Screening of Some Selected Medicinal Plants of Nepal for Their Antioxidant and Anticancer Activities and Identification of Active Compounds

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SCREENING OF SOME SELECTED MEDICINAL PLANTS OF NEPAL FOR THEIR ANTIOXIDANT AND ANTICANCER ACTIVITIES AND IDENTIFICATION OF ACTIVE COMPOUNDS

A THESIS SUBMITTED TO

CENTRAL DEPARTMENT OF CHEMISTRY INSTITUTE OF SCIENCE AND TECHNOLOGY TRIBHUVAN UNIVERSITY NEPAL

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMISTRY

BY KHAGA RAJ SHARMA JUNE 2017

DECLARATION

Thesis entitled “Screening of some selected medicinal plants of Nepal for their antioxidant and anticancer activities and identification of active compounds” is being submitted to the Central Department of Chemistry, Institute of Science and Technology (IOST), Tribhuvan University, Nepal for the award of the degree of Doctor of Philosophy (Ph.D.), is a research work carried out by me under the supervision of Dr. Surya Kant Kalauni, Central Department of Chemistry, Tribhuvan University. This research is original and has not been submitted earlier in part or full in this or any other form to any university or institute, here or elsewhere, for the award of any degree.

…………………… Khaga Raj Sharma

TRIBHUVAN UNIVERSITY CENTRAL DEPARTMENT OF CHEMISTRY KIRTIPUR, KATHMANDU, NEPAL

RECOMMENDATION

This is to recommend that Mr. Khaga Raj Sharma has completed thesis entitled “Screening of some selected medicinal plants of Nepal for their antioxidant and anticancer activities and identification of active compounds” for the award of Doctor of Philosophy (Ph.D.) degree in Chemistry under my supervision. To the best of my knowledge, this work has not been submitted for any other degree.

He has fulfilled all the requirements laid down by the Institute of Science and Technology (IOST), Tribhuvan University, Kirtipur for the submission of the thesis for the award of Ph.D. degree.

Date: June 11, 2017

------Surya Kant Kalauni, Ph.D. Supervisor Central Department of Chemistry Tribhuvan University Kirtipur, Kathmandu, Nepal

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CERTIFICATE OF APPROVAL

On the recommendation of assistant professor Dr. Surya Kant Kalauni, this Ph.D. thesis is submitted by Mr. Khaga Raj Sharma entitled “Screening of some selected medicinal plants of Nepal for their antioxidant and anticancer activities and identification of active compounds” is approved by Central Department Research Committee (CDRC), Institute of Science and Technology, Tribhuvan University for the award of Doctor of Philosophy (Ph.D.) degree in chemistry.

Date: June 11, 2017

------Prof. Megh Raj Pokhrel, Ph.D.

Head

Central Department of Chemistry Tribhuvan University Kirtipur, Kathmandu, Nepal

ACKNOWLEDGEMENTS

The present study which focused on search of active plant extracts and isolated pure compounds against pancreatic cancer which ultimately causes diabetes. The anticancer drugs known till now are found totally ineffective for treatment of pancreatic cancer. In this context, this research is an attempt made to identify an effective anticancer, antidiabetic, and antioxidant compounds. The first and the most important contribution made on this dissertation is by Dr. Surya Kant Kalauni, Assistant Professor at Central Department of Chemistry, Tribhuvan University without which the completion of this research would not have been possible. Therefore, it is my pleasure to express profound gratitude to my supervisor Dr. Surya Kant Kalauni for his continuous guidance and encouragement which enabled me to successfully complete this research study. I would like to express my sincere gratitude to Prof. Dr. Megh Raj Pokhrel, Head Central Department of Chemistry, Tribhuvan University for giving the permission to conduct this research work. I am grateful to Prof. Dr. Kedar Nath Ghimire, former head Central Department of Chemistry, for the Precious suggestions and inspiration in this research work. I am grateful to Prof. Dr. Muhammad Iqbal Choudhary, HEJ Research Institute of Chemistry (ICCBS) International Center for Chemical and Biological Sciences, University of Karachi, Pakistan for his kind supervision, valuable guidance and precious suggestions during the period of collaborative research work. I wish to express my sincere thank to assistant professor Dr. Achyut Adhikari, HEJ Research Institute of Chemistry for his help in laboratory works and identification of isolated compounds. I am grateful to Dr. Suresh Awale Frontier Research Core for Life Science University of Toyama, Japan for his valuable supports during my research work. Similarly, I wish to express my sincere thank to assistant professor Dr. Yuba Raj Pokharel, South Asian University New Delhi India for his kind support during my research period. I would like to extend my sincere gratitude to all the members of research committee Institute of Science and Technology and research committee of Central Department of

Chemistry Tribhuvan University. Similarly, I would like to extend my sincere gratitude to Prof. Dr. Mohan Bikram Gewali, Prof. Jay Krishna Shrestha, Prof. Dr. Rhiddi Bir Singh, Prof. Dr. Rameshwar Adhikari, Prof. Dr. Amar Prasad yadav, Prof. Dr. Ram Chandra Basnyat, Prof. Dr. Jagadeesh Bhattarai, Prof. Dr. Mina Rajbhandari, Prof. Dr. Paras Nath Yadav, Prof Dr. Vinaya Kumar Jha, Prof. Dr. Armila Rajbhandari, for encouragement and valuable suggestions in this research work. I am grateful to the department heads and all the teaching and non-teaching staffs of Central Department of Chemistry, Central Department of Biotechnology, Central Department of Microbiology and Central Department of Botany Tribhuvan University for laboratory facilities. I am grateful to the Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo Sri Lanka for the cytotoxicity assay. I am thankful to Nepal Academy of Science and Technology (NAST) for providing Ph.D. fellowship. I am grateful to the campus chief, head of chemistry department and all faculty members of Birendra Multiple Campus Bharatpur Chitwan for their kind support in my research work. I am grateful to Rita Chhetry and Dhan Raj Kandel National Herbarium and plant resources Godawari Lalitpur for identification of plants. Finally, I would like to express my deepest gratitudes to my parents with whom I have unforgettable memories, who taught me the lesson of hard work in life and who have supported me in every moment, and my both elder brothers, who cared me like parents. I feel very happy to thank my two lovely sons Sugam Sharma and Sajal Sharma for bring joy and happiness during my research work. I am grateful to brother Dr. Tika Ram Gautam, Central Department of Sociology, Tribhuvan University for his valuable suggestions, guidelines and overall support in my research work. I would like to thank to my wife Mathura Sharma (Lamsal) for her cordial help and support including management of time. Finally, I wish to thank all the persons who directly or indirectly helped me to complete this research work.

Khaga Raj Sharma Date: June 11, 2017

ABSTRACT

People of Nepal have been using various medicinal plants, available in different regions, as medicine in the treatment of different diseases throughout the history. Those medicinal plants possess unique and valuable secondary metabolites which are responsible for the therapeutic values. Very few natural compounds identified until today are found effective against pancreatic cancer to some extent. However, all the cancer drugs discovered are found completely ineffective against the pancreatic cancer. Therefore, the present study aims to explore the medicinal value of these traditionally used medicinal plants with the principles of natural product chemistry in order to isolate the active compounds against pancreatic cancer. For this purpose, 50 medicinal plants were collected from different regions of Nepal which were further screened at first using different methods of bioassay followed by fractionation and isolation in the bioactive plant extracts. Thus the focus of this study is an isolation of active compound from selected medicinal plant extracts against pancreatic cancer which ultimately controls diabetes so that it can be recommended for drug discovery process.

The method of screening were DPPH radical scavenging and preferential cytotoxicity assay against pancreatic cancer PANC-1 cell lines under nutrient deprived condition (NDM). Radical scavenging assay indicated fifteen plant extracts were found as potent antioxidant with high value of total phenolic and flavonoid content. Medicinal plant extracts were tested against microorganisms such as E. coli, Salmonella typhi, Staphylococcus aureus and Bacillus subtilic in order to explore the antibacterial activity of plant extracts. Out of fifty medicinal plants, sixteen medicinal plants showed antimicrobial activity against these organisms.

The plant extracts of Bridelia retusa and Scoparia dulcis were selected as potent for isolation of pure compounds by chromatographic techniques. Eight compounds (1-8) from the dichloromethane and hexane soluble fraction of Scoparia dulcis Linn and three compounds from ethyl acetate soluble fraction of Bridelia retusa were isolated. Structure of isolated compounds was elucidated by modern spectroscopic techniques; 1H-NMR, 2D-NMR, mass, UV and IR spectroscopy. Isolated compounds were further tested for antidiabetic, antioxidant, immunomodulatory and anticancer activity.

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Coixol (1), glutinol (2), glutinone (3), friedelin (4), betulinic acid (5) and tetratriacontan-1-ol (6) isolated from the plant Scoparia dulcis Linn were evaluated for their insulin secretion activity on isolated mice islets and MIN-6 pancreatic β-cell line, and coixol (1) and glutinol (2) were found to be potent and mildly active respectively. Coixol (1) was further evaluated for insulin secreting activity on MIN-6 pancreatic β- cell line. Coixol (1) was subjected to cytotoxicity assay against MIN-6 and 3T3 cell lines that was found to be non-toxic. The insulin releasing activity of coixol (1) and glutinol (2) supported the ethno-botanic uses of Scoparia dulcis as an antidiabetic agent. To the best of our knowledge this is the first report of the insulin secreting activity of some major constituents of an anti-diabetic plant Scoparia dulcis.

Betulinic acid (5) isolated from hexane soluble fraction of methanolic extract of Scoparia dulcis was found potent cytotoxic against breast cancer cell line MCF-7 and

MDA-MB-231 with IC50 value 13.65 ppm. Betulinic acid (5) also showed 100 percent preferential cytotoxicity against pancreatic cancer cell (PANC-1) and (PSN-1) at a concentration of 31.60 μM and 3.893 μM respectively under NDM. Among all tested natural compounds isolated from S. dulcis, glutinone (3) exerted potent inhibition of oxidative burst from whole blood cells. Glutinone (3) showed potent inhibitions of intracellular reactive oxygen species (ROS), when tested on zymosan activated isolated human PMNS using luminol as probe. Glutinone (3) also showed inhibition on the production of proinflammatory cytokine TNF-α and weak inhibition was observed when it was tested for IL-1β and NO (Nitric oxide). Current study demonstrated the anti-inflammatory potential of glutinone and it may be the lead compound for further drug discovery process.

Tambulin (9) isolated from Bridelia retusa showed high antioxidant activity in DPPH radical scavenging assay (IC50 166.15±1.92 SEM [μM] and the radical scavenging activity 86.03 percent. Tambulin (9) is reported first time from the plant Bridelia retusa which showed potent immunomodulatory activity. Tambulin (9) has potent antiurease activity (IC50 41.82±1.60 SEM [µM] as compared to the standard thiourea

(IC50 21.00±0.11 SEM [µM].

Keywords: antiausterity; betulinic acid; Bridelia retusa; coixol; glutinone; PANC-1; Scoparia dulcis; tambulin; anticancer; antidiabetic; antioxidant.

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TABLE OF CONTENTS

Title page………………………………………………………………………………i Declaration………...... ii Recommendation ...... iii Certificate of approval ...... iv Acknowledgements ...... v Abstract…… ... ………………………………………………………………………vii Table of contents..…... …………………..……...……………………………………ix List of abbreviations……………………………………………………………….....xv List of tables… ...... xviii List of figures...... xix CHAPTER 1 INTRODUCTION 1- 7 1.1 General introduction………………………………………………… ....…………1 1.2 Rationale ……………………………………………………...…………………...6 1.3 Objectives ...... 7 1.3.1 General objective...………………...... 7 1.3.2 Specific objectives……………………………………………………………… 7 1.4 Hypothesis...... 7 CHAPTER 2 LITERATURE REVIEW 8- 48 2.1 Oxalis corniculata (From Syangja)…………………………………………… .....8 2.2 Drymaria diandra ...... 9 2.3 Melia azedarach...... 9 2.4 Cyperus rotundus ...... 10 2.5 Cissampelos pareira ...... 11 2.6 Coccinia grandis ...... 12 2.7 Euphorbia hirta ...... 12 2.8 Cynodon dactylon ...... 13 2.9 Ageratum houstonianum ...... 14 2.10 Curcuma angustifolia...... 15 2.11 Strychnos nux vomica ...... 15 ix

2.12 Shorea robusta ...... 16 2.13 Acacia catechu ...... 18 2.14 Lyonia ovalifolia ...... 18 2.15 Pterocarpus santalinus ...... 20 2.16 Desmostachya bipinnata ...... 20 2.17 Aegle marmelos ...... 21 2.18 Mahonia napaulensis ...... 22 2.19 Phyllanthus emblica ...... 23 2.20 Berberis aristata ...... 24 2.21 Tinospora sinensis ...... 25 2.22 Cuscuta reflexa ...... 26 2.23 Leucas cephalotes ...... 27 2.24 Drynaria propinqua ...... 28 2.25 Tinospora cordifolia ...... 28 2.26 Centella asiatica ...... 29 2.27 Asparagus filicinus...... 30 2.28 Justicia adhatoda (From Chitwan)…………………….…………………… .....30 2.29 Litsea cubeba ...... 31 2.30 Oxalis cornicullata (From Chitwan)..…………………………………………..31 2.31 Justicia adhatoda (From Syangja)……………………………………………...32 2.32 Cleistocalyx operculatus ...... 32 2.33 Bauhinia variegata...... 34 2.34 Pogostemon amaranthoides ...... 35 2.35 Betula alnoides...... 35 2.36 Bergenia ciliata ...... 37 2.37 Periploca calophylla ...... 38 2.38 Astilbe rivularis ...... 39 2.39 Piper mullesua ...... 39 2.40 Bombax ceiba ...... 39 2.41 Calotropis gigantea ...... 40 2.42 Annona reticulata...... 41 2.43 Mimosa pudica ...... 42 2.44 Ziziphus mauritiana ...... 43 2.45 Cascabela thevetia ...... 44 x

2.46 Achyranthes bidentata ...... 44 2.47 Callicarpa sp...... 45 2.48 Cinnamomum tenupile…………………………………………………………..45 2.49 Bridelia retusa ...... 46 2.50 Scoparia dulcis...... 48 CHAPTER 3 MATERIALS AND METHODS 58- 82 3.1Selection of medicinal plants ...... 58 3.2 General experimental conditions………………………………………………....59 3.2.1 Physical constants ...... 59 3.2.2 Spectroscopic technique...... 59 3.2.3 Chromatography and staining ...... 60 3.2.4 Equipments ...... 60 3.2.5 Chemicals ...... 60 3.2.6 Phytochemical screening ...... 61 3.2.6.1 Alkaloids ...... 61 3.2.6.2 Flavonoids ...... 61 3.2.6.3 Steroids ...... 61 3.2.6.4 Terpenoids...... 61 3.2.6.5 Reducing sugars ...... 61 3.2.6.6 Glycosides ...... 61 3.2.6.7 Polyphenols ...... 62 3.2.6.8 Tannins ...... 62 3.2.6.9 Cardiac glycoside ...... 62 3.2.6.10 Anthraquinone...... 62 3.21.6.11 Saponins ...... 62 3.2.6.12 Carotenoids ...... 62 3.2.7 Antioxidant activity (DPPH radical scavenging assay)..………………………62 3.2.8 Total polyphenol content determination ...... …….63 3.2.9 Total flavonoid content determination ...... 64 3.2.10 In - Vitro antimicrobial activity ...... 65 3.2.10.1 Preparation of culture media ...... 65 3.2.10.2 Nutrient agar (NA) ...... 65

