraction to Rhyzopertha dominica and Callosobruchus analis, and chloroform to Callosobruchus analis respectively.

Remaining samples were inactive in the above mentioned assay.

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Table-3.17: Insecticidal activity of the MBHE and fractions (% Mortality)

Name of insect MBHE n-Hexane Chloroform Ethyl acetate n-Butanol Aqueous Positive Negative control control Tribolium castaneum 10 00 40 00 00 00 100 00 Rhyzopertha dominica 00 20 00 10 00 00 100 00 Callosobruchus analis 20 00 10 10 00 00 100 00

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Chapter: 3 Results & Discussion

Tribolium castaneum 50

Percentmortality 10

MBHE Aqueous n-Hexane n-Butanol ChloroformEthyl acetate

Rhyzopertha dominica 25

Percentmortality 5

MBHE Aqueous n-Hexane n-Butanol ChloroformEthyl acetate

Callosobruchus analis 25

Percentmortality 5

MBHE Aqueous n-Hexane n-Butanol ChloroformEthyl acetate

Fig-3.24: Insecticidal activity of the MBHE and fractions.

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3.3.1.7. Urease inhibitory assay

The results of urease inhibitory potential of MBHE, fractions and isolated compounds are showed in Table-3.18 and Figure-3.25. All the samples exhibited inhibitory potential against urease enzyme. The MBHE showed a mild (17.9%) inhibitory activity. Ethyl acetate fractions showed a maximum (61.7%) inhibitory potential with

IC50 of 151.3 ± 1.40. n-Butanol fraction moderately (35.3%) inhibited the enzyme, while n-hexane, aqueous and chloroform fractions didn’t show any significant activity. According to the results compound 9 showed maximum (98.6%) inhibition with IC50 of 51.6±1.46, followed by compound 4 (40.9%), while compound 1 and 6 didn’t show any appreciable inhibitory potential.

Urease is an enzyme that catalyzes the hydrolysis of urea, lead to the production of ammonia. This enzyme is naturally produced by various pathogens especially Heli- cobacter pylori265. Production of ammonia has a primary role in the survival of H. pylori. It makes the environment favourable for H. pylori in the stomach, also weaken the mucus membrane, enabling the pathogen to get penetrated. Production of ammonia makes the environment basic in kidney, causes the precipitation of salts.

Precipitation of salts such as calcium oxalate lead to the formation of kidney stones266.

Studies suggested that urease inhibitors solubilise the kidney stones267. Traditionally fruit of Monotheca buxifolia has been used in treating various ailments such as gastritis and urinary tract infections162,268. In the current study it is shown that MBHE, subsequent fractions and isolated compounds have a marked inhibitory potential against urease enzyme. The use of Monotheca buxifolia in folk medicine for the treatment of gastritis and urinary tract infection can be validated through this research work.

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Table-3.18: Urease inhibitory potential of the MBHE and fractions

Compound Concentration (mM) % Inhibition IC 50 MBHE 0.2 17.9 – n-Hexane 0.2 7.2 – Chloroform 0.2 19.7 – Ethyl acetate 0.2 61.7 151.3±1.40 n-Butanol 0.2 35.3 – Aqueous 0.2 14.4 – Compound 1 0.5 7.3 – Compound 4 0.5 40.9 – Compound 6 0.5 8.6 – Compound 9 0.25 98.6 51.6±1.46

100

Percentinhibition

MBHE AqueousComp-1Comp-4Comp-6Comp-9 n-Butanol ChloroformEthyl acetate

Fig-3.25: Urease inhibitory potential of the MBHE, fractions and isolated compounds.

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3.3.1.8. Acetylcholineesterase inhibitory assay

Acetylcholineesterase inhibitory assay was performed for MBHE and four isolated compounds as shown in the Table-3.19 and Figure-3.26. The tested samples didn’t show any appreciable inhibition against acetylcholineesterase enzyme.

The enzyme acetylcholineesterase (ACh-E) is an ultimate target to design a coherent drug and for the innovation of mechanism-based inhibitors. ACh-E has a primary role in the break down and hydrolysis of neurotransmitter acetylcholine (ACh). ACh-E inhibitors are considered to be the most advanced and highly effective approach to

269 manage the cognitive. symptoms of Alzheimer disease (AD) . ACh-E also has significant therapeutic uses in diseases like ataxia, senile dementia and Parkinson’s disease270. Currently available ACh-E inhibitors that are used in the management of

AD are tacrine, donepezil, eserine, rivastigmine, and galanthamine271-272. Majority of them are discovered from natural sources. These drugs are having critical limitations in their clinical uses due to potential side effects and short-half-lives.