3.2.10.3 Preparation of mueller hinton agar (MHA) ...... 66 3.2.10.4 Preparation of standard culture inoculums ...... 66 3.2.10.5 Transfer of the bacteria on the petriplates ...... 66 3.2.10.6 Antibacterial test ...... 66 3.2.10.7 Antimicrobial screening ...... 67 3.3 Preferential cytotoxicity against PANC-1 cancer cell line ...... 68 3.4 Isolation of pure compounds from Scoparia dulcis Linn………………………..69 3.4.1 Collection of plant samples ...... 69 3.4.2 Extraction and isolation of pure compounds…………………………………..69 3.5 Isolation of pure compounds from Bridelia retusa………………………………..72 3.5.1 Plant materials ...... 72 3.5.2 Extraction ...... 72 3.5.3 Isolation of pure compounds from bark extract of Bridelia retusa………… ....72 3.5.3.1 Coixol (1) ...... 74 3.5.3.2 Glutinol (2)...... 74 3.5.3.3 Glutinone (3) ...... 74 3.5.3.4 Friedelin (4) ...... 75 3.5.3.5 Betulinic acid (5)...... 76 3.5.3.6 Tetratriacontan-1-ol (6) ...... 76 3.5.3.7 β-sitosterol (7) ...... 77 3.5.3.8 Sigmastanone (8) ...... 77 3.5.3.9 Tambulin (9) ...... 78 3.5.3.10 β-sitosterol glucoside (10)...... 78 3.6 Biological assay of isolated pure compounds……………………………………79 3.6.1 Anti diabetic activity of coixol (1) ...... 79 3.6.1.1 Islets isolation and insulin secretion assay...... 79 3.6.1.2 MIN-6 cell culture and insulin secretion assay ...... 79 3.6.1.3 Toxicity assay ...... 80 3.6.2 Immunomodulatory activity of glutinone (3) ...... 80 3.6.2.1 Determination of ROS by chemiluminescence assay ...... 80 3.6.2.2 Nitric oxide (NO) assay ...... 81 3.6.2.3 Cytokine assay ...... 81 3.6.3 Cytotoxicity against MCF-7 (breast cancer) cell lines ...... 81 3.6.3.1 Cell culture ...... 81 xii

3.6.3.2 Cytotoxic assay ...... 82 3.6.4 Urease inhibition assay………………………………………………...... 82 CHAPTER 4 RESULTS AND DISCUSSION 83- 119 4.1 Results and discussion ...... 83 4.2 The yield percentage of plant extracts ...... 83 4.3 Phytochemical screening of plant extracts……………………………………….84 4.4 Antioxidant activity (DPPH radical scavenging assay) ...... 84 4.5 Total phenolic content...... 87 4.6 Total flavonoid content ...... 89 4.7 Preferential cytotoxicity against pancreatic cancer cell lines (PANC-1)………...91 4.8 Antimicrobial activity ...... 92 4.9 Anti-microbial screening of plant extracts……………………………………….93 4.10 Antimicrobial activity of screened plant extracts ...... 95 4.11 Structure elucidation of isolated pure compounds……………………………...97 4.11.1 Coixol (1) ...... 97 4.11.2 Glutinol (2)…....…………………………………………………………...... 98 4.11.3 Glutinone (3) ...... 99 4.11.4 Friedelin (4) ...... 101 4.11.5 Betulinic acid (5)...... 102 4.11.6 β-sitosterol (7) ...... 103 4.11.7 Sigmastanone (8) ...... 104 4.11.8 Tambulin (9)………….………...…………………………………………. ..104 4.11.9 3-O-β-D-glucopyranosyl-β-sitosterol glucoside (10)……………………… .105 4.12 Biological activity of isolated pure compounds……………………………….106 4.12.1 Insulin secretory activity of coixol (1)……………………………………...106 4.12.2 Coixol (1) exerts an exclusive glucose dependent insulinotropic effect in βTC- 6 cells ...... 108 4.12.3 The clinical effect and safety ...... 109 4.12.4 Immunomodulatory activity of glutinone (3) ...... 110 4.12.5 Cytotoxicity of betulinic acid (5) against breast cancer cell lines ...... 113 4.12.6 Preferential cytotoxicity of pure compounds against pancreatic cancer cell line (PANC-1) and PSN-1………………………………...... 114

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4.12.7 Antioxidant activity of tambulin (9)……………………………………….. .118 4.12.8 Urease activity of tambulin (9)……………………………………………. ..118 4.12.9 Immunomodulatory activity of tambulin (9)………………………………. .119 CHAPTER 5 CONCLUSIONS AND RECOMMENDATION 120-121 5.1 Conclusions ...... 120 5.2 Recommendation ...... 121 CHAPTER 6 SUMMARY 123 REFERENCES ...... 126 APPENDICES ...... 146 Appendix 1a: Research paper published in International Journals ...... 146 Appendix 1b: Research Paper Published in National Journals…………………….147 Appendix 1c: Paper presented in national and international seminar/workshop…...148 Appendix 1d: Poster Presented in national and international seminar/workshop…. 150 Appendix 2a: Seminar attended ...... 151 Appendix 2b: Letter of invitation as fellow researcher in HEJ Research Institute of Chemistry, ICCBS, University of Karachi, Karachi Pakistan…...... 153 Appendix 2c:Letter of recommendation/Participation in different academic activities in HEJ Research Institute of Chemistry ICCBS, University of Karachi, Pakistan...... ….154 Appendix 3: List of studied plants with their family, local name, English name, yield percentage, parts used and therapeutic uses………………...... 155 Appendix 4: Antioxidant screening of plant extract (DPPH radical scavenging assay)…………………………………………………………...... 156

Appendix 5: Total phenolic, flavonoid content and free radical scavenging (IC50)...163 Appendix 6: Total phenolic content (standard callibration curve for gallic acid)…..164 Appendix 7: Cytotoxicity (breast cancer) of compounds glutinone, betulinic acid, sigmastanone, friedelin and coixol……………………………… ...... 164 Appendix 8: List of identified plants used in the study ...... 166 Appendix 9: List of spectra of isolated pure compounds……………………… ...... 169

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LIST OF ABBREVIATIONS

LC50 Lethal Concentrations

IC50 Inhibitory Concentration

PANC- 1 Pancreatic Cancer Cell Line

DMEM Dulbecco’s Modified Eagle Medium

NDM Nutrient Deprived Medium

DCM Dichloromethane

BHA Butylated Hydroxyanisole

BHT Butylated Hydroxytoluene

PG Propyl gallate

MeOH Methanol

QE Quercetin Equivalent

GAE Gallic Acid Equivalent

EtOH Ethanol

MHA Mueller Hinton Agar

NA Nutrient Agar

MIN Mouse Insulinoma Pancreatic beta cells

SEM Scanning Electron Microscope

MMPs Matrix Metalloproteinases

PA Pyrolizidine Alkaloid

TAF Total Alkaloids Fraction

MTAF Modified Total Alkaloid Fraction

HFLS-RA Human Fibroblast-Like Synoviocytes-Rheumatoid Arthritis

HCC Hepatocellular Carcinoma

FRAP Ferric Reducing Antioxidant Power

TPC Total Phenolic Content

RP-HPLC Reverse Phase High Performance Liquid Chromatography

DAD Diode Array Detector

1D-NMR 1 Dimensional Nuclear Magnetic Resonance

HSQC Heteronuclear Single-Quantum Correlation

HMBC Heteronuclear Multiple Bond Correlation

ESI-MS Electrospray Ionization- Mass spectroscopy

GC-FID Gas Chromatography- Flame Ionization Detector

GC-MS Gas Chromatography- Mass Spectrometry

DPPH 2,2-diphenyl-1-picrylhydrazyl

CPAE Cissampelos pareira Aqueous Extract

RAPD Random Amplified Polymorphic DNA

MCK-7 Muscle Creatine Kinase 7

HPTLC High Performance Thin Layer Chromatography

MTAF Modified Total Alkaloids Fraction

DEM Digital Elevation Model

EPE Ethanolic phyllanthus emblica

MPE Methanolic phyllanthus emblica

AST Aspartate aminotransferase

ALT Alanine aminotransferase

PC12 Pheochromocytoma

HCC Hepatocellular Carcinoma

SGPT Serum Glutamate Pyruvate Transaminase

SGOT Serum Glutamate Oxaloacetate Transaminase

GOT Glutamic Oxaloacetic Transaminase

GPT Glutamic Pyruvic Transaminase

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GC-FID Gas Chromatography-Flame Ionization Detector

STZ Streptozotocin

TAA Total Antioxidant Activity

SPEt Scoparia dulcis Plant Extract

HRTEM Transmission Electron High-resolution Microscopy

XRD X-ray diffraction

Au-QD Gold Quantum Dots

FBS Fetal Bovine Serum

DEPT Distortionless Enhancement by Polarization Transfer

BB Broad Band EI-MS Electron Ionized Mass Spectrometry TNF-α Tumor Necrosis Factor alpha L-NMMA Monomethyl L-Arginine Acetate PMNs Polymorphonuclear leukocytes

MCF-7 Michigan Cancer Foundation-7

TBARS Thiobarbituric acid reactive substances

MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide

ELISA Enzyme Linked Immunosorbent Assay

BSA Bouvine Serum Albumin

HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid

PMA Phorbol Myristate Acetate

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LIST OF TABLES

Table 1: Total phenolic content in potent antioxidant plant extracts 88

Table 2: Total flavonoid content in potent antioxidant plant extracts 90

Table 3: Preferential cytotoxicity against pancreatic cancer cell lines 92

Table 4: Microbial screening of plant extracts zone of inhibition (ZOI) mm 93

Table 4.1: Microbial screening of plant extracts zone of inhibition (ZOI) mm 94

Table 4.2: Microbial screening of plant extracts zone of inhibition (ZOI) mm 94

Table 5: Antimicrobial activity of screened plant extracts zone of inhibition (ZOI) mm 95 Table 5.1: Antimicrobial activity of screened plant extracts zone of inhibition

(ZOI) mm 96

Table 6: Antimicrobial activity of drugs (positive control) against the microorganisms, ZOI mm 96 Table 7: 1H and 13C- NMR and chemical shift value of coixol (MeOD, ppm,

500 MHz) 97

1 13 Table 8: H and C- NMR and chemical shift value of glutinol (CDCl3 ppm,

500 MHz) 98

1 13 Table 9: H- and C-NMR chemical shift value of glutinone (CDCl3, ppm, 500 MHz 100

1 13 Table 10: H- and C-NMR chemical shift value of friedelin (CDCl3 ppm, 500 MHz) 101

Table 11: Effect of glutinone (3) on nitric oxide (NO), proinflammatory cytokines, TNF-α and IL-1β 112

Table 12: Cytotoxicity of compounds against MCF-7 (Breast cancer) cell lines 113 Table 13: Immunomodulatory activity of tambulin on ROS with respect to Ibuprofen 119

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LIST OF FIGURES

Figure 1: Cell survival under insufficient blood supply 3

Figure 2: Preferential cytotoxicity under nutrient deprived conditions 3

Figure 3: Preferential cytotoxic activity test against PANC-1 cell lines 68

Figure 4: Fractionation of crude methanolic extract of S. dulcis 70

Figure 5: Isolation of coixol (1) from dichloromethane fraction of Scoparia dulcis 71

Figure 6: Isolation of compounds 2, 3, 4, 5, 6, 7, and 8 from dichloromethane and hexane fraction of Scoparia dulcis 71

Figure 7: Fractionation of crude methanolic extract of Bridelia retusa bark 73

Figure 8: Isolation of compounds 1, 2 and 3 from ethyl acetate fraction of Bridelia retusa bark 73

Figure 9: Yield percentage of plant extracts 83

Figure 9a: Yield percentage of plant extracts 84

Figure 9b: Yield percentage of plant extracts 84

Figure 10: Calibration curve of standard ascorbic acid 85

Figure 11: Free radical scavenging activity of active selected plant extracts 86

Figure 12: Free radical scavenging activity and concentration of plant extracts 86

Figure 12a: Free radical scavenging activity and concentration of plant extracts 87

Figure 13: Calibration curve of standard gallic acid 87

Figure 14: Calibration curve of standard quercetin 89

Figure 15: Structure of coixol (1) 97

Figure 16: Structure of glutinol (2) 99

Figure 17: Structure of glutinone (3) 99

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Figure 18: Structure of friedelin (4) 102

Figure 19: Structure of betulinic acid (5) 103

Figure 20: Structure of β- sitosterol (7) 104

Figure 21: Structure of sigmastanone (8) 104

Figure 22: Structure of tambulin (9) 105

Figure 23: Structure of 3-O-β-D-glucopyranosyl-β- sitosterol glucoside (10) 106

Figure 24: Effect of compounds 1-6 (A), and dose response of compound (1) (B) on glucose stimulated insulin secretion from isolated mice islets 107

Figure 25: Showing coixol exerts an exclusive glucose-dependent insulinotropic effect in βTC-6 109

Figure 26: Effect of compounds on luminol enhanced oxidative burst using zymosan activated whole blood, readings presented as mean ± SD of three determinations 112 Figure 27: Cytotoxicity of compounds against MCF-7 (Breast cancer) cell lines 113

Figure 28: Preferential cytotoxicity of betulinic acid (5) and isolated pure

compounds against pancreatic cancer (PANC-1) cell line 115

Figure 29: Preferential cytotoxicity of betulinic acid (5) and isolated pure

compounds against pancreatic cancer (PSN-1) cell lines 116

Figure 30: Preferential cytotoxicity of betulinic acid (5) against pancreatic

cancer (PANC-1) cell line in dose dependent manner 117

Figure 31: DPPH radical scavenging activity of tambulin (9) and standard BHT 118

Figure 32: Urease inhibitory concentration and percentage inhibition of

tambulin (9) and standard thiourea 119

CHAPTER 1 INTRODUCTION

1.1 General introduction

Nepal is known as the country of green forest. Since very past there was a saying that Hariyo Ban Nepalko Dhan (Green forest is the wealth of Nepal). Nepal is also a country of diversity from social, cultural, geographical as well as flora and fauna features. Nepal’s contrasting feature basically emanate from its diverse geographical variation. The rich geographical variation of Nepal has caused the wider variation in biodiversity. Biodiversity commonly denotes the variety of species and the multiplicity of various forms of life (Bhattarai, 1991). However, in the context of this study diversity refers to varieties of plants including medicinal plants available in diverse ecological regions of Nepal. Nepal Himalayas is known as a rich source for valuable medicinal plants since vedic periods.