Focusing on importance of the Ach-E inhibitors detailed studies are needed to discover novel, highly potent, safe and effective Ach-E inhibitors. Extracts of a variety of medicinal plants are already been screened, as previously stated, many of them have been confirmed for potent Ach-E inhibitor activity273. In traditional medicine the fruit of M. buxifolia is used as laxative and or purgative, focusing these qualities the current study was aimed to investigate if such activity is due to inhibition of ACh-E. ACh-E inhibitors directly increase the net level of ACh by inhibiting its hydrolysis. Rise in ACh level in turn enhances all cholinergic activities including spasmogenic effect on smooth muscles of gastrointestinal tract leading to laxation or purgation. In the above mentioned assay all the samples didn’t show any significant

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Chapter: 3 Results & Discussion

ACh-E inhibitory activity. Yet it is clear that the laxative activity of Monotheca buxifolia fruit is due to mechanisms other than ACh-E inhibition.

Table-3.19: Acetylcholineesterase inhibition of MBHE and isolated compounds.

Sample Concentration % Inhibition IC 50 MBHE 100µg/mL 7.180 ± 0.193 >100 Compound 1 20 µg/mL 6.038 ± 0.352 >100 Compound 4 20 µg/mL 3.050 ± 0.306 >100 Compound 6 20 µg/mL 2.043 ± 0.554 >100 Compound 9 20 µg/mL 1.407± 0.525 >100

Percentinhibition 2

MBHE Comp-1 Comp-4 Comp-6 Comp-9

Fig-3.26: Acetylcholineesterase inhibition of MBHE and isolated compounds.

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3.3.1.9. Anticancer activity (PC-3 cell lines)

Results of anticancer activity against prostate cancer (PC3) cell line are shown in

Table-3.20 and Figure-3.27. Compound 9 showed maximum inhibition (55.56%).

Compound 5 mildly (29.99 %) inhibited the growth followed by compound 1

(24.03%), 4 (13.74%) and 6 (12.53%). In case of compound 7 least anticancer activity

(10.29%) was observed.

The currently available treatment of cancer is highly expensive, having potential side effects and/or unsuccessful in majority of conditions. Discovering anticancer drugs with the aim to be highly effective, safe, inexpensive and easily available is highly desired. MTT colorimetric procedure is easy and quick method for screening samples for their anticancer potential. A variety of anticancer compounds have been isolated from plants such as taxol from the bark of Taxus brevifolia, and vinca alkaloids from

Catharanthus roseus274. In the current procedure the above six compounds (3 new and

3 reported) were investigated for anticancer potential against PC3 cell lines.

Compound 9 showed maximum inhibition of more than 50%. Although this study doesn’t provide a mechanism oriented data, still it can be claimed that compound 9 may be recommend in the treatment of prostatic cancerous conditions. Other compounds didn’t show any appreciable activity.

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Table-3.20: Percent growth inhibition of the compounds against PC3 cell lines

Compound Concentration % Inhibition Comments 1 1µM 24.03 Inactive 4 1µM 13.79 Inactive 5 1µM 29.99 Low 6 1µM 12.53 Inactive 7 1µM 10.29 Inactive 9 1µM 55.56 Good

Percentinhibition

Comp-1 Comp-4 Comp-5 Comp-6 Comp-7 Comp-9

Fig-3.27: Percent growth inhibition of the compounds against PC3 cell lines.

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3.3.1.10. α-Chymotrypsin inhibitory assay

α-Chymotrypsin inhibition assay was evaluated for MBHE, fractions and six compounds. Results are shown in Table-3.21 and 3.22 and Figure-3.28 and 3.29 respectively. A mild inhibition (20.0, 19.1 and 18.1%) was observed in case of compound 8, n-hexane and ethyl acetate fraction respectively, while remaining samples were inactive in the stated assay.

α-Chymotrypsin, a digestive enzyme produced in the pancreas. It is secreted in inactive form (pro-enzyme) to the lumen of small intestine where it is converted into active form. It has a primary role in the metabolism of protein. It can degrade any protein, yet rise in its level can cause ulceration of small intestine. α-Chymotrypsin inhibitors are used in such condition to avoid ulceration275.

In traditional medicine fruit of Monotheca buxifolia is used in the treatment of various gastrointestinal tract (GIT) problems including ulcers and gastritis. The current study was aimed to evaluate α-chymotrypsin inhibition potential of this fruit. In the above mentioned assay all the samples were inactive, hence it is proved that the gastro-protective activity of this fruit follow mechanism other than α-chymotrypsin inhibition.

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Table-3.21: α-Chymotrypsin inhibition assay of MBHE and fractions.

S.No Sample Concentration Inhibition (%) Inhibition IC 50 1 MBHE 1 mM 1.6 >100 2 n-Hexane 1 mM 19.1 >100 3 Chloroform 1 mM 6.8 >100 4 Ethyl acetate 1 mM 18.1 >100 5 n-Butanol 1 mM 12.3 >100 6 Aqueous 1 mM 2.1 >100

Table-3.22: α-Chymotrypsin inhibition assay of compounds.

S.No Sample Concentration Inhibition (%) Inhibition IC 50 1 Compound 1 1 mM 6.4 >100 2 Compound 4 1 mM 0.9 >100 3 Compound 5 1 mM 4.4 >100 4 Compound 6 1 mM 2.1 >100 5 Compound 7 1 mM 1.7 >100 6 Compound 9 1 mM 20.0 >100

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Percentinhibition

MBHE Aqueous n-Hexane n-Butanol ChloroformEthylacetate

Fig-3.28: α-Chymotrypsin inhibition assay of MBHE and fractions.