Plants have a complex chemical defense system, which is based on the production of a large number of chemically diverse compounds. These medicinal plants possess to have important medicinal values as mentioned in Ayurveda. Ayurvedic medical system originated and developed in the Indian sub-continent, which is perhaps the oldest traditional medicinal system in the world having its origin in the vedic period (1500-900 BC) (Borthakur, 2008). There are two types of Ayurvedic physicians; Vaidya and Kaviraj, in Nepal. Vaidya are trained in the ayurvedic colleges and universities and Kaviraj learn the knowledge and skill of the profession from their father or from their gurus (Ragavan, Surulinathi & Neelakandan, 2012). Indigenous and local communities have been using traditional and indigenous knowledge for centuries under local laws, customs and traditions (Bhattarai, 1992) in practicing medicinal plants.

Medicinal plants are those that have recognized for medicinal use. They range from those used in the production of mainstream pharmaceutical products and in herbal medicine preparations (Odebiyi & Sofowora, 1978). Plants with such medicinal quality are available all over the world but differ in types and nature. They also differ in their recognition as well as in knowledge and practice in different parts of the world.

The knowledge of medicinal plant has been continuously handing over from generation to generation. The major medicinal plants systems being practiced in Nepal are allopathic, homeopathic, ayurvedic, tibetan, unani and traditional faith healing (Russell, 2002). The importance of these medicinal plants in human life has generated a lot of interests into researches on their effects on human and livestock. Traditionally the medicinal plants have been used for the treatment of diseases such as, asthma, tuberculosis, dysentery, hyperglycemia, cancer, fever, intestinal complaints, sleep disturbances and inflammation (Russell, 2002). In developed countries, demand of medicinal plants or herbs largely reflects the growing interest of consumers in natural health enhancement agents, whereas in developing world, because of limited availability and high cost of modern medicines and traditional beliefs, the medicinal plants continued to be used in medicinal practices (Kalisdha, Balasubramani, Surulinathi & Amsaveni, 2013). Large number of chemical compounds are derived and isolated from plants, animals and microorganisms. Natural compounds such as quinine from Cinchona bark, morphine and codeine from the latex of the Opium poppy, digitoxin from digitalis leaves, atropine and hyoscine from species of the solanaceae continue to be in clinical use.

Natural compounds, also known as secondary metabolites, isolated from plants have therapeutic value such as cyclosporine (immunosuppression), mevinolin (hypercholesterolaemia), avermectin (parasitic disease), artemisinin (malaria), vinblastine, vincristine and taxol (cancer) (Egwaikhide, Okeniyi & Gimba, 2007). Among them cancer is the uncontrolled growth of cells coupled with malignant behavior, invasion and metastasis. Cancer is thought to be caused by the interaction between genetic susceptibility and environmental toxins. There are five major histological classes of cancer such as carcinoma, sarcoma, myeloma, leukemia and lymphoma (Awale & Lu, 2006). Among the several types of cancer, pancreatic cancer is the most serious form of cancer that shows resistance to almost all known chemotherapeutic agents because of resistance of the cells to apoptosis. Almost all patients of pancreatic cancer develop metastasis and die within a short period of time after the diagnosis. The survival rate of this cancer is five year which is the lowest among the survival rates of all other kinds of cancer (Awale et al., 2006). This lowest level of survival is associated with till known anticancer drugs are completely ineffective for this cancer because human pancreatic cells are known to exhibit marked

2 tolerance to nutrition starvation that enables them to survive for prolonged period of time even under extremely nutrient deprived conditions. It is hypothesized that elimination of tolerance to nutrient deprived conditions might be an approach for the treatment of pancreatic cancer (Awale, Feng, Onozuka, Esumi, Tezuka & Kadota, 2008). Pancreatic cancer cell can tolerate to nutrient starvation by austerity and angiogenesis (Thygesen, Thulin, Mortensen, Skibsted & Molgaard, 2006). In this regard, in order to discover drugs against pancreatic cancer different branches of chemistry have been actively engaged with. The present study is also the result of an inspiration from the discovery of several drugs from natural sources.

Cancer Ischemia Austerity 1 Angiogenesis 2

Hypoxia Response Metabolism Change

Glycolysis Angiogenesis Tolerance to nutrient starvation Get nutrient supply

Survival

Figure 1: Cell survival under insufficient blood supply (Source: Esumi et al., 2006)

Targeting Cancer Cells Tolerance to Nutrient Starvation

Anti-Austerity Strategy

Compounds

Ordinary medium

- Toxicity + Toxicity

Nutrient Deprived Medium Conventional drugs

- Toxicity + Toxicity

Invalid New strategy

Figure 2: Preferential cytotoxicity under nutrient deprived conditions (Source: Esumi et al., 2006)

This study follows the path of austerity hypothesizing that it brings changes in metabolism of cells that enables the cells to tolerate nutrition starvation which ultimately helps in cell survival. In such nutrition deprived condition the isolated natural compounds from the potent plant extract may inhibit the proliferation of pancreatic cancer cells. It may enhance the secretion of insulin through pancreatic beta (β) cells which directly contributes in controlling diabetes. This indicates the positive relationship between pancreatic cancer and diabetes. Therefore curing pancreatic cancer through isolated natural compounds in austerity condition also contributes in curing diabetes.

American Society of Clinical Oncology (ASCO) annual meeting highlighted and supported the positive association between pancreatic cancer and diabetes. It is further supported that diabetes appears to be a moderate risk factor for pancreatic cancer with 40 percent higher risk seen in diabetic than non-diabetic patients (Everhart, 1995 & Huxley, 2005). It shows there is unique relationship between pancreatic cancer and diabetes. Those with duration of diabetes of 2-8 years were at highest risk being 1.8 times as likely to develop pancreatic cancer as non-diabetics. No increased risk was observed for those with the longest duration of diabetes greater than nine years (Everhart, 1995 & Huxley, 2005). Most of the anticancer drugs are found as antioxidants too. Oxidative stress causes damage to many components of human cells such as proteins, lipids, and DNA and is involved in carcinogenesis. Nutrients with antioxidant properties may protect against oxidative stress which further prevents the patient from pancreatic cancer (Han, Ye & wang, 2013). Many medicinal plants contain large amount of antioxidants such as polyphenols, vitamin C, vitamin E, β-carotene, lycopins, lutin and other carotenoids which play important roles in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxide. The phytoconstituents which are phenols, anthraquinones, alkaloids, glycosides, flavonoids and saponins are antibiotic principles of plants (Arkemase, Kayode & Ajiboye, 2011).

People have been using medicinal plants since ancient times to treat and manage diabetes mellitus in traditional medical systems in many cultures throughout the world. Medicinal plants are continuously playing an important role in the management of diabetes mellitus, especially in developing countries even today, particularly in those places where many people do not have access to conventional antidiabetic therapies

(Kumar, Dhiman, Choudhary & Chikara, 2014). In developed countries the use of antidiabetic herbal remedies is reported to have been declining since the introduction of insulin and synthetic oral hypoglycemic agents during the early part of the twentieth century. Diabetes mellitus is one of the common metabolic disorders. Almost 1.3 percent of the population is suffering from this disease throughout the world and number of diabetic patient is increasing by seven percent per year. Insulin and oral hypoglycemic agents like sulphonylureas and biguanides are still the major players in the management but there is quest for the development of more effective antidiabetic agents (Zhang & Sun, 2015). Scholars in natural product chemistry are carrying out different research on exploring effective anticancer and antidiabetic agents (Kalauni, Choudhary, Shaheen, Manandhar, Rahman, Gewali & Khalid, 2001). This study is also one of the attempts made in exploring anticancer agents in the field of natural product chemistry.

In the present study fifty medicinal plants were collected from the different regions of Nepal based on knowledge provided by the ethno-botanical users and traditional healers. These medicinal plants were screened for several bioassays such as phytochemical tests, antioxidant activities, and preferential cytotoxicity against pancreatic cancer cell line PANC-1. This screening was focused on pancreatic cancer cell line PANC-1 because till now known anticancer drugs are totally ineffective for treatment of pancreatic cancer and several studies have explored the relationship between diabetes and pancreatic cancer which is a unique relationship (Li, Yeung, Hassan, Konopleva & Abbruzzese, 2009). Diabetes is thought to be both a potential cause and effect of pancreatic cancer. In order to better understand these diseases and how they are associated, more research needs to be done. It is also found that pancreatic cancer occurs with increased frequency among persons with long-standing diabetes. It has been proved from a practice of diabetes drug. Diabetic patients who had taken metformin had a significantly lower risk of pancreatic cancer compared with those who had not taken metformin. This difference remained statistically significant when the analysis was restricted to patients with a duration of diabetes >2 years or those who never used insulin (Li et al., 2009). Therefore, the Scoparia dulcis and Bridelia retusa were selected, for the purpose of this study, as the potent plant for isolation and identification of active compounds against pancreatic cancer which ultimately controls diabetes.

1.2 Rationale

As mentioned earlier Himalayan country Nepal is rich in medicinal, endemic and poisonous plants. These plants might have a number of bioactivity such as antibacterial, antidiabetic, antioxidant, anti-cancer etc. However, the information based on research work is limited in this area. Some biochemical analysis has been done in some plants outside the country. And within Nepal only a few ethno botanical works related to listing of the plant name with its description and uses have been done. This can be found in different publications including master’s dissertations at Central Department of Botany and Chemistry at Tribhuvan University. But important is to find out the biochemical uses of such plants for the human welfare on the one hand and conservation and sustainable use of such plants species on the other. Nepalese medicinal plants often show potent antioxidant, antidiabetic, anticancer activity and can be used for the management of various ailments. These anticancer drugs are used for treatment of different cancer diseases. Among the several types of cancer, pancreatic cancer is the most serious form of cancer that originates in the tissue of the pancreas and shows resistance to almost all known chemotherapeutic agents due to resistance of the cells to apoptosis. Pancreatic cancer is found as the lowest five year survival rates and also one of the major health problems that remains unresolved till now as mentioned earlier.

Sometimes diabetes seems to be an early manifestation of pancreatic cancer. Therefore, it is important to identify whether diabetes is an independent risk factor for pancreatic cancer or it is a consequence of it. Pancreatic cancer progresses without signiﬁcant early symptoms and is generally diagnosed at late stages. Diabetes mellitus is one of the common metabolic disorders. Almost 1.3 percent of the population is suffering from this disease throughout the world and number of diabetic patients is increasing by seven percent per year (WHO, 2004). Insulin and oral hypoglycemic agents like sulphonylureas and biguanides are still the major players in the cure of diabetes. There is quest for the development of more effective natural anti-diabetic agents because the insulin and hypoglycemic have relatively larger complications. Recently, some medicinal plants have been reported to be useful in diabetes and have been used empirically as antidiabetic and antihyperlipidemic remedies. Despite the presence of known antidiabetic medicine in the pharmaceutical market, diabetes and the related complications continued to be major medical problems.

Antihyperglycemic effects of these plants are attributed to their ability to restore the function of pancreatic tissues by causing an increase in insulin output or inhibit the intestinal absorption of glucose or to the facilitation of metabolites in insulin dependent processes. Therefore, the isolation and characterization of compounds against pancreatic cancer has significant role in diabetes as well.

1.3 Objectives

1.3.1 General objective

The general objective of this study is to screen some selected Nepalese medicinal plants collected from different regions of Nepal through preferential cytoxicity against PANC-1 cell and antioxidant activity by DPPH radical scavenging bioassay methods for isolation and structural elucidation of active compounds from those selected plants (Scoparia dulcis and Bridelia retusa) and their biological activities against diabetes, pancreatic cancer and antioxidant potential.

1.3.2 Specific objectives

The specific objectives of this study are as follows: a) To screen some selected medicinal plants for antioxidant and preferential cytotoxicity against pancreatic cancer cell (PANC-1) collected from different regions of Nepal and to isolate active compounds. b) To carry out phytochemical study on Scoparia dulcis and Bridelia retusa of Nepalese origin for isolation of bioactive secondary metabolites against pancreatic cancer and diabetes. c) To test antidiabetic, antioxidant, anticancer, immunomodulatory and antiurease activity of isolated compounds. d) To recommend active compounds for drug discovery processes.

1.4 Hypothesis

Medicinal plants collected from different regions of Nepal, as recommended by ethno- botanical users and traditional healers, are rich in secondary metabolites with bioactive constituents such as, antidiabetic, antioxidant, anticancer and immunomodulatory which can be used in drug discovery process against disease like pancreatic cancer that ultimately leads to diabetes or vice versa.

CHAPTER 2

LITERATURE REVIEW

This chapter deals with the previous works done by various scholars particularly focusing on the collected medicinal plants for this study which were recommended by ethno-botanical users, traditional healers and old people having experiences on such plants which are useful in curing different diseases including jaundice and diabetes which ultimately lead to pancreatic cancer. The following sections review the previous works in the order of plant selection basis; first selection first review.

2.1 Oxalis corniculata

The Oxalis corniculata, locally known as Chariamilo, is usually available in many parts of Nepal. It belongs to family Oxalidaceae. The plant collected for the purpose of this study was from Syangja district. It has been traditionally used by people of rural community when they suffer from stomachache. Ibrahim, Hussain, Imran and Mahoob (2013) reported that the ethanol extract of the plant contain flavonoids, alkaloids, tannins and phenols. The report further mentions that a new flavonoid glucoside was isolated from the ethyl acetate soluble fraction of the whole plant along with the luteolin-7-O-β-D-glucoside and β-sitosterol-3-O-β-D glucoside, which is reported for the first time. Some other scholars have further tested such compounds against the microorganism. Mukherjee, Koley, Berman, Mitra, Datta, Ghosh, Paul and Dhar (2013) have tested the compounds and reports that the extract exhibited numerous pathogenic bacteria like Staphylococcus aureus, Escherichia coli. The compounds such as β-sitosterol, betulin, 4-hydroxybenzoic acid, ethyl gallate, 5-hydroxy-7,8-dimethyl flavones, 5-hydroxy- 3’,4’,6,7,8-pentamethoxyflavone, 7,5-dimethoxy-3,5,2-trihydroxy flavones, 4’,5’- dihydroxyl-3,6,7-trimethoxy flavone, apigenin-7-O-β-D-glucoside and 3’,5,7-trihydroxy-4-methoxyflavon-7-O-β-D-glucopyranoside (Sayani, Hemanta, Soumik, Soma, Sanjukta, Santinath & Pubali, 2013).

Betulin Ethyl gallate

2.2 Drymaria diandra

The plant Drymaria diandra, locally known as Abhijalo, is usually found in shady and moist place in many regions of Nepal. It belongs to family Caryophyllaceae. The plant was collected from Chitwan district. It has been traditionally used by the people during their nose problems (sinusitis). The stem of Drymaria diandra were evaluated for their phytochemical constituents like total phenols, orthodihydric phenol, flavonols, tannins and antioxidant activity against 2,2-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radical, nitric oxide radical and anti lipid peroxidation activity. Two compounds were isolated by repeated column chromatography like 6-carboxymethyl- 5,7,4’-trihydroxyflavones and 1-O-β-D-glucopyranosyl(2S,R,4E,8E)-Z-N-(2’-hydrox- ypalmitoyloctadecasphinga-4,8-dienine. Four compounds were isolated from Drymaria diandra. The compounds were identified as 3-acetyloleanolic acid, cordatanine, β-sitosterol and β-daucosterol by spectral analysis (Xueqiong, Meihong, Yabin & Zhongtao, 2005).

3-acetyloleanolic acid

2.3 Melia azedarach

The plant Melia azedarach, commonly known as Bakaino, is usually found in many parts of Nepal. It belongs to family Meliaceae. The sample plant collected for this

9 study was from Chitwan district. This plant has been traditionally used as insecticides to kill insects. Hexane extract of the fruits of Melia azedarach Linn exhibited cytotoxic activities against leukemia (HL 60), lung (A 549), stomach (Az 521), and breast (SK-BR-3) cancer cell lines with IC50 values in the range of 2.9-21.9 µg/mL. Three new limonoids, 3-deacetyl-4’-demethylsalanin (5,1), 3-deacetyl-28-oxosalannin and 1-detigloylohchinolal, along with 16 known limonoids and one known triterpenoid were isolated from hexane soluble fraction (Xin, Masahiro, Yasuhiro, Takashi, Jie, Motohiko & Rima, 2014).

2.4 Cyperus rotundus

The plant Cyperus rotundus, locally known as Mothe jhar, is found in different regions of Nepal. It belongs to family Cyperaceae. The plant collected during the sample collection of this study was from eastern part of Chitwan district. It has been traditionally used when people suffer from pain and vomiting. Hashmat, Sofi, Aziz and Azad (2014) reported Cyperus rotundus, a cosmopolitan weed, is found in all tropical subtropical and temperate regions of the world. In India it is commonly known as Nagarmotha and it belongs to the family of Cyperaceae. The major chemical components of this herb are essential oils, flavonoids, terpenoids, sesquiterpenes, cyprotene, cyperene, aselinene, rotundene, valencene, cyperol, gurjunene, trans- calamenene, cadalene, cyperotundone, mustakone, isocyperol and acyperone. It has been already reported that it possesses pharmacological activities such as, anthelminthic, analgesic, antiinflammatory, antidysenteric, antirheumatic activities. Antimicrobial activity showed that Staphylococcus aureus was the most inhibited bacteria for the whole essential oil (Ismahen, Koubaier, Ahmed, Herve, Moncef & Nabiha, 2014).

Ismahen et al. (2014) isolated thirty four compounds from this plant and the main constituent were cyperene (14.78%), β-cyperone (14.41%), α-cyperone (12.57%) in cyperus rotundus Wen. and the relative contents of ten main constituents in Cyperus rotundus collected from Shandong and Hainan were different.

β-cyperone α-cyperone

2-hydroxy-4-methoxy-1,2,3,4-tetrahy- 3,4-dihydroxyphenyl-1,2,3,4- dronaphthalen-1-yl benzene-1,2-diol tetrahydronaphthalene-1,3-diol

2.5 Cissampelos pareira

The plant Cissampelos pareira is locally named as Batulpate. It is cosmopolitan in availability. It belongs to family Menisermaceae. The plant was collected from Chitwan district for the purpose of this study. It has been traditionally used in different communities for the treatment of headache. GC-MS analysis of the petroleum ether extract showed the presence of ten compounds of which five are nitrogenous compounds. Chloroform extract of Cissampelos pareira showed the presence of ten compounds of which eight are nitrogenous compounds. Similarly, MeOH extract of C. pareira showed the presence of eight compounds of which seven are nitrogenous in nature. Some of the nitrogenous compounds identified from this plant are aziridine, azocine, boraneamine, 1-(2-(2-hydroxy ethoxy) ethyl piperazine, 3-[1-aziridinyl] propoxy]-2,5-dimethyl pyrazine (Thavamani, Mathew & Dhanabal, 2014).

Cissampelos pareira was traditionally used as an antidiabetic agent in streptozotocin- nicotinamide induced diabetic male mice. Antidiabetic effect of aqueous extract of C. pareira leaves was evaluated at 250 mg/kg and 500 mg/kg body weight dose in male albino mice over the period of 14 days. Random blood glucose level and body weight were observed periodically. No significant changes were observed in the body weight and organs. C. pareira was capable in reducing diabetic attritions so it might be a valuable candidate for diabetes treatment (Yadav, Thomas, Shiny, Srivastav, Rai & Mishra, 2013).

Azocine Aziridine 1-(2-hydroxy-2-ethoxy) ethyl piperazine piperazine

Caroline (2013) reported that percent phagocytosis of peritoneal macrophages was significantly enhanced in normal and hyperglycemic Cissampelos pareira Linn. leaf extract treated rats. The result showed C. pareira aqueous leaf extracts play an important role in stimulation of immune response. Singh, Duggal and Katekhaye (2010) reviewed that Cissampelos pareira was a significant medicinal plant of herbal materia medica. It was used in the treatment of wide range of disease in traditional medicine Ayurveda and western herbalism (Thavamani et al., 2014). The review summarises ethnopharmacological investigations carried out on the plant with special reference to isoquinoline alkaloids.

2.6 Coccinia grandis

Coccinia grandis Linn, commonly known as Kunruk, is usually available in many regions of Nepal. The plant belongs to family Cucurbitaceae. The plant collected for the present study was from Chitwan district. Traditionally, it has been used by the peoples when they suffer from leprosy. Plant is a perennial dioecious herb with heteromorphic sex chromosomes has a quality of model plant for analysis of sexual evolution in angiosperms. Screening of genomic DNA with RAPD primers was used for sex diagnosis and gender specificity of C. grandis (Bhowmick, Kumar, Satyabrata, Sanghamitra, Sumita & Raj, 2014).

2.7 Euphorbia hirta

The plant Euphorbia hirta is locally known as Dudhejhar. It is mainly available in terai region of Nepal. The plant belongs to family Euphorbiaceae. The plant collected for the purpose of this study was from Chitwan district. The plant has been traditionally used for the treatment of skin diseases. The phytochemical screening of methanolic extract of stem of Euphorbia hirta revealed the presence of triterpenoid. The isolated compound from this plant have been established as 13-α-methyl-27- norolean-14-en-3β-ol namely teraxerol. The compound teraxerol showed the anti- asthmatic activity carried out on histamine induced bronchospasm in guinea pigs significantly inhibited the contractile effect of histamine (Saxena & Tiwari, 2014).

Qualitative phytochemical test and quantitative estimation of total flavonoid and phenol content was carried out on ethanol, methanol and water extract of E. hirta whole plant. TLC analysis showed presence of quercetin, ferulic acid and gallic acid in total flavonoid fraction of E. hirta whole plant (Bigoniya, Agrawal & Verma, 2013).

Presence of myricitrin, quercitrin, kaempferol, luteolin and gallic acid like polyphenolic compounds in E. hirta indicated the potential scavenging effect as important determinant of wound healing property.

Bigoniya et al. (2013) reported that Euphorbia hirta (Euphorbiaceae) has antimicrobial, antifungal, antiviral, antiinflammatory, antiarthritic and antioxidant effect with presence of polyphenolic and flavonoid compound lead to us to evaluate the wound healing activity of enriched flavonoid fraction (Ping, Yuet, Chen & Sasidharan, 2013).

Kaempferol Luteolin

2.8 Cynodon dactylon

Cynodon dactylon, locally known as Dubo is usually found in almost all ecological regions of Nepal. The plant belongs to family Poacceae. In this study, the plant was collected from Chitwan district. It has been traditionally used when people suffer from stomachache. It is commonly known as Doob in India is a weed and has been regarded to posses various medicinal properties. It possesses much therapeutics well as decorative values and other unexplored potentials. The aqueous plant extract is used as anti-inflammatory, diuretic, antiemetic and purifying agent. C. dactylon has been used an antidiabetic agent in traditional system of medicine in India. Aqueous extract of C. dactylon revealed the presence of alkaloid and carbohydrates in chloroform extract, alkaloid, carbohydrates, saponins, tannins and terpene in methanol extract, glycosides, carbohydrates, saponins and tannin in ethanol extract and carbohydrates and fixed oils in petroleum ether extracts. The aqueous extract contained carbohydrates and tannins (Jurry, Gupta & Mishra, 2013).

Solanki and Nagori (2013) reported that Cynodon dactylon possessed various medicinal properties such antiarrhythmic, anticonvulsive, antidiabetic, antidiarrheal, antiepileptic, antihypertensive, anti-inflammatory, antiulcer and many more (Solanki

13 et al., 2013). The whole plant affords carbohydrates, alkaloids, flavonoids, phytosterols, β-sitosterol, glycosides, proteins and triterpenes.

Apigenin Luteolin

Jurry et al. (2013) reported that the qualitative phytochemical analysis of Cynodon dactylon Linn showed the alkaloids, anthraquinone, flavonoids, saponins, steroids, terpenoids and tannins as main constituents of the aqueous and alcoholic extract of whole plant.

Vitexin Orientin

2.9 Ageratum houstonianum

Ageratum houstonianum is locally known as Gandhe jhar. It is mainly found in all regions of Nepal. The plant belongs to family Asteraceae. It has been traditionally used by local peoples for killing insects. Rizvi, Danish, Khan, Sibhghatulla, Deboshree and Kamal (2014) have reported anticancer compounds from Ageratum houstonianum 1,2-benzenedicarboxylic acid bis(2-ethylhexylphenyl) ethanone and 6-vinyl-7- methoxy-2,2-dimethyl chromene isolated from methanolic extract of leaves of Ageratum houstonianum.

6-vinyl-7-methoxy-2,2-dimethyl chromene

The study reported that 1,4-cyclohexylphenyl ethanone isolated from this plant is a more efficient inhibitor of human MMP-2 and MMP-9 enzymes compared to the other natural compounds. Four pyrolizidine alkaloids (PA) were isolated from Ageratum houstonianum (Quijano, Calderon, Gomez, Federico & Edgar, 1985).

1,4-cyclohexylphenyl ethanone

2.10 Curcuma angustifolia

Curcuma angustifolia is locally known as Beshar. It is usually found in many parts of Nepal. It belongs to family Zingiberaceae. The plant was collected from Daman, Makawanpur district of Nepal. The plant has been traditionally used by the peoples when they suffer from stomachache and constipation. Rajila, Liji, Sindur and Suganyadevi (2013) reported that amylase was produced by Aspergillus niger utilizing Curcuma angustifolia as a carbon source in submerged fermentation. The effect of varying pH of the medium, temperature carbon and nitrogen sources on the production of α amylase was investigated. The maximum activity of α-amylase was recorded after seven days of submerged fermentation at pH 5 and room temperature 28 oC. The enzyme produced by Aspergillus niger can be used in industrial process after characterization. The maximum amylase activity was recorded as 345 U/ mg (Rajila et al., 2013).

2.11 Strychnos nux vomica

Strychnos nux vomica is locally known as Kuchila. It is usually available in many parts of Nepal, particularly in eastern part. It belongs to family Loganiaceae. The plant

15 was collected from Daman, Makawanpur districts for this study. It has been traditionally used by the peoples to kill feral mammals and rodents. Chen, Qu, Wang, Peng, Cai, Gao and Cai (2014) have reported that strychnine and brucine in the seeds of Strychnos nux vomica tested for toxicity and pharmacokinetics of TAF (Total Alkaloids Fraction) and MTAF (Modified Total Alkaloid Fraction) to know antitumor, analgesics and antiinflammatory activities (Fang, Chen, Ma, Zhang, Chi & Feing, 2013).

Strychnos alkaloids, strychnine and brucine have obviously inhibitory effect on HFLS- RA proliferation and brucine showed a better inhibitory effect than strychnine with the decreasing concentration (Fang et al., 2013). Different concentration of strychnos alkaloids showed inhibitory effect on (Fibroblast like Synoviocytes-Rheumatoid Arthritis) HFLS-RA (Patel, Duraiswamy & Dhanabal, 2012).

Strychnine Brucine

Brucine is an alkaloid derived from the seeds of Strychnos nux vomica Linn. Which have long been used as a traditional medicine for the treatment of hepatocellular carcinoma (HCC) in China (Shu, Mi, Cai, Zhang, Yin, Yang & Deng, 2013). Brucine is a source of antimetastasis activity against HCC.

2.12 Shorea robusta

Shorea robusta is popular plant locally known as Sal. It is usually found in many parts of hill and terai regions of Nepal. It belongs to family Dipterocarpaceae. The plant was collected from Chitwan district for the purpose of this study. The plant has been traditionally used by the peoples for treatment of different diseases and for healing wounds. This plant is not only available in Nepal but also in India and Bhutan. It is mostly found in the plains and lower foothills of the Himalayas including along the valleys. Shorea robusta has been traditionally used for various ailments.The leaves

16 and barks are used to treat wounds, ulcers, leprosy, cough, gonorrhea, earache and headache. The bark is also used for treat diarrhea, dysentery and vaginal discharges. The fruits are useful in tubercular ulcers, seminal weaknesses, burning sensation and dermopathy (Sharma, Payal & Dobhal, 2014). Shorea robusta contains ursolic acid and α-amyrenone, α and β-amyrin. Bark contains ursonic acid and oleanane, shoreaphenol. Seed contains hopeaphenol and leucoanthocyanidin. The isolation of β- amyrin, friedelin, β-sitosterol, and dihydroxyisoflavone from mature leaves was also reported (Rajesh, Dixit, Irchhaiya & Singh, 2013).

Friedelin Ursolic acid

β-sitosterol α-amyrin

Dihydroxyisoflavone Leucoanthocyanidin

2.13 Acacia catechu

Acacia catechu is locally known as Khayar which is usually found in the most parts of Nepal. It belongs to family Fabaceae. For this study the plant was collected from Chitwan district. It has been traditionally used by the peoples of local communities when they are suffered from stomachache and indigestion. Phytochemical studies were carried out in different parts of three Acacia species viz. A. catechu, A. nilotica and A. leucophloea using chromatogram and spectrophotometric analysis. Various extract like alcoholic, aqueous, hydroalcoholic were compared both qualitative and quantitative. The different extracts showed that they are rich sources of phenolic compounds. The invention relates to an Acacia catechu based health tea capable of alleviating stomatitis. The health tea has the advantages of good health care functions, and is capable of nourishing kidney invigorating spleen, and alleviating stomatitis (Sulaiman, Gopalkrishnan & Balachandran, 2014).

5-hydroxy-2-[2-(4-hydroxyphenyl) (2S,3S)-3,7,8,3',4'-pentahydroxyflavane acetyl]-3-methoxylbenzoic acid

4-hydroxyphenyl ethanol 3,3',5,5',7-pentahydroxyflavane

Acacia catechu stem bark extracts have been used traditionally as anti-inflammatory, immunomodulatory, hepatoprotective, antioxidant, antimicrobial and antitumor activities (Nutan, Manoj, Charlene, Shrestha, Rawat, Singh & Kumar, 2013).

2.14 Lyonia ovalifolia

Lyonia ovalifolia is locally known as Aanger which is usually distributed in many parts of the Nepal. The plant belongs to family Ericaceae.The plant was collected for

18 the purpose of this study was from Syangja district. It has been traditionally used for skin diseases and stomachache. Phytochemical studies on the branches and leaves of Lyonia ovalifolia yielded a new grayanane diterpenoid lyonin together with two known compounds (Wu, Li, Wang, Chen & Luo, 2011). Five new lignans ovalifolinins were isolated from the wood of Lyonia ovalifolia (Kashima, Yun, Sooklna, Kunuji, Lnoue & Ovafolinins, 2010).