Percentinhibition

Comp-1 Comp-4 Comp-5 Comp-6 Comp-7 Comp-9

Fig-3.29: α-Chymotrypsin inhibition assay of compounds.

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3.3.1.11. Cytotoxic activity (NIH-3T3 cell lines)

Cytotoxicity potential of six compounds was evaluated against NIH 3T3 cell lines.

Results are shown in Table-3.23 and Figure-3.30. A significant cytotoxic behaviour was observed in all samples except compound 1, which showed a minimum (5.35%) cytotoxicity. Compound 6 and 9 were highly toxic with a percent inhibition of 67.82 and 66.90%, respectively. Moderate (41.20, 44.66 and 45.31%) cytotoxic potential was noticed in case of compound 5, 7 and 4 respectively. Discovering new drugs with the aim to be more potent, safe and effective is critical need of the day. For such purposes every new compound is screened for various biological activities. Before going to in-vivo procedures, compounds should be screened for its cytotoxic potential.

Previously MTT assay has already been reported for its effectiveness in evaluating samples for their cytotoxic potential. Compounds with least inhibitory potential in the mentioned experiment are claimed to be safe. Hence it is concluded that compound 1 is safe and can be implicated in in-vivo protocols.

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Table-3.23: Percent growth regulation potential against NIH 3T3 cell lines

Compound Concentration % Inhibition Comments 1 1 mM 5.35 Non- Cytotoxic 4 1 mM 45.31 Cytotoxic 5 1 mM 41.20 Cytotoxic 6 1 mM 67.82 Cytotoxic 7 1 mM 44.66 Cytotoxic 9 1 mM 66.90 Cytotoxic

% Cytotoxicity effect on NIH 3T3 cell lines 80

Percentregulation

Comp-1 Comp-4 Comp-5 Comp-6 Comp-7 Comp-9

Fig-3.30: Percent growth regulation potential against NIH 3T3 cell lines.

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Chapter: 3 Results & Discussion

3.3.1.12. Protein antiglycation assay

MBHE and fractions were evaluated for the protein antiglycation potential. The results are shown in Table-3.24 and Figure-3.31. From the results it is clear that ethyl acetate and chloroform fraction were moderately active in the stated bioassay with percent inhibition of 19.49 and 15.09%, other samples didn’t show any appreciable results.

Glycation is a spontaneous, non-enzymetic reaction of protein with reducing sugar.

Normally glycation and antiglycation are going side by side in the body and such processes are normal and may not be noticed during normal conditions. In aging process, diabetes mellitus and/or Alzheimer’s disease, the process of protein glycation suc- ceeds the antiglycation276. Agents like aminoguanidines inhibit glycation process by interfering with the active carbonyl groups of intermediates277. Glycation occurs by the formation of Schiff’s base (the reaction of carbonyl group of reducing sugar with amino group of protein). Glycated proteins may undergo further reactions to form cross-linked, complex heterogeneous and fluorescent molecules that are known as advanced glycated end products. The chemistry of advanced glycated end products may be characterized as, such products can be fluorescent cross-linked compounds such as pentosidine, nonfluorescent crosslinked compounds such as arginine-lysine imidazone cross link or non cross-linked compounds like pyrraline. Because of significant therapeutic potential antiglycating agents are of quite interest to be discovered. These agents may help in combating complication related to diabetes, aging processes and other diseases in which glycation of protein overcome the antiglycation278. Keeping in view the prime importance of antiglycating agents the current study was aimed to evaluate the antiglycation potential of M. buxifolia fruit.

Ethyl acetate and chloroform fraction showed a mild antiglycating potential of 19.49

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Chapter: 3 Results & Discussion and 15.09% respectively, while rest of the samples were fairly inactive in the above mentioned assay. Further studies on sub fractions of ethyl acetate and chloroform fraction can lead to the discovery of lead antiglycating agent.

Table-3.24: Protein antiglycation potential of MBHE and fractions

Codes Concentration % Inhibition Active/Inactive MBHE 1 mM 3.79 Inactive n-Hexane 1 mM 1.53 Inactive Chloroform 1 mM 15.09 Inactive Ethyl acetate 1 mM 19.49 Inactive n-Butanol 1 mM 4.61 Inactive Aqueous 1 mM 9.84 Inactive

Percentinhibition

Crude Aqueous n-Hexane n-Butanol Chloroform Ethyl acetate

Fig-3.31: Protein antiglycation potential of MBHE and fractions.

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Chapter: 3 Results & Discussion

3.3.1.13. Immune modulatory assay

Immune modulatory assay was performed for six compounds. Results are shown in

Table-3.25 and Figure-3.32. Compound 7 showed potential suppressive activity, while other samples were inactive in the above assay. Compared to the normal peptide loading value represented as geometric mean (geo. mean) (42.14) compound 7 shows significant suppression value of geo. mean (33.94).