Flavonoid compounds were isolated from the leaves of L. ovalifolia collected from various places in Japan were studied by paper chromatography. The major flavonoid components of the leaves of L. ovalifolia were quercetin 3-O-α-L-rhamnoside, quercetin 3-O-β-D-galactoside and quercetin-3-O-β-D-glucuronide, quercetin, P- coumaric and caffeic acids were also identified (Sakakibara, Hotta & Yasue, 1974).

p-Coumaric acid Caffeic acid

The leaves of Lyonia ovalifolia, supplied from Nepal, were isolated aliphatic higher hydrocarbons, esters, β-sitosterol, ursolic acid, oleanolic acid, maslinic acid, quercetin, eriodictyol, astilbin, β-sitosteryl β-D-glucoside, glucose and xylose.

Maslinic acid Eriodictyol

Astilbin Oleanolic acid

2.15 Pterocarpus santalinus

Pterocarpus santalinus Linn is locally known as Raktachandan which is usually available in many parts of Nepal. It belongs to family Fabaceae. The plant collected for the purpose of this study was from Chitwan district. The plant has been traditionally used for skin care and as cooling agent. Extensive literature survey showed that the plant was claimed to have antipyretic properties for management of fever. Phytochemical evaluation revealed the presence of tannins, flavonoids, terpenoids, steroids, alkaloids, glycosides, saponins and resin (Wu, Hwang, Chen, Ohkoshi, Kuo-Hsiung &Yang, 2011). One new phenanthrenedione, pterolinus, and one chalcone pterolinus was isolated from the heartwood extract of P. santalinus (Arokiyaraj & Perinbam, 2010).

Leaves of P. santalinus were exhaustively extracted in different solvents like hexane, ethyl acetate and methanol in ascending order of the polarity. All these extracts were subjected to antifungal screening and phytochemical analysis. MeOH extract of Pterocarpus santalinus (leaves) was evaluated for HPTLC finger print, phytochemical analysis and antioxidant activity. Leaf and stem bark extract of Pterocarpus santalinus (Fabaceae) showed great spectrum antibacterial activity against Gram positive and Gram negative organism (Manjunatha, 2006).

2.16 Desmostachya bipinnata

Desmostachya bipinnata Linn is locally known as Kush. It is usually available in many parts of Nepal. The plant belongs to family Poaceae. The plant collected for the purpose of this study was from Syangja district. It has been traditionally used as medicine to treat diarrhea, indigestion and asthma. The plant is called Kusha in Sanskrit a sacred grass which was used extensively in India during Vedic period. It is used in India as traditional Indian medicine to treat microbial infection in combination with other herbs. Isolated compound such as β-sitosterol-D-glucopyranoside was the bioactive compound identified to have the best antimicrobial activity (Rahate, Rajasekaran & Manju, 2011).

D. bipinnata Linn. has been traditionally used to treat various disorders such as asthma, kidney stone, diarrhea and wound healing. Phytochemical screening and chromatography revealed the presence of glycosides, steroids, flavonoids, coumarins and alkaloids (Singh, Vikas & Bhandari, 2014). Subramanian, Manikandaraja and

Sivasubramanian (2014) have reported that the methanolic extract exhibited scavenging activity towards superoxide and ABTS due to the presence of relatively high total phenol and flavonoid content. New xanthenes were isolated from the methanolic extract of D. bipinnata through repeated silica gel and octadecyl silica gel column chromatography. The structure of compound was determined to be 2,6- dihydroxymethoxy-3H-xanthen-3-one.The flavonoid compound 4’-methoxyquercetin- 7-O-glucoside isolated from ethylacetate fraction might be useful as a chemopreventive agent for peptic ulcer in H-pyloric infected individuals, after its clinical valuation (Guleria, Tiku, Singh, Koul, Gupta & Rana, 2013).

Isobornyl acetate Camphene

2.17 Aegle marmelos

Aegle marmelos, commonly known as Bael, is usually distributed in many parts of Nepal. It belongs to family Rutaceae. The plant was collected from Chitwan district for the purpose of this study. The plant has been traditionally used as medicine to treat constipation and gastrointestinal problems. It is an important medicinal plant in the traditional Indian system of medicine, the Ayurveda. The extract is also useful in ophthalmia, deafness, inflammations, catarrh, diabetes, and asthmatic complaints. The fruits are used in treating diarrhea, dysentery, stomach ache, and cardiac ailments. Scientific studies have validated many of Baels ethnomedicinal properties and its potential antimicrobial effects, hypoglycemic, astringent, antidiarrheal, antidysenteric, demulcent, analgesic, antiinflammatory, antipyretic, wound healing, insecticidal, and gastroprotective properties (Baliga, Thilakchand, Rai & Venkatesh, 2013).

Leaves, fruits, stem, bark and roots of Aegle marmelos have been used in ethnomedicine to exploit its medicinal properties including astringent, antidiarrhoel, antidysentric, demulcent, antipyretic, antimicrobial, anticonvulsant, hepatoprotective, antioxidant, and analgesic, wound healing and antiinflammatory activities. Recent advances prove that compounds isolated from Bael have been active against several

21 major diseases including cancer, immunomodulatory, cardiovascular diseases (Shrinath, Ramdas, Ponadka, Suresh & Ponemone, 2013). Major phenolics determination using RP-HPLC in analyzed species were gallic acid, chlorogenic acid, p-hydroxy benzoic acid, caffeic acid, vanillic acid, syringic acid, p-coumaric acid and ferulic acid (Baliga et al., 2013).

Syringic acid 4-Hydroxy-3-methoxy benzoic acid

Ferulic acid Chlorogenic acid

2.18 Mahonia napaulensis

Mahonia napaulensis, locally known as Jamanemandro, is usually available in many parts of Nepal. It belongs to family Berbeidaceae. The plant was collected from Kathmandu district for the purpose of this study. It has been traditionally used for dyeing. The antifungal activity of the methanolic extract of Mahonia napaulensis leaves was evaluated with four species of common pathogenic fungi such as Colletotrichum capsici, Leptosphaerulin trifoli, Alternaria brassicicola and Helminthosporium solani. The antifungal textile dyeing was also carried out with aqueous extract of stem and leaves of Mahonia and the dyed fabric was tested against fungal species Trichoderma for its antifungal activity in vitro. Mahonia extract showed substantial antifungal activity of 83.33 percent for Leptosphaerulina trifolii and Alternaria brassicicola by 80 ppm dose in 24 h and 46 percent antifungal activity in Mahonia dyed pieces in broth against Trichoderma (Nguyen, Tran, Hoang, Chau, Ninh & Phan, 2009).

From the wood of Mahonia napaulensis, two bisbenzyl isoquinolines homoaromoline and isotetrandrine were isolated by using various chromatographic techniques.

2.19 Phyllanthus emblica

Phyllanthus emblica, locally known as Amala, is widely distributed in many parts of Nepal. It belongs to family Phyllanthaceae. The plant was collected from Chitwan district for the purpose of present study. It has been traditionally used to treat stomachache and diarrhea. The plant posses a vast ethnomedicinal history and represents a large group of phytochemical reservoir of medicinal uses. It is one of the ingredients used from time immemorial in various ancient literatures like in "Ayurveda" and "Charka Samhitha" as a potential ingredient for various ailments. The fruit is studied for various phytochemical constituents like quercetin, gallic acid, tannins, flavonoids, pectin and Vitamin C and also contains various polyphenolic compounds (Deepak & Gopal, 2014).

Many pharmacological studies also have exhibited proven results for antioxidant, anticarcinogenic, antitumor, antigenotoxic, anti inflammatory activities supporting its traditional uses. GC-MS analysis of ethyl acetate extract of the bark portion of the P. emblica could be a possible source of extinguish therapeutically useful products (Gupta & Gupta, 2014).

Vitamin c Gallic acid Quercetin

HPLC analysis, showed gallic acid and vanillic acid are major phenolic compounds isolated from methanolic and ethanolic extract. Both EPE and MPE inhibited tyrosinase activity stronger than the ethanolic extract of P. emblica fruit (Zhang, Liang, Zhao, Hong, Wang & Cen, 2013).

Vanillic acid Gallic acid 23

Ten compounds were isolated from Phyllanthus emblica and have been determined as methyl gallate, quercetin, quercetin-3-O-α-L-rhamnoside, naringenin-7-O-β-D-glucopyranoside, 3,4,8,9,10-pentahydroxydibenzopyran-6-one, 3,4,3'-tri-O-methylellagic acid, lupeol, lup-20,29-en-3β,3-O-diol, betulin and gallic acid (Sripanidkulchai & Junlatat, 2014).

2.20 Berberis3,4,8,9,10 aristata-pentahydroxydibenzo 3,4,3'-tri-O-methylellagic acid pyran-6-one

2.20 Berberis aristata

Berberis aristata, locally known as Chutro, is usually available in many parts of Nepal. The plant belongs to family Berberidaceae. The plant collected for the purpose of this study was from Kathmandu district. It has been traditionally used by the peoples of rural areas of Nepal when they suffer from stomachache and Jaundice. It is also known as Indian Berberi, Daruharidra, Daruhaldi, Darvi and Chitra. The plant is used as anti-pyretic, anti-bacterial, antimicrobial, anti-hepatotoxic, antihyperglycemic, anti-cancer, anti-oxidant and antilipidemic agent. B. aristata extracts and its formulations are also useful in the treatment of diarrhea, hemorrhoids, gynaecol disorders, HIV-AIDS, osteoporosis, diabetes, eye and ear infections, wound healing, jaundice, skin diseases and malarial fever (Srivastava, Khatoon, Ajay, Mehrotra & Pushpangadan, 2001).

Saied, Batool and Naz (2007) have evaluated the phytochemical, antidiabetic, and cytoprotective properties of Berberis aristata DC. (Berberidaceae) root extracts. Administration of ethanol extract of B. aristata roots in diabetic rats showed dose dependent reduction in hyperglycemia. The levels of serum total cholesterol, triglyceride, AST (Aspartate Aminotransferase), ALT (Alanine Aminotransferase), serum creatinine and blood urea were significantly decreased in diabetic rats when compared with diabetic control rats (Kakkar & Singh, 2007).

Berberine

Four alkaloids, pakistanine (1), 1-O-methylpakistanine, pseudopalmatine chloride and pseudoberberine chloride were isolated for the first time from Berberis aristata (Srivastava et al., 2001).

2.21 Tinospora sinensis

Tinospora sinensis, locally named as Gurjokolahara, is widely available in many parts of Nepal. It belongs to family Menisermaceae. The plant is collected from Syangja district. It has been traditionally used as medicine when people suffer from vomiting. Their belief is that it stops accessive bleeding after child birth. The plant is large, glabrous, deciduous climbing shrub belonging to the family Menispermaceae. Some bioactive polyphenol compounds are absorbed from the gut in their native or modified form. Polyphenols exhibit a wide range of biological effects as a consequence of their antioxidant properties (Seghal & Majumdar, 2014).

4-methylheptadec-6-enoic acid ethyl ester

Chloroform extract yielded an amorphous substance, m.p. 90-4 °C, water extract yielded three fractions on an alumina chromatographic column. One of the fractions was physiologically active (Myocardium) and yielded with orange red crystals, m.p. 114-15 °C, with picric acid, yellowish brown crystals, m.p. 121-2 °C and with auric chloride, brown crystals, m.p. 170-3 oC (Singh, Pandey, Srivastava, Gupta, Patro & Ghosh, 2003).

3-hydroxy-2,9,11-trimethoxy-5,6-dihydro isoquino[3,2]-isoquinolinylium

2.22 Cuscuta reflexa Cuscuta reflexa, locally known as Aakashbelli, is widely available in many parts of Nepal. It belongs to family Convolvulaceae. It is a golden yellow, leafless, perennial, parasitic herb which was collected from Syangja district for the purpose of this study. It has been traditionally used by local peoples when they suffer from jaundice. Cuscuta reflexa has been investigated for antispasmodic, hemodynamic, anticonvulsant, antisteroidogenic, antihypertensive, muscle relaxant, cardio tonic, antiviral, antibacterial, antioxidant, cholinergic, diuretic and hair growth activities (Paudel, Prabodh, Shrestha & William, 2014). Many chemical constituents have been isolated from C. reflexa such as cuscutin, amarbelin, β-sitosterol, stigmasterol, kaempferol, dulcitol, myricetin, quercetin, coumarin and oleanolic acid (Patel, Sharma, Chauhan & Dixit, 2012).

Dulcitol Myricetin

Oleanolic acid Kaempferol

Cuscutin Coumarin

Biological screening for antimicrobial activities did not show appreciable activity against either Gram-positive (Bacillus cereus and Staphylococcus aureus) or Gram- negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. Mitra, Chang and Yoo (2011) have reported that Kaempferol, a strong antioxidant, was extracted from the methanolic extract.

2.23 Leucas cephalotes

Leucas cephalotes, locally known as Bishmara, is usually available in shady place in many parts of Nepal. The plant belongs to family Ranunculaceae. The plant was collected from Syangja district for the purpose of this study. It has been traditionally used by local peoples for killing insects. Antariksh, Pradhan, Tyagi and Pradeep (2010) reported that Leucas cephalotes Roth. Spreng (Lamiaceae) is the well known herb in the Ayurvedic and Modern systems of medicine, to cure various disorders. Powdered plant material analyzed for the two major attributes, Pharmacognostic parameters and phytochemical characterization followed by the quantitative analysis of tannins and flavonoids. Antimicrobial activity on the toluene and methanolic extracts was also performed. The methanolic extract of Leucas Cephalotes has exhibited significant analgesic and anti-inflammatory effects, which were comparable with standard drugs (Baburao, Reddy, Rama, Parameshwar, Narsimha & Ravi, 2009).

L. cephalotes leaves were extracted by soxhlet extractor using different organic solvents like hexane, dichloromethane, methanol and ethylacetate. Among these tested organic extracts, hexane and methanolic extracts showed prominent antibacterial activity.

β- sitosterol Oleanolic acid

2.24 Drynaria propinqua

Drynaria propinqua locally known as Commeri, is highly distributed in shady place in many parts of Nepal. The plant belongs to family Polypodiaceae. The plant was collected from Syangja district for the purpose of present study. The plant has been traditionally used by local peoples for treatement of bone fracture and headache. Liu, Xiao & Fang (1992) reported four compounds isolated from the rhizomes of Drynaria propinqua. One of them was a new natural product, namely propinqualin, (-)- epiafzelechin-3-O-β-D-allopyranoside. The other three were 4-O-β-D-glucopyranosyl caffeic acid, β-sitosterol-3-O-β-D-glucopyranoside and sucrose. The rhizome of Drynaria propinqua contained propinqualin and (E)-4-O-β-D-glucopyranosyl caffeic acid was identified as epiafzelechin-3-O-β-D-allopyranoside (Saha, Guria, Singha & Kumarmaity, 2013). A new flavanol glycoside, (-)-epiafzelechin-3-O-β-D-allopyranoside was isolated from the rhizomes of D. propinqua (Liu, Xiao & Feng, 1994).

2.25 Tinospora cordifolia

Tinospora cordifolia, commonly named as Gurjogano, is usually available in many parts of Nepal. It belongs to family Menispermaceae. The plant was collected from Syangja district for the purpose of this study. It has been traditionally used by local peoples of rural community when they suffer from chest pain and jaundice. The plant is a genetically diverse, large, deciduous climbing shrub with greenish yellow typical flowers, found at higher altitude. A variety of active components derived from the plant like alkaloids, steroids, diterpenoid lactones, aliphatics, and glycosides have been isolated from the different parts of the plant body, including root, stem, and whole plant. Recently, the plant is reported for its medicinal properties like antidiabetic, antiperiodic, antispasmodic, antiinflammatory, antiarthritic, antioxidant, antiallergic, antistress, antileprotic, antimalarial, hepatoprotective, immunomodulatory and

28 antineoplastic activities. The plant possesses antioxidant, antihyperglycemic, antineoplastic, antistress, antidote, antispasmodic, antipyretic, antiallergic, antileprotic antiinflammatory, antihyperlypidaemia, Immunomodulatory properties. Bioprosp- ecting studies of Tinospora cordifolia have revealed three constituents they are cycloeuphordenol, cyclohexyl-11-heneicosanon and 2-hydroxy-4-methoxybenzaldehy- de (Sharma, Gupta, Singh & Batra, 2010).

2-hydroxy-4-methoxy -benzaldehyde

2.26 Centella asiatica

Centella asiatica, locally known as Ghottapre, is usually found in many parts of Nepal. It belongs to family Mackinlayaceae. It was collected from Kaski district. It has been traditionally used as medicine for brain stimulating and wound healing. Three new pentacyclic triterpenoids, named centella saponin, centella saponin J and centella saponin E, together with three known compounds were isolated from the whole plants of Centella asiatica. A variety of active constituents with wide range of pharmacological actions have been reported with Centella asiatica. It is reported that Centella asiatica bears significant analgesic and antiinflammatory activities (Shao, Yang, Gao, Cheng, Weng & Kong, 2014).

Dextropropoxyphene

Acetic acid and its derivatives are the most common triterpenoids in traditional popular medicinal herb Centella asiatica, and have been reported to possess various pharmacological activities such as antiinflammatory, anticancer and antidepressant (Shao et al., 2014).

2.27 Asparagus filicinus

Asparagus filicinus, locally known as Kurilo, is distributed in shady and moist places of different parts of Nepal. The plant belongs to family Asparagaceae. The plant was collected from Syangja district. It has been traditionally used as medicine against stomachache. Asparagusin was isolated from the roots of Asparagus filicinus. The compound exhibited a cytotoxic activity on PC12 cells. Three new steroidal saponins were isolated from the root of A. filicinus a folk medicine of Yunnan Province, China, used for the treatment of bronchitis, pneumonitis and cough. The structures of isolated compounds were established as sarsapogenin-3-O-β-D-xylopyranosyl (1,4)- β-D-glucopyranoside, sarsapogenin-3-O-β-D-xylopyranosyl, (1,4)α-L-arabinopyranosyl, (1,6)-β-D-glucopyranoside, and (25S)-5β-furost-3β,22,26-triol-3-O-β-D-xylopyranosyl, (1,4)α-L-arabinopyranosyl, (1,6)-β-D-glucopyranoside-26-O-β-D-glucopyranoside (Cong, Ye & Che, 2000).

Glucuronic acid

Two new spirostanoides, filiasparosides, one new furostanoside, filiasparoside, and one new ecdysterone, stachysterone, acetonide, together with six known steroidal saponins, asparagusin, filiasparoside, filiasparoside, aspafilioside, aspafilioside, and filiasparoside were isolated from the roots of Asparagus filicinus (Wu, Cheng, Zuo, Wang, Li, Zhang, Wang & Ye, 2010).

2.28 Justicia adhatoda

Justicia adhatoda, locally known as Asuro, is available in many parts of Nepal. The plant belongs to family Acanthaceae. The plant was collected from Chitwan district. It has been traditionally used as medicine against cough, cold and asthma. Justicia adhatoda Linn contains alkaloids like vasicine, vasicinone and deoxyvasicine. These alkaloids give the plant its expectorant activity, antispasmodic, antiseptic and antihelmintic properties. Alkaloids were extracted with methanol, quantified and identified by color reactions, thin layer chromatograms (TLC), high performance liquid chromatogram (HPLC) and fourier transform IR spectroscopy (FT-IR) using

30 vasicine as standard (Rashmi & Linu, 2012). α-naphthyl acetic acid and mannitol was used as elicitors to improve the productivity of useful metabolite, vasicine for archiving high concentration in Justicia adhatoda Linn. cell suspension cultures.

2.29 Litsea cubeba

Litsea cubeba, locally named as Sidharlo, is distributed in shady and moist places of different parts of Nepal. It belongs to family Lauraceae. The plant was collected from Syangja district. It has been traditionally used as medicine when they suffer from asthma, backpain and digestive ailments. The major compounds in L. cubeba essential oil were β-myrcene, D-limonene, eucalyptol, citronellal 3,7-dimethyl-2,6- octadienal and linalool, β-caryophyllene (You & Yan, 2013). Litsea cubeba oil was extracted with different organic solvent and theirs in vitro antioxidant activity were evaluated by DPPH tests. Base on the GC-MS peak area normalization method, the relative contents of main compounds were obtained. These results demonstrated the solvent extraction exhibited significant antioxidant activity similar to synthetic antioxidants BHA and large variance in the antioxidant result among the different solvent. With the high polar methanol as extracted medium, the oil had the most significant antioxidant activity. Twelve compounds were isolated from alcoholic extract of Litsea cubeba Lour. The compounds were 4,4-dimethyl-1,7-heptanedioic acid, (-) divanillyltetrahydrofuran, bis(2-ethylhexyl)phthalate, (+)-9-O-feruloyl-5,5- dimethoxy lariciresinol, N-methyl laurotetanine, isocorydine, dihydrodehydro- diconiferyl, N-trans-sinapoyltyramine, fumaric acid, trans-N-p-coumaroyl tyramine and decane (You et al., 2013).

trans-N-p-coumaroyltyramine Boldine

2.30 Oxalis corniculata

Oxalis corniculata locally known as Chariamilo, is usually distributed in shady and moist places in many parts of Nepal. The plant belongs to family Oxalidaceae. The plant was collected from Syangja district for the purpose of this study. It has been

31 traditionally used by the local and trival peoples when they suffer from stomachache and high fever. The compounds such as β-sitosterol, betulin, 4-hydroxybenzoic acid, ethyl gallate, 5- hydroxy-7,8-dimethyl flavones, 5-hydroxy-3’,4’,6,7,8-pentamethoxyflavone, 7,5-dimethoxy-3,5,2-trihydroxy flavones, 4’,5’-dihydroxy-3,6,7-trimethoxy- flavone, apigenin-7-O-β-D-glucoside and 3’,5,7-trihydroxy-4-methoxyflavon-7-O-β- D-glucopyranoside have been isolated from this plant (Mukherjee et al., 2013).

Betulin 4-hydroxybenzoic acid 5- hydroxy-3',4',6,7,8- Pentamethoxy flavone 2.31 Justicia adhatoda

Justicia adhatoda, locally named as Asuro, is widely available in many parts of Nepal. The plant belongs to family Acanthaceae. It was collected from Syangja district. It has been traditionally used as medicine to kill insects. Justicia adhatoda Linn contains alkaloids like vasicine, vasicinone and deoxyvasicine. These alkaloids give the plant its expectorant activity, antispasmodic, antiseptic and antihelmintic properties. Alkaloids were extracted with methanol, quantified and identified by color reactions, thin layer chromatograms (TLC), high performance liquid chromatogram (HPLC) and fourier transform IR spectroscopy (FT-IR) using vasicine as standard (Rashmi & Linu, 2012). α-naphthyl acetic acid and mannitol was used as elicitors to improve the productivity of useful metabolite, vasicine for archiving high concentration in Justicia adhatoda Linn cell suspension cultures.

2.32 Cleistocalyx operculatus

Cleistocalyx operculatus, locally known as Kyamuno, is often found in many parts of Nepal. It belongs to family Oxalidaceae. The plant was collected from Syangja district. It has been traditionally used when peoples suffer from muscular swelling. Chun-Lin, Xuan-Gan and Huang (2013) have reported that the antioxidant activity and the protective effect of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone

(DMC), the main compound from the buds of Cleistocalyx operculatus, on human umbilical vein endothelial cells against cytotoxicity induced by H2O2.

Yoon, Kim, Hwan-Won, Oh, Dao and Thuong (2012) have isolated 7-hydroxy-5- methoxy-6,8-dimethylisoflavone, 5,7-dihydroxy-6,8-dimethyldihydroflavonol, 2,7-dihydroxy-5-methoxy-6,8-dimethylflavanone, 4,2',4'-trihydroxy-6'-methoxy-3',5'-dimethylchalcone, 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone, 7-hydroxy-5-methoxy-6,8-dimethylfavanone, 2',4'-dihydroxy-3'-methyl-6'-methoxychalcone, 6-formyl-8-methyl-7-O-methylpinocembrin, (2S)-8-formyl-5-hydroxy-7-methoxy-6-methyl- flavanone, 5,7-dihydroxy-6,8-dimethyl flavanone and 2,2',4'-trihydroxy-6'-methoxy- 3',5'-dimethylchalcone from Cleistocalyx operculatus (Min, Thu, Nguyen, Jang & Hung, 2008).

7-hydroxy-5-methoxy-6,8 6-formyl-8-methyl-7-O- dimethylisoflavone methylpinocembrin

Four new flavonoids, 3'-formyl-4',6',4-trihydroxy-2'-methoxy-5'-methylchalcone, 3'- formyl-6',4-dihydroxy-2'-methoxy-5'-methylchalcone,4'-O-β-D-glucopyranoside, (2S) -8-formyl-6-methylnaringeni, and (2S)-8-formyl-6-methylnaringenin-7-O-β-D-glucopyranoside were isolated from the buds of C. operculatus (Zhang & Lu, 1990).

Nine constituents have been isolated from the flower bud of C. operculatus after the removal of essential oils by steam distillation. Eight of them were identified as 2',4'- dihydroxy-6'-methoxy-3',5'-dimethylchalcone, 5,7-dihydroxy-6,8-dimethylflavone, 7- hydroxy-5-methoxy-6,8 dimethyl flavanone, ethyl gallate, gallic acid, ursolic acid, β- sitosterol and cinnamic acid (Zhang & Lu, 1990).

5,7-dihydroxy-6,8-dimethylflavone 7-hydroxy-5-methoxy-6,8 dimethyl flavanone 2.33 Bauhinia variegata

Bauhinia variegata, locally named as Koiralo, is available in many parts of Nepal. The plant belongs to family Fabaceae. The plant was collected from Syangja district for the purpose of present study. It has been traditionally used by local peoples when they suffer from asthma and gastric. Pandey (2015) reported different phytoconstituents from this plant that makes it remarkable for its use by traditional practitioners. The hydromethanolic extract of B. variegata were evaluated against Gram-positive and Gram-negative bacteria by using disc diffusion assay. Phytochemical screening of all extracts showed the presence of alkaloids, steroids, phenolic compounds, tannins, saponin, carbohydrates, proteins, amino acids and organic acids.

This plant has been used as a traditional medicine for treatment of stomach disease and lung disease in Yunnan province. Previously, several components including flavonoids, terpenoids and alkaloids were reported from rhizomes and stems of B. variegata. Systematic phytochemical investigation in the flower of this plant led isolation of eight known compounds such as isoliquiritigenin, naringenin, kaempferol, kaempferol-7-O-β-D-glucopyranoside, kaempferol-3-O-β-D-glucopyranoside, caffeic acid, catechin, and kaempferol-3-O-L-rhamnopyranoside (Divya & Anita, 2012).

Naringegenin Kaempferol-3-O-L-rhamnopyranoside

Negi, Sharma, Pant and Singh (2012) have reported the total phenolics, flavonoids, and tannins in the petroleum ether, ethyl acetate, methanol, and aqueous extract of the stem bark of Bauhinia variegata. The methanol extract showed highest concentration of phenolics, flavonoids, and tannins with petroleum ether extract reporting the least. Previous results clearly indicated that B. variegata was a rich source of phenolics compounds as the basis of its traditional use in different systems of medicines (Negi et al., 2012).

The antimicrobial activities of extract were tested and compared by agar well diffusion method against human pathogens such as Escherichia coli, Streptococcus mutans, Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa. Bioactive compounds revealed by phytochemical screening were saponins, tannins, flavonoids, cardiac glycosides and steroids. Free radical scavenging capacity was evaluated by reducing power assay which demonstrated a correlation between concentration of extract and antioxidant potential (Liao & Li, 2013).

2.34 Pogostemon amaranthoides

Pogostemon amaranthoides, locally named as Rudilo, is usually available in shady and moist places in many parts of Nepal. The plant belongs to family Labiatae. The plant was collected from Syangja district. It has been traditionally used when they suffer from cough and cold. It is also used as spices, natural flavor, raw material for essential oil industry and other medicinal purpose. A wide range of phytochemical constituents have been isolated from Patchouli which possesses activities like antimicrobial, cytotoxic, antiemetic, analgesic, antimutagenic and antiinflammatory activity. Based upon the given significant information, Pogostemon cablin can be developed into novel natural medicine (Chakrapani, Venkatesh, Chandra, Singh, Arun, Kumar, Amareshwari & Rani, 2013).

2.35 Betula alnoides

Betula alnoides, locally known as Sour, is found in many parts of Nepal. It belongs to family Batulaceae. The bark of the plant was collected from Manang district. It has been traditionally used when people suffer from jaundice and stomachache. Nineteen polymorphic microsatellite markers were isolated from this species, which displayed three to twelve alleles per locus. These markers would be useful tools in genetic resource assessment, molecular marker-assistant breeding, parentage analysis and

35 genetic diversity studies for this species (Sur, Pandit, Battacharyya, Kumar, Ashok, Chatttopadhyay & Mandal, 2002).

The essential oil obtained by steam distillation of fresh bark from B. alnoides was contained methyl salicylate (99.4 percent) as its major component. Eight other constituents were present of which six trace compounds were identified (Ghimire, Tamang, Yu Chang, Jung & Chung, 2012). Dried bark from B. alnoides (Betulaceae) collected in the Son La province of North Vietnam yielded lupeol, 3-O- acetoxyoleanolic acid, betulinic acid, and betulin (Kamperdick, Thuy, Van Sung & Adam, 1995).

Lupeol 3-O-acetoxyoleanolic acid

Betula alnoides has been widely used in local traditional medicinal treatment for a variety of diseases, wounds and to cure diabetes. It is reported that 80 percent methanolic extracts exhibited high 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. In addition, both the 80 percent methanolic extract and EtOAc fraction exhibited more potent reducing activity than did butylated hydroxyanisole (BHA) and trolox. Methanolic extract and EtOAc fraction showed higher levels of antimicrobial activity than did other fractions. The methanolic extract had the most powerful α- glucosidase inhibitory effect. The results suggest that bark extracts of B. alnoides could be a potential source of natural antioxidants and for treating pathogenic diseases (Dung, Moi & Leclercq, 1995). The antiinflammatory activity of Betula alnoides extract was evaluated in acute and sub acute inflammation models (Guo, Zeng, Zhou & Zhao, 2008). The extract was also evaluated for antiinflammatory activity in sheep RBC induced sensitivity and in membrane stabilization models.