Targeted immune suppression play a vital role in the treatment of numerous diseases caused by abnormal immune responses such as cancers and auto-immune diseases279.

Majority of the available drug target the T-cells receptors to prevent the release of inflammatory cytokines and growth factors 280. Here we report a new way of treatment by targeting antigen peptide loading at the surface of antigen-presenting cells (APC) leading to complete suppression of immune response.

From the current study it is clear that compound 7 can be used as an immune modulatory agent.

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Table-3.25: Immune modulatory activity of compounds.

Code Concentration Geometric Comments mean 1 1 µM 37.25 Inactive 4 1 µM 38.53 Inactive 5 1 µM 36.60 Inactive 6 1 µM 38.24 Inactive 7 1 µM 33.94 Suppressor 9 1 µM 40.58 Inactive Control 1 µM 42.14 -

suppressor

Geo.mean 10

Control Comp.1 Comp.4 Comp.5 Comp.6 Comp.7 Comp.9

Fig-3.32: Immune modulatory activity of compounds.

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Irfan Irfan

Fig-3.33: The effect of compound 7 on cell surface peptide loading of MHC-II molecules. HLA-DRB1-expressing cells were incubated with respective peptide anti- gens (5 ng/mL) in the absence (uncatalyzed) or presence (catalyzed) of compound 7 (500 µM). Representative histogram plots show the fluorescence recorded in the absence (control) or presence of compound 7.

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3.3.2. In-vivo activities

3.3.2.1. Acute toxicity

In-vivo acute toxicity was performed for MBHE before executing any other in-vivo protocol in order to find out the percent toxicity and highest toxic dose. Results are shown in Table-3.26. From the results it is clear that MBHE was non toxic up to 2000 mg/ kg body weight. All the animals were alive and no abnormal behaviour was observed even after 24 hours of dose administration.

The acute toxicity test is of prime importance. It is performed to find out the toxic dose or any abnormal behaviour observed during the procedure. It is also significant to be performed before any other in-vivo protocol, as to find out the safe dose. The reason for death observed during any other protocol can be subsided from the toxicity caused by extract.

Table-3.26 Evaluation of in-vivo acute toxicity of MBHE

Treatment No. of Animal alive No. of Animal dead (mg/kg) after 24 hrs after 24hrs 100 All Nil 300 All Nil MBHE 500 All Nil 1000 All Nil 1500 All Nil 2000 All Nil Normal Saline 10 All Nil

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3.3.2.2. Antipyretic activity

Brewer’s yeast induced significant increase (p < 0.001) of body temperature and the temperature remained elevated throughout the 5 hours study period, as shown in

Table-3.27 and Figure-3.34. After 1 hour of treatment, significant reduction

(p < 0.001) in pyrexia was noticed with MBHE at doses of 100 and 150 mg/kg.

Almost, similar protective effect was observed after 2 hour of treatment with MBHE at 100 mg/kg (p < 0.01) and 150 mg/kg (p < 0.001). After 3 hours, less significant antipyretic effect was observed with the 100 mg/kg (p < 0.05) and 150 mg/kg

(p < 0.01) doses of MBHE and this effect persisted till the 5th hour. The 50 mg/kg dose of MBHE was only effective at 2nd hour, at which a less significant reduction

(p < 0.05) of pyrexia was observed. The standard acetaminophen at 150 mg/kg produced a robust antipyretic effect (p < 0.001) throughout the five hours study period.

Brewer’s yeast is an exogenous pyrogen which produces pathogenic fever by binding to lipopolysaccharide binding protein and results in the release of different cytokines like interleukin 1 (IL-1), IL-6, tumor necrosis factor alpha and prostaglandins 281.

These pro-inflammatory mediators cross the blood brain barrier and act on hypothalamus causing the release of prostaglandin E2 which is produced through the action of cyclo-oxygenase-2 and thus increasing the body temperature282. In this study, potential antipyretic was observed for the 100 and 150 mg/kg doses of MBHE and the effect was similar to that of acetaminophen (150 mg/kg).

In folk medicine fruit of M. buxifolia is used as antipyretic. Keeping in view the ethno-medicinal use the current experiment was designed to evaluate its antipyretic potential. Hence, antipyretic potential of the fruit can be validated through this study.

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Chapter: 3 Results & Discussion

Table-3.27: Antipyretic activity of MBHE against Brewer’s yeast induced pyrexia in mice

Group 1st hr (oF) 2nd hr (oF) 3rd hr (oF) 4th hr (oF) 5th hr (oF) 1 100.4 ± 0.358 100.5 ± 0.346 100.0 ± 0.335 100.4 ± 0.265 100.5 ± 0.341 2 96.18 ± 0.757*** 96.62 ± 0.755*** 95.50 ± 1.023*** 96.07 ± 0.720*** 95.23 ± 0.731*** 3 97.93 ± 0.449 98.20 ± 0.634* 98.58 ± 0.838 99.27 ± 0.860 99.88 ± 0.812 4 96.02 ± 0.884*** 97.18 ± 0.570** 97.68 ± 0.244* 97.72 ± 0.342* 97.42 ± 0.709* 5 93.70 ± 0.753*** 96.93 ± 0.584*** 96.48 ± 0.303** 97.33 ± 0.547** 96.35 ± 0.837**

Values are expressed as mean ± SD. ANOVA followed by Dunnett’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001

compared to brewer’s yeast alone treated group (n = 6).