2.36 Bergenia ciliata

Bergenia ciliata, locally known as Pakhanvedh, is mostly found in hilly regions of Nepal. The plant belongs to family Saxifragaceae. The plant was collected from Manang district. It has been traditionally used as important medicine when people suffer from stomachache and urination trouble due to stone formation in the body. Bergenia species are evergreen herb belonging to the family saxifragaceae. The rhizomes of these plants are used in the indigenous system of medicines. There are three species of Bergenia, namely B. ligulata, B. ciliata and B. stracheyi. The rhizome and other parts of B. ligulata is used in urinary bladder stone, antilithic activity diuretic activity, antibradykinin activity, antiviral activity, antipyretic activity, antibacterial, anti inflammatory, hepatoprotective activity, insecticidal activity, α-glucosidase activity and all these activities of the plant is due to presence of its constituents like β- sitosterol, tannic acid, stigmasterol, gallic acid, bergenin, (+)-afzelechin, (+)-afzelechintetraacetate, (+)-5,7,4'-trimethoxyafzelechin, (+)-tetra- methoxyazelechin, (+)-3-acetyl-5,7,4'-trimethoxyafzelechin. The second species is B. ciliata, have antitussive, antiulcer, antioxidant, antibacterial, hypoglycemic and toxic activity (Ruby, Chauhan, Sharma & Dwivedi, 2012).

Bergenia ciliata was analyzed for its biochemical composition and active medicinal components. The analysis of plant revealed presence of fair amount of biomolecules namely carbohydrates, total sugars and amino acids in both rhizome and leaves. Photosynthetic Pigments (chlorophyll-a, chlorophyll-b and carotenoids) were analyzed which showed their co-relationship with medicinal components (Rajkumar, Guha, Ashok & Mathew, 2010). The previous study was aimed to evaluate antioxidant activity of methanolic and aqueous extracts of Bergenia ciliata. Free radical scavenging potential of the extract revealed that both extracts to be active radical scavengers, reducing (Fe+3-Fe+2) power and lipid peroxidation. Inhibition efficiency (TBARS assay) of both extract was also evaluated and showed promising activity in preventing lipid peroxidation and might prevent oxidative damages to biomolecules (Bashir, Rafiq, Hai, Khan & Sheikh, 2011).

Catechin Bergenin Gallic acid

2.37 Periploca calophylla

Periploca calophylla, locally known as Shikari lahara, is usually available in hilly regions of Nepal. The plant belongs to family Asclepiadaceae. The plant was collected from Manang district. The plant has been traditionally used by local peoples when they suffer from high fever and headache. Two oligosaccharides were isolated from the chloroform extract of P. calophylla and their structures were identified as 4- O-acetyl-β-cymaropyranosyl, (1,4)-O-β-D-cymaropyranosyl, (1,4)-O-β-D-canaropyr- anosyl, (1,4)-O-β-D-cymaropyranosyl, (l,4)-O-oleandronic acid, lactone, and perisa- ccharide (Deepak & Khare, 1986).

Eight compounds were isolated from the chloroform fraction of P. calophylla, including periplocin, periplogenin, periplocoside, 2α,3β,23-trihydroxy-12-ene-28-ca- rboxylic acid, glycoside, ursolic acid, β-sitosterol and daucosterol (Guo & Zhou, 2005).

Eight glycosides were isolated from n-butanol fraction of Periploca calophylla and identified as periplocin, kaempferol-3-α-D-arabinoside, kaempferol-3-O-β-D-g luco- side, 3',4',5,7-tetrahydroxyflavanone, 2(S)-3'-O-β-D-glucopyranoside, (+)-syringare- sinol-4'-O-β-D-monoglucoside, 1-sinapoylglucoside, erigeside, 2,6-dimethoxy-4-hy- droxyphenol-1-O-β-D-glucoside. All the compounds were isolated first time from Periploca calophylla (Sethi, Deepak & Khare, 1988). Calocinin was isolated from twigs of P. calophylla and its structure established as 3-O-β-L-2,6-dideoxyfuco- pyranoside. A pregnane glycoside of boucerin named locin was isolated from the dried twigs of P. calophylla and its structure was established as boucerin-3-O-β-D- digitoxoside (Long, Xu, Zhang, Tan & Sun, 2012).

2.38 Astilbe rivularis

Astilbe rivularis, locally named as Thulookhati, is available in hilly regions of Nepal. The plant belongs to family Saxifragaceae. The plant was collected from Manang district. It has been traditionally used as medicine when they have problems of dysentery and diarrhea. Bioassay guided fractionation led to the isolation of arbutin, bergenin and abergenin derivatives. Bergenin, β-sitosterol, and astilbic acid were newly isolated from A. rivularis, whereas flavonoids and triterpenoids were previously described. Bergenin exhibited significant antifeedant activity against two lepidopterous insects (Tandon, Shukla, Tripathi & Sharma, 1996). Extraction of the aerial parts of A. rivularis yielded β-amyrin, β-sitosterol, β-peltoboykinolic acid, astilbic acid, quercetin, and bergenin (Rajbhandari, Lalk, Mentel & Lindequist, 2011).

Quercetin

2.39 Piper mullesua

Piper mollesua, locally named as Pipala, is usually found in shady and moist places in many parts of Nepal. The plant belongs to family Piperaceae. It was collected from Syangja district. The plant has been traditionally used in curing of asthma and bronchitis. Hieu, Thang, Hoi and Ogunwande (2014) have reported the main constituents of P. boehmeriaefolium such as benzyl benzoate, benzyl alcohol, 2- hydroxybenzoic acid, phenylmethyl ester and 2-butenylbenzene. The leaf of P. maclurei was characterized by higher amount of (E)-cinnamic acid and (E)-nerolidol. Moreover, (Z)-9-octadecanoic acid methyl ester, (E)-cinnamyl acetate, phytol and (E)-cinnamaldehyde were the major compounds identified in the stem oil.

2.40 Bombax ceiba

Bombax ceiba, locally known as Simal, is widely distributed in hilly and terai regions of Nepal. The plant belongs to family Bombacaceae. It was collected from Chitwan district. It has been traditionally used for the treatment of diarrhea, burns and

39 dysentery. The plant is a large, briefly deciduous tree occurring in warm monsoon forests in southern Asia. It is one of the world’s most spectacular flowering trees famous for large, showy, six-inch flowers with thick, waxy, red petals that densely clothe leafless branch tips in late winter and early spring. Joshi, Devkota & Shoji (2014) have isolated two new aromatic compounds from stem bark of Bombax ceiba along with five known compounds shamiminol, O-epicatechin-7-O-β-xylopyranoside, O-catechin-7-O-β-xylopyranoside, (+)-isolarisiresinol-9-O-β-glucopyranoside and (+)-lyoniresinol-9-O-β-glucopyranoside.

2.41 Calotropis gigantea

Calotropis gigantea, locally known as Aak, is usually available in many parts of Nepal. The plant belongs to family Apocynaceae. The plant was collected from Chitwan district. It has been traditionally used to cure swelling and paralysis. The plant is distributed in Himalaya region and all over India. This plant is widely used in different types of activity. The GC-MS concluded that Petroleum ether extract is having sixteen compounds. The preliminary phytochemical screening revealed that the presence of alkaloids, steroids, triterpenoids and resins (Shirsat, Pal, Buchake, Gupta & Bais, 2014).

Calotropis gigantea Linn is popularly known as the swallow-wort or milkweed and is used as one of the most important drug in traditional system of medicine to treat various ailments. Dhivya and Manimegalai (2013) reported the presence of alkaloids, tannins, phenol, flavanoids, sterols, anthraquinones, proteins and quinones in the flower extract. The GC-MS analysis of the ethanolic extract revealed the presence of fourteen major compounds. This study forms a basis for the biological characterization and importance of the compounds identified and creates a platform to screen many bioactive components to treat many diseases.

Sureshkumar (2013) reported the phytochemical property of Calotropis gigantea, commonly known as wasteland weed or milkweed. Acetone, alcoholic and

Chloroform extract of the plant were reviewed by using GC-MS. The maxium number of compounds was recorded in the chloroform extract (Mohaimenul, Ismail, Abu, Abdul, Rowshahul & Rezeul, 2012).

di-(2-ethylhexyl) phthalate

Mohaimenul et al. (2012) prepared methanol, n-hexane and ethyl acetate extracts from the root bark of Calotropis gigantea. Phytochemical study on the ethyl acetate extract of the root bark of Calotropis gigantea led to the isolation of two compounds α-taraxerol and β-sitosterol acetate.The cytotoxicity of prepared extracts and isolated compounds against brine shrimp nauplii (Artemia salina) were also evaluated and among the samples ethyl acetate extract showed better activity (Mohaimenul et al., 2012).

2.42 Annona reticulata

Annona reticulata, locally named as Sarifa, is usually available in many parts of Nepal, particularly in Tarai region. The plant belongs to family Annonaceae. It was collected from Chitwan district. It has been traditionally used as medicine when people suffer from stomachache due to intestinal worms. The plant is called bullocks- heart in English and Ramphal in Hindi and Marathi and it has various pharmacological activities such as antioxidant, anticancer, analgesic and CNS depressant, antimalarial, antihelmintic, in syphilis and few more. Some compounds have been isolated and reported from the extract of various parts of the plant possessing good pharmacological activity. The studies performed on the seed and root extract also evidenced that the same compound causes cell death in various cancer cell lines (Thang, Kuo, Yang & Wu, 2013).

Kaurenoic acid Iiriodenine Norushinsunine

Chemical investigation of the leaves of Annona reticulata has resulted in the identification of nine compounds including annonaretin, a new triterpenoid. The purified compounds exhibited significant nitric oxide inhibition (Araya, Maeda, Hara, Prasad, Begum, Sahai & Fujimoto, 2012).

Eight bioactive annonaceous tetrahydrofuranic acetogenins were isolated from the seeds. Among them, four acetogenins, asimicin, annonacin-10-one, squamostatin, 10- hydroxyasimicin and squamocin-Z, were determined from the seeds of the plant for the first time together with annonacin, murisolin, bullatacin, squamocin. A. reticulata seeds are a promising source of tetrahydrofuranic acetogenins possessing wide spectral bioactivity (Chavan, Shamkuwar, Damale & Pawar, 2014).

Hydrodistillation oil obtained from the leaves of Annona reticulata Linn. grown in Nigeria was analyzed by capillary GC and GC/MS. Thirty nine components were characterized. These consisted of eighteen monoterpenes amounting to 29.0 percent, sesquiterpenes totaling 52.9 percent and one aromatic esters making up 10.9 percent (Ogunwande, Ekundayo, Nureni & Kasali, 2006).

2.43 Mimosa pudica

Mimosa pudica, locally known as Lajjawati, is widely distributed in many parts of Nepal. The plant belongs to family Fabaceae. It was collected from Chitwan district. It has been traditionally used when people suffer from nerve problems and healing wounds. The whole plant of Mimosa pudica is well-known for its medicinal properties in traditional system of medicines. Rani, Sharma and Vasudeva (2012) have reported the pharmacognostical examination viz; morphology, microscopical characters, loss on drying, of ash values, extractives values, foreign organic matter and crude fiber content. Preliminary phytochemical screening, elemental analysis and microbial contamination of powdered drug were also carried out (Rani et al., 2012).

The whole plant of Mimosa pudica is very useful for various pharmacological and biological activities. Mostly root and leaves of Mimosa pudica were showed maximum pharmacological activities as antidiabetic, antioxidant, antihepatotoxin and wound healing activity (Azmi, Singh & Akhtar, 2011).

The ethanolic extract of Mimosa pudica at 200 and 400 mg/kg has significantly inhibited ulcer formation. There was a significant dose-dependent decrease in the ulcerative lesion index produced by all the three models in rats as compared to the standard drug lansoprazole. The reduction in gastric fluid volume total acidity and an increase in the pH of the gastric fluid in pylorus ligation rats proved the antisecretory activity of Mimosa pudica leaves (Khalid, Kumar, Singh, Setty, Reddy, Narasimha & Hakeemuddin, 2011).

2.44 Ziziphus mauritiana

Ziziphus mauritiana, locally known as Bayar, is widely available in hilly and terai regions of Nepal. The plant belongs to family Rhamnaceae. The plant was collected from Chitwan district. It has been traditionally used as medicine when people suffer from gastrointestinal problems and stomachache. Antioxidant properties of fruits of Ziziphus mauritiana were determined by DPPH radical scavenging activity, reducing power assay, superoxide anion radical activity, total phenolic and flavonoid content (Kavitha, Kuna, Supraja, Sagar, Blessy & Prabhakar, 2014).

The nutritional components of Ziziphus mauritiana were studied and analysed. There were seven kinds of essential amino acids in total amino acids. The fruits are not only contained high levels of Ca and Mg, but also contained the trace elements including Fe, Cu, Mn, Zn and Se that were all essential to human (Deng, Shen & Deng, 2013).

Nine phenolic acids like ferulic acid, chlorogenic, venillic, caffeic, vanillin, o-and-p coumaric acids, Protocatechuic, P-hydroxybenzoic acid were extracted separated and quantified by HPLC-DAD. Identification of phenolic acids was achieved by comparision of retention times, UV, and mass spectral data with authentic standard compounds. p-coumaric acid was predominant phenolic acid. From dry fruits of Ziziphus mauritiana four phenolic acid namely hydroxybenzoic acid, vanillin, ferulic acid, and o-coumaric acid were obtained in intermediate amounts. It showed Ziziphus mauritiana fruits are good natural source of phenolic acids (Memon, Muhammad & Luthria, 2012).

Ferulic acid Caffeic acid Vanillin

2.45 Cascabela thevetia

Cascabela thevetia, locally named as Karbir, is distributed in many parts of Nepal. The plant belongs to family Apocynaceae. The plant was collected from Chitwan district. The plant has been traditionally used as medicine when people suffer from chest pain. Microwave assisted bio-based green synthesis of highly monodispersed spherical Gold Quantum Dots (Au-QD) using the Cascabela thevetia flower extract was reported. The synthesized material exhibited the surface plasmon resonance at 520 nm. The transmission electron micrographs of the nanoparticles showed the formation of spherical nanoparticles. The material synthesized was characterised by HRTEM, Electron Diffraction and XRD. The biomaterial functioned both as reducing and stabilizing agent (Choudhury, Paul & Das, 2012).

2.46 Achyranthes bidentata

Achyranthes bidentata, Blume locally named as Datiwon, is widely distributed in many parts of Nepal. The plant belongs to family Amaranthaceae. It was collected from Syangja district. It has been traditionally used as medicine when people suffer from toothache and inflammatory. The plant is widely distributed in Asian countries like India, Korea, Japan, Nepal and China. The root of A. bidentata has been prescribed in the Chinese Pharmacopeia as an important herbal medicine and its multiple pharmacological effects, such as antiosteoporosis, antitumor, antiinflammatory and immunomodulatory activities are well documented. Previous phytochemical investigations of A. bidentata have reported eight phytoecdysteroids, including two new ones, (25S)-20,22-O-(R-ethylidene) inokosterone and 20,22-O-(R- 3-methoxy carbonyl) propylidene-20-hydroxyecdysone, and six known phytoecdysteroids (Zhong-Yu Zhou, Yong Cao, Wei-Min Zhang & Jian-Wen Tan, 2012).