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Chapter: 3 Results & Discussion

After one hour After two hour 150 150

***

*** 100 *** 100

50 50

BodyTemperature

0 0

saline dose 1 dose 2 dose 3 saline dose 1 dose 2 dose 3 standard standard

After three hour After four hour 150 150

*** ***

*** *** 100 *** 100 ***

50 50

BodyTemperature BodyTemperature

0 0

saline dose 1 dose 2 dose 3 saline dose 1 dose 2 dose 3 standard standard

After five hour 150

***

*** 100 ***

BodyTemperature

saline dose 1 dose 2 dose 3 standard

Fig-3.34: Antipyretic activity of MBHE against Brewer’s yeast induced pyrexia in mice.

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Chapter: 3 Results & Discussion

3.3.2.3. Analgesic activity

As shown in Figure-3.35, significant attenuation (p < 0.01) of acetic acid induced visceral pain was demonstrated by MBHE at doses of 50 and 100 mg/kg. However, the antinociceptive effect was highly significant (p < 0.001) at 150 mg/kg. Similarly, the standard diclofenac sodium at 50 mg/kg produced significant amelioration

(p < 0.001) of acetic acid induced writhes.

The nociceptive response in the acetic acid induced writhing test results from the production of prostaglandins through the action of cyclooxygenases283. The liberated prostaglandins stimulate sensory pathways in the mouse peritoneum and incite viscero-somatic reflexes manifested as strong abdominal constrictions or writhes284.

The acetic acid induced writhes are sensitive to various analgesics. MBHE when administered at doses of 50, 100 and 150 mg/kg depressed the acetic acid induced writhes and the antinociceptive effect was analogous to that of the standard diclofenac sodium (50 mg/kg).

Fig-3.35: Antinociceptive activity of MBHE in acetic acid induced abdominal constriction assay. ANOVA followed by Dunnett’s post hoc test. *p < 0.05,

**p < 0.01, ***p < 0.001 compared to saline treated group. n = 6.

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Chapter: 3 Results & Discussion

3.3.2.4. Carrageenan induced paw edema

As shown in Table-3.28, carrageenan alone treated animals exhibited significant

(p < 0.001) paw edema after 1 hour of administration and this condition remained persistent for 5 hours. Pre-treatment with MBHE significantly alleviated the carrageenan elicited paw edema after 1 and 2 hours at doses of 50 mg/kg (p < 0.01),

100 mg/kg (p < 0.001 and (p < 0.01) and 150 mg/kg (p < 0.001). After 3 and 4 hours, highly significant reduction (p < 0.001) of paw edema was produced by all the tested doses, however; a less significant reduction (p < 0.01) was observed with these doses after 5 hours of carrageenan administration. Moreover, aspirin (standard drug) at a dose of 150 mg/kg produced moderate to highly significant alleviation of paw edema after 1 hour (p < 0.01) and 2-5 hours (p < 0.001) duration of challenge with carrageenan.

Protocol of carrageenan-induced rat paw edema is a widely used test to determine the anti-inflammatory activity of both natural and synthetic compounds. Edema formation due to carrageenan administration in mouse paw is a biphasic event. The initial phase lasting about 1–5 hours is predominately characterized by a non-phagocytic edema and has been attributed to the action of various mediators including histamine, serotonin and bradykinin on vascular permeability285. The initial phase is followed by a second phase having duration of 2–5 hours and results from overproduction of prostaglandins. It has been reported that the second phase of edema is sensitive to drugs like hydrocortisone, phenylbutazone and indomethacin261. In our study, we have observed a robust anti-inflammatory activity of MBHE at all the tested doses (50, 100 and 150 mg/kg) and was comparable to the cyclooxygenase inhibitor, aspirin (150 mg/kg).

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Chapter: 3 Results & Discussion

Table 3.28: Anti-inflammatory activity of Monotheca buxifolia (MBHE) against carrageenan induced paw edema in mice

Group 1st hr 2nd hr 3rd hr 4th hr 5th hr 1 0.235 ± 0.012 0.227 ± 0.026 0.240 ± 0.011 0.210 ± 0.016 0.230 ± 0.036 2 0.340 ± 0.016### 0.370 ± 0.028### 0.395 ± 0.025### 0.490 ± 0.057### 0.440 ± 0.028### 3 0.255 ± 0.028** 0.250 ± 0.035*** 0.252 ± 0.020*** 0.325 ± 0.052*** 0.305 ± 0.019*** 4 0.262 ± 0.022** 0.282 ± 0.012** 0.287 ± 0.025*** 0.292 ± 0.015*** 0.352 ± 0.029** 5 0.227 ± 0.040*** 0.277 ± 0.035** 0.290 ± 0.046*** 0.287 ± 0.037*** 0.347 ± 0.029** 6 0.210 ± 0.028*** 0.257 ± 0.026*** 0.287 ± 0.022*** 0.272 ± 0.044*** 0.330 ± 0.038** Values are expressed as mean ± SD. ANOVA followed by Tukey’s post hoc test. ###p < 0.001 compared to group 1, **p < 0.01,

***p < 0.001 compared to group 2. N = 6.

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Chapter: 3 Results & Discussion

3.3.2.5. Prokinetic and Laxative activity

3.3.2.5.1. Effect of MBHE on charcoal meal intestinal transit

Result of the charcoal meal transit activity is shown in Table-3.29 and Figure-3.36. It is clear from the results that MBHE exhibited dose dependent rise in the impetus movement of charcoal meal. The movement of charcoal through the small intestine after the treatment with MBHE was 73.45 and 80.29% at 100 and 300 mg/kg respectively, while in the CCh, atropine and saline treated groups the movement of charcoal was 86.65, 21.80 and 45.80% respectively. When the MBHE (both doses) and CCh given to the groups pre-treated with atropine (I.P.) to study their influence on charcoal transit, all the prokinetic responses were significantly reduced. Distance travelled by charcoal was reduced to 24.94, 29.86 and 42.12% in groups 6, 7 and 8 respectively. Yet, it is clear that the excitatory effects of MBHE are partially antagonized by atropine.

3.3.2.5.2. Laxative activity

Results of laxative activity are shown in Table-3.30. It is evident from the results that animals of group-4 and 5 (MBHE 50 and 100 mg/kg) produced 73.79 and 85.52% wet faeces respectively, group-3 (CCh 1mg/kg I.P.) produced 87.42%, atropine and saline treated groups didn’t produce any wet faeces. The production of wet faeces in groups pre-treated with atropine was significantly reduced. Animals of group-6 (CCh and atropine) produced 33.67% wet faeces. In groups-7 and 8 (atropine + MBHE 50 and

100 mg/kg) the animals produced 36.31 and 50.59% wet faeces.

Keeping in view the folkloric use of Monotheca buxifolia fruit as laxative and purgative, the current study was designed to evaluate the laxative and prokinetic

151

Chapter: 3 Results & Discussion potential of the fruit. It is evident from the results that MBHE enhance the impetus movement of charcoal meal in the small intestine and also raised the production of wet faeces, therefore showing pro-kinetic and laxative activities, almost similar to

CCh (positive control). The intestinal excitatory responses of the extract were partially antagonized by atropine, a standard anti-cholinergic drug, demonstrating the presence of acetylcholine (ACh)-like compounds in accumulation to some other gut stimulating agents286. ACh is a typical para-sympathomimetic drug and prototype agent for stimulation of smooth muscles of gastro-intestinal tract by activating the muscarinic receptors. Therefore the presence of compounds having ACh-like effect partially explains the medicinal use of the fruit as laxative and purgative.

In the charcoal diet transit test MBHE (100 and 300 mg/kg) produced a significant effect (73.45 and 80.29% respectively) by enhancing the travel of charcoal through small intestine comparable to standard drug i.e. CCh (86.655%). The prokinetic response of MBHE (100 and 300 mg/kg) was partially reduced (29.86 and 42.12%) in groups pre-treated with atropine.

Similarly in laxative activity the groups treated with MBHE (100 and 300mg/kg) showed significant production of wet faeces (73.79 and 85.52% respectively) comparable to groups treat with CCh (87.42%), while the production of wet faeces in groups pre-treated with atropine were reduced to 36.31 and 50.59% by treating with

MBHE 100 and 300 mg/kg.

Yet it is clear from the results the MBHE contains some of the components behaving exactly similar to ACh while some other mechanisms of gut stimulation may also be involved as the affect was partially antagonized by atropine. Further studies are

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Chapter: 3 Results & Discussion warranted, such as in-vitro multiples models prokinetic assay to elucidate the exact mechanism of action of MBHE as laxative and purgative.

Table-3.29: Effect of atropine on the prokinetic effect of MBHE in mice.

Group Treatment Distance travelled by charcoal 1 Saline 45.80 ± 4.62 2 CCh 86.65 ± 2.44*** 3 Atropine 21.80 ± 2.32 4 MBHE 100mg 73.45 ± 2.96*** 5 MBHE 300 mg 80.29 ± 5.54*** 6 CCh+ Atropine 24.94 ± 2.63*** 7 MBHE 50 + Atropine 29.86 ± 2.98 8 MBHE 100 + Atropine 42.12 ± 3.94##

Values are expressed as mean ± SEM. ANOVA followed by Tukey’s post hoc test. ***p < 0.001 compared to group 1. ##p < 0.01 compared to group 3. (n = 6)

100

***

80 ***

travelled by charcoal meal travelledcharcoal by

Percent intestinelenght small of 0

atr CCh saline ext-100 ext-300 atr+CCh atr+ext-100atr+ext-300

Fig-3.36: Effect of atropine on the percent prokineteic effect of MBHE in mice.

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Chapter: 3 Results & Discussion

Table- 3.30: Effect of atropine on the percent laxative effect of MBHE in mice.

Group no Treatment Dose Defecation/group Number of wet % of wet faeces (mg/kg) faeces/group 1 Saline 10 4.17 ± 0.4 0 0 2 Atropine 10 1.83 ± 0.3 0 0 3 Carbachol 1 12.33 ± 0.42*** 10.83 ± 0.79*** 87.42 ± 4.472 4 MBHE 100 7.17 ± 0.70* 5.33 ± 0.70*** 73.79 ± 4.47 5 300 10.83 ± 1.04*** 9.00 ± 0.73*** 85.52 ± 3.99 6 Carbachol + Atropine 1 + 10 3.17 ± 0.40 1.00 ± 0.26 33.67 ± 6.87 7 MBHE +Atropine 100 + 10 6.17 ± 0.54### 2.17 ± 0.70 36.31 ± 11.13 8 300 + 10 8.00 ± 0.58### 4.00 ± 0.58### 50.59 ± 7.39 Values are expressed as mean ± SD. ANOVA followed by Tukey’s post hoc test. *p < 0.05, ***p < 0.001 compared to group 1###p < 0.001 compared to group 2. n = 6.

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Chapter: 3 Results & Discussion

3.3.2.6. Hepatoprotective activity

3.3.2.6.1. Effect of hydroethanolic extract on biochemical parameters

The results of hepatoprotective effects of MBHE on isoniazid (INH) and rifampicin

(RIF) intoxicated rats are shown in Table-3.31, administration of INH + RIF (50 mg/kg/day each) significantly (p < 0.001) raised SGPT, SALP, SGOT, SB with respect to normal. By treating with MBHE at a dose of 150 mg per kg and 300 mg per kg body weight, one hour before INH + RIF administration appreciably (p < 0.05 to p

< 0.001) confined the rise of SGPT, SGOT, SB, and SALP comparable to group treated with silymarin. TP level in the blood significantly (p < 0.001) decreased in rats given INH + RIF, while treated with MBHE (both doses) significantly (p < 0.001) recovered the total protein level, comparable to group treated with silymarin.

3.3.2.6.2. Effect of hydroethanolic extract on liver histology

After 21 days of INH + RIF treatment, the central veins of the hepatic lobules were congested with red blood cells and their epithelium were disrupted. The hepatocytes were depleted of glycogen. The sinusoidal spaces were dilated and were infiltrated by large number of lymphocytes. Increase numbers of focal aggregations of lymphocytes with necrotic hepatocytes were present around central vein. Although the liver retained its characteristic lobular appearance, however the hepatocytes appeared necrotic and exhibited ballooning degeneration. Pre-treatment with MBHE and silymarin for 21 days provided protection against INH + RIF induced hepatotoxicity as no significant histopathological changes were observed in the liver (Figure-3.37 and

Table-3.32).

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Chapter: 3 Results & Discussion

Metabolites of certain drugs can be electrophilic substances or free-radicals that initiates or catalyze different types of reactions i.e. glutathione depletion; binding with lipids and/or peroxidation of lipids results in oxidative stress. Drug induced hepatotoxicity occurs in a variety of mechanisms such as membrane disruption or cellular necrosis, which may be resulted from binding of drug or its metabolite to cellular proteins, making new adducts that serves as targets for immune system and activates immunological reactions287. Other causes might be the inhibition of drug metabolism pathways. Interruption in the bile flow or disruption of filaments at sub-cellular level of bile duct causes abnormal or disturbed bile secretions, leading to jaundice and minimal cellular injury. Programmed cell death or apoptosis due to tissue necrosis factor or FAS pathways and inhibition of mitochondrial functions lead to accumulation of reactive oxygen species and subsequently lipid peroxidation and cell death288.

INH and RIF are the first line antitubercular drugs and are used as standard hepatotoxic in various experiments. Administration of INH and RIF causes changes in both morphology and cellular function of liver. In the current study, Sprague Dawly rats were given INH and RIF (50 mg/kg per day orally for 21 days) to induce hepatotoxicity. Three folds rise in transaminases level in the serum of animals was a biochemical warning of hepatic injury. INH and RIF are potent hepatotoxic drugs, but the exact mechanism is still unclear. INH is converted to acetyl-isoniazid via hepatic

N-acetyltransferase-2, which in turn hydrolyzed to acetylhydrazine. Acetylhydrazine is oxidized by cytochrome P450 to form certain hepatotoxic intermediates. Hydrazine, either as direct (from INH) or indirect (from acetyl hydrazine) induces CYP2E1287,289.

The best role of CYP2E1 is the production of reactive oxygen species which may be responsible for the hepatotoxicity caused by isoniaizd290. RIF exaggerate the INH

156

Chapter: 3 Results & Discussion hepatotoxicity possibly by increasing the production of hydrazine or inhibition of bile pathway291-292. In this study, RIF and INH significantly increased the serum levels of

ALT, AST, ALP and billirubin while it decreased the level of TP. However, treatment with M. buxifolia extract at doses of 150 and 300 mg/kg significantly decreased the

INH and RIF induced elevated serum levels of ALT, AST, ALP, billirubin and TP, and this protective effect was comparable to the standard hepatoprotective drug, thus proving the hepatoprotective effect of M. buxifolia. The biochemical investigation was corroborated by histopathological findings, which showed certain morphological changes. Treatment with M. buxifolia at both doses (150 and 300 mg/kg) significantly inhibited the morphological changes. These observations strongly supported the hepatoprotective potential of M. buxifolia fruit against INH and RIF induced hepatotoxicity in rats.

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Chapter: 3 Results & Discussion

Table-3.31: Hepatoprotective activity of MBHE by isoniazid and rifampicin induced hepatotoxicity in rats

Groups SGPT SGOT BILLIRUBIN TP ALP IU/L IU/L mg/dl (g/dL) IU/L Normal 48.33 ± 2.02 84.17 ± 3.89 0.46 ± 0.03 7.82 ± 0.32 119.5 ± 2.54 TC 140.8 ± 3.10*** 131.2 ± 3.06*** 1.23 ± 0.04*** 3.58 ± 0.10*** 184.7 ± 3.82*** TCE1 114.7 ± 4.09*** 115.7 ± 3.58* 0.93 ± 0.05*** 5.70 ± 0.26*** 120.3 ± 4.16*** TCE2 62.5 ± 2.79*** 91.33 ± 3.56*** 0.73 ± 0.07*** 6.76 ± 0.16*** 114.8 ± 3.51*** TCStd 54.33 ± 2.33*** 85.50 ± 3.19*** 0.47 ± 0.01*** 7.48 ± 0.19*** 118.2 ± 4.72***

Data presented as Mean ± SEM *** = (p < 0.001), ** = (p < 0.01), * = (p < 0.05)

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Chapter: 3 Results & Discussion

Table 3.32: Effect of MBHE on the severity of isoniazid and rifampicin induced hepatotoxicity after 21 days of treatment

Group Group Group Histopathological findings Group NC Group TC TCE1 TCE2 TCSTD Glycogen depletion – + + + – – Congestion + + + + – + + Endothelium disruption – + + + – – – Sinusoidal dilatation – + + – – – Hydropic degeneration – + + – – – Cytolysis – + – – – Lymphocytic infiltration – + + + – – Perivenular necrosis – + + – – – Lymphoid aggregates in the – + + + – – – portal tract

(–) none; (+) mild; (++) moderate; (+++) severe

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Chapter: 3 Results & Discussion

Fig-3.37: Histopathological evaluation of isoniazid and rifampicin induced hepatotoxicity pre-treated with MBHE for 21 days (H & E; × 400 original magnification). (A): Photomicrograph of a section of liver from a rat treated with isoniazid plus rifampicin showing congestion of the central vein (CV) with disruption of its endothelium (large arrow), dilatation of the sinusoidal spaces

(asterisk), infiltration (arrow head) as well as aggregation (bar) of lymphocytes around the central vein and necrosis of hepatocytes (small arrows). Normal histology of central vein (CV) with intact endothelium (large arrow), hepatocytes (small arrows) and sinusoidal spaces (asterisk) lined by endothelial cells (arrow heads) were observed in groups of rats treated with (B): MBHE (150 mg/kg) (C): MBHE (300 mg/kg) and (D): silymarin (100 mg/kg) one hour before administration of isoniazid plus rifampicin.

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Conclusion

4. Conclusion

In this study, the in-vitro biological assays, antibacterial, phytotoxic, cytotoxic, urease inhibition, anticancer (PC3 cell lines) and immune modulatory activities were more momentous as compared to antifungal, insecticidal, α-chymotrypsin inhibition, and protein antiglycation activities. However, there was found no significant leishmanicidal and acetylcholineesterase inhibitory activity. Moreover, significant in-vivo antipyretic, analgesic, anti-inflammatory, hepatoprotective, prokinetic, and laxative activities were exhibited by Monotheca buxifolia fruit.

In addition, the phytochemical investigation led to the isolation of 4 new, 1 first time isolated from natural sources and 4 known compounds, belonging to alkaloids, flavonoids, triterpenoids, pyrone and glycosoides.

In light of the above findings, the fruit of Monotheca buxifolia can be used in traditional medicine as analgesic, antipyretic, laxative, hepatoprotective, anticancer, and phytotoxic.

However it also may be suggested to evaluate Monotheca buxifolia fruit as antibacterial in gastritis, urinary tract infection, and general infections by considering its urease inhibitory activity. The newly isolated molecules from Monotheca buxifolia fruit may act as a good source for pharmacologically active compounds having therapeutic use(s).

161

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