2.47 Callicarpa sp.

Callicarpa sp., locally named as Dhaichamle, is available in hilly and terai regions of Nepal. The plant belongs to family Labiatae. The plant was collected from Chitwan district for the purpose to test in the present study. The plant has been traditionally used by local peoples when they suffer from rheumatism and stomach trouble. Martha, Blanca, Maria, Anthony, Richard and Jordi (2008) have reported endophytic fungus isolated from the leaves of Callicarpa acuminata (verbenaceae) resulted in the isolation of four naphthoquinone spiroketals, including three new compounds and palmarumycin. The biological activity of isolated compounds were tested against three endophytic fungi (Colletotrichum sp., Phomopsis sp. and Guignardia manguifera) isolated from the same plant species and against four economically important phytopathogenic microorganisms. The new spiroketals displayed significant growth inhibition against all the phytopathogens.

2.48 Cinnamomum tenupile

Cinnamomum tenupile, locally named as Sugandha kokila, is widely distributed in many parts of Nepal. The plant belongs to family lauraceae. The plant was collected from Chitwan district for the purpose of present study. It has been traditionally used by peoples for imparting odour in different components. Kumar, Ninan, Kuttan and Maliakel (2014) reported the plant extract could be used for their relative antihyperglycemic effects in comparison with standard aqueous extract containing eighteen percent polyphenol content and 0.8 percent coumarin. De-coumarinated extracts were found to be safe and showed 3.4 fold enhancements in relative lowering of blood sugar levels as compared to the standard cinnamon extracts when administered to streptozotocin induced diabetic rats.

Huang, Xu, Liu, Zhang and Hu (2014) have reported trans cinnamaldehyde as the major compound from Cinnamomum cassia bark. Anti bacterial activity of essential oil extracted from bark was studied against four food related bacteria. The essential oil was found active against Stayphylococcus aureus and significant effect on growth rate of surviving S. aureus and Escherichia coli.

2.49 Bridelia retusa

Bridelia retusa, locally known as Gayo, is widely distributed in hilly region of Nepal. The plant belongs to family Euphorbiaceae. It was collected from Syangja district for the purpose of present study. It has been traditionally used by the peoples to kill worms of livestock and healing of wounds. Tatiya, Tapadiya, Kotecha and Surana (2011) reported acetone extracts shown highest polyphenol content with highest antioxidant activity and potent natural antimicrobial agent.

Raja and Srilakshmi (2010) reported that an aqueous ethanol extract of Bridelia retusa exhibited highest in-vitro hepatoprotective effects as evident from the significantly reduced serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) into the incubation medium of rat hepatocytes with carbon tetrachloride (CCl4), over the other organic extracts

(chloroform, ethylacetate, and methanol). CCl4 administered produced a marked elevation in the serum levels of GOT, GPT, lactate dehydrogenase, alkaloid phosphatase, bilirubin, thiobarbituric acid reactive substances, and decreased in the levels of reduced glutathione, superoxide dismutase, catalase, glutathione-S- transferase, glutathione reductase, glutathione peroxidase, and total protein content. A flavonoid was isolated from the benzene fraction of ethanolic leaves extract of Bridelia retusa found to show strong antimicrobial activity against human pathogenic bacteria (Adhav, Solanki, Patel & Gharia, 2002). Antifungal activity guided fractionation of stem bark of Bridelia retusa against Cladosporium cladosporioides, furnished new bisabolane sesquiterpenes, (E)-4-(1,5-dimethyl-3-oxo-1-hexenyl) benzoic acid, (E)-4-(1,5-dimethyl-3-oxo-1,4-hexadienyl) benzoic acid, (R)-4-(1,5- dimethyl-3-oxo-4-hexenyl) benzoic acid and (-)isochaminic acid, together with the known (R)-4-(1,5-dimethyl-3-oxohexyl) benzoic acid, 5-allyl-1,2,3-trimethoxy- benzene, (+)sesamin and 4-isopropylbenzoic acid (Jayasinghe, Kumarihamy, Jayarathna, Nishantha & Gayathri, 2003).

Phytochemistry of Bridelia cambodiana

Extensive studies on Bridelia cambodiana have been carried out which led to the isolation of a large number of compounds. The survey of some compounds on this genus till date is presented below:

Friedelin 24-Methyllanosta-9(11), 25-dien-3-one

24,24-Dimethyllanosta-9(11), 25-dien-3-one 24-Methyl-5α-lanosta-9(11), 25-dien-3α-ol

Lupeol Betulinic acid

α-amyrin Oleanolic acid

Maslinic acid Sigmasterol

2.50 Scoparia dulcis

Scoparia dulcis Linn locally named as Chinijhar, is widely distributed in terai region of Nepal. It belongs to family Scorphulariaceae.The plant was collected from Chitwan district. It has been traditionally used as medicine when people suffer from stomachache and jaundice.The plant is also known as sweet broomweed Mithipatti and Bana Dhania in Western Orissa. The plant is also known as 'GhodaTulsi' in Hindi. S. dulcis is rich in flavones, terpenes and steroids. Main chemical constituents such as scoparic acid, scopadulcic acid A and B, scopadulciol, scopadulin and ammelin have been shown to contribute to the observed medicinal effect of the plant. Some aspects of the several speculated pharmacological properties of S. dulcis have been validated by scientific research, which includes the presence of hypoglycemic and antitumor promoting compound. It also has antimicrobial and antifungal effects as well as antihyperlipidemic action (Muthumani, Chiristina, Venkataraman, Meera, Abraham, Devi, Kameswari & Eswara priya, 2010).

Sharma and Shah (2010) reported that the antihyperglycemic effects of flavonoids from methanolic extract of aerial parts of Scoparia dulcis leaves in normal, glucose loaded and streptozotocin induced diabetic rats. The extract exhibited significant hypoglycemic activity in all three animal models when compared with a standard antidiabetic agent Glibenclamide. The hypoglycemia produced by the extract may be due to increased uptake of glucose at tissue level and or increase in pancreatic β-cell function or due to inhibition of intestinal glucose absorption of glucose. The findings of the previous study suggested that the methanolic extract of Scoparia dulcis produced significant antihyperglycemic activity in STZ induced diabetic rat which is comparable to Glibenclamide (Latha, Pari, Ramkumar, Rajaguru, Suresh, Dhanabal, Sitasawad & Bhonde, 2009).

Scoparia dulcis has been documented as a traditional treatment of diabetes. The insulin secretagogue action of Scoparia dulcis plant extract (SPEt) was further investigated using isolated pancreatic islets from mice (Hayashi, Goton, Kiyoshi, Okamura & Asamizu, 1994). It is revealed the possible therapeutic value of Scoparia dulcis for the better control, management and prevention of diabetes mellitus progression.

Scoparia dulcis plant extract is tried for prevention and treatment of diabetes mellitus. Oral administration of an aqueous extract of Scoparia dulcis plant (200 mg/kg body wt.) for six week to diabetic rats significantly increased the plasma insulin and plasma antioxidants and significantly decreased lipid peroxidation (Begum, Nahar & Mosihuzzaman, 2002).

Three new acetylated flavonoid glycosides 5,6,4'-trihydroxyflavone-7-O-α-L-2,3-di- O-acetylrhamnopyranosyl-(1-6)-β-D-glucopyranoside, apigenin-7-O-α-L-3-O-acetylrhamnopyranosyl-(1-6)-β-D-glucopyranoside and apigenin-7-O-α-L-2,3-di-O-acetylrhamnopyranosyl(1,6)-β-D-glucopyranoside were isolated from Scoparia dulcis together with the known compound eugenyl β-D-glucopyranoside (Hayashi, Okamura, Tamada, Iida & Fujita, 1993).

Dichloromethane, 1-butanol and an aqueous part of a methanol extract of Scoparia dulcis Linn was tested for antibacterial, antifungal, insecticidal and toxicity activities. All the extracts were active against three bacteria, namely Klebsiella pneumoniae,

Proteus mirabilis and Streptococcus pyogenes (Hayashi, Uchida, Hayashi, Niwayama & Morita, 1988).

A new chemotype was found in S. dulcis from Taiwan, China and Thailand based on the diterpene compound and characterized by the presence of scopadulciol and scopadiol.

Two new diterpenes, scoparinol and dulcinol, closely related to scopadulcic and scoparic acids, were isolated from S. dulcis (Mahato, Das & Sahu, 1981).

The structure and stereochemistry of scopadulin a novel aphidicolane-type diterpene isolated from S. dulcis were established from spectral data and single crystal x-ray analysis of its acetone solvate (Ramesh, Nair, Ramachandran & Sankara, 1979).

Hymenoxin, isolated from the whole plant of S. dulcis, showed cytotoxicity against cultured human cells (Chiu-Ming & Ming-Tyan, 1976).

New diterpenoids were isolated from whole plant of Scoparia dulcis Linn. Scrophuraliaceae and their structures were elucidated mainly by means of 2- dimensional NMR spectroscopy and NOE difference spectra (Mishra, Behera, Jha, Panda, Mishra, Pradhan & Choudhary, 2011).

D-Mannitol, coixol and betulinic acid were isolated from the roots of S. dulcis and their structures confirmed by identification with known samples (Ediriweera, Jayakody & Ratnasooriya, 2011).

Betulinic acid Coixol

The triterpenoids of S. dulcis were identified as friedelin, glutinol, α-amyrin, betulinic acid, ifflaionic acid, and dulcioic acid by spectral and chemical means (Latha, Pari, Ramkumar, Rajaguru, Suresh, Dhanabal, Sitasawad & Bhonde, 2009).

The Et2O fraction obtained from 90 percent EtOH extract of S. dulcis fresh leaves gave two flavones identified as 7-O-methylscutellarein and scutellarein. The flavonoid glycoside in the EtOAc fraction was scutellarein-7-O-β-D-glucuronide S. viscoa leaves gave the flavones diosmetin.

The benzoxazolinone and ifflaionic acid were isolated from the roots of S. dulcis and their structures were determined on the basis of their IR, UV, 1H-NMR, and mass

52 spectra and by identification of their acetylation and methylation derivatives (Latha et al., 2009).

The hypoglycemic activity of methanol extract of Scoparia dulcis was performed on both in vitro and in vivo models along with determination of total extractable polyphenol. Methanol extract of Scoparia dulcis contains 4.9 percent and water extract contains 3.2 percent of total extractable polyphenol. The antioxidant activity showed very promising result in both the tested methods that is 2,2-diphenyl-1-picrylhydrazyl and ferric ion reducing capacity. The antioxidant activity is directly correlated to the antidiabetic potential of drug. The two enzymes (amylase and glycosidase) found in intestine are responsible for the increasing postprandial glucose in body. In vitro model was performed on these enzymes and the results showed that methanol extract of Scoparia dulcis was effective to check the postprandial glucose level. The in vivo hypoglycaemic activity of methanol extract of Scoparia dulcis was performed on streptozotocin induced diabetes mellitus showed significant inhibition of blood glucose level as compared to control and similar to that of standard glibenclamide (Ahsan, Islam, Gray & Stimson, 2003). The overall data potentiates the traditional value of Scoparia dulcis as an antidiabetic plant.

Phytochemistry of Scoparia dulcis

Since the first phytochemical investigation on the plant Scoparia dulcis in 1988, extensive studies on this plant have been carried out which led to the isolation of a large number of compounds. The survey of some compounds on this genus till date is presented below:

Scopadulcis acid A Scopadulcis acid B

Scoparic acid A Scoparic acid B

Apigenin Scoparic acid C

Acacetin Alpha amyrin

Betulinic acid Benzoxazolone

Hymenoxin Cirsimarin

Cynaroside Coumaric acid

Cirsitakaoside Gentisic acid

Glutinol Iffaionic acid

β-sitosterol Dulcitol

Vitexin Benzoxazin

In overall, the literatures, collected through scientific finder (at HEJ Research Institute of Chemistry, ICCBS, University of Karachi, Pakistan), on fifty selected medicinal plants showed that all medicinal plants were not found as the sources of secondary metabolites useful to anticancer and antidiabetes. However, very few among them; Scoparia dulcis and Bridelia retusa were rich in secondary metabolites against diabetes. Rest of the plants, out of collected fifty, were explained as rich plants for antimicrobial and antioxidant activity. Therefore, the plants Scoparia dulcis and Bridelia retusa were further subjected to test for anticancer and antioxidant activity through preferential cytotoxicity against PANC-1 cell and DPPH radical scavenging bioassay screening methods.

CHAPTER 3

MATERIALS AND METHODS

3.1 Selection of medicinal plants

People of Nepal have been using medicinal plants as medicine in the treatment of various diseases throughout the history. However, various plants in different ecological belts of Nepal which possess medicinal compounds have not been well explored based on natural product chemistry. Considering this fact in mind it aimed to screen some selected medicinal plants to extract secondary metabolites, to test antibacterial, antioxidant and preferential cytotoxicity against pancreatic cancer cell line (PANC-1), to determine total phenolics and flavonoid contents in the potent antioxidant extracts and finally to isolate the compounds from the active plant extract.

In order to achieve the goal, different medicinal plants were collected from different parts of Nepal. For the purpose, it was decided to meet traditional healer and practicioners who have experiences and working with medicinal plants used as medicine on Jaundice, gastritis and diabetes. On the basis of information provided by these people a list of fifty different medicinal plants available in different regions of Nepal was made. The areas where these medicinal plants were available also reported by these traditional healers and local practicioners. Literature survey and interview with the experienced people were additional basis for plant collection in this study. These people reported Chitwan, Makawanpur, Kathmandu, Manang, Kaski and Syangja district as the areas where the medicinal plants were available.

Fifty medicinal plants were collected from Chitwan, Makawanpur, Kathmandu, Manang, Kaski and Syangja district of Nepal. Most of the plant collected on the basis of experiences shared by traditional healers and the peoples of different communities who have been using these plants in curing diseases like diabetes, jaundice, gastritis, etc. Some plants were collected based on ethnobotanical uses.

Scoparia dulcis is an edible perennial medicinal herb which is extensively being used all over the world for treatment of different diseases such as diarrhea, coughs and fever in Bangladesh (Zulfiker, Ripa, Rahman, Ullah, Hamid, Khan & Rana, 2010). It is also used for hypertension, diabetes, antipyretic, analgesic and for skin wounds in Brazil

58 and only for the treatment of hypertension in China (Hayashi et al., 1994). Indian people are using this plant in dysentery, jaundice, toothache, and stomach problems (Satyanarayana, 1969) and in similar practice in Thailand (Panyaphu, Sirisaarad, Naubol, Nathakarnkitkal, Chansakaow & Vanou, 2012). Nepali people also have been using it against diabetes and headache (Manandhar, 1993). Hence, all these results support for collection of this plant to search of cytotoxic agent against pancreatic cancer which ultimately lowers diabetes.

Insulin secretory activity and cytoprotective role of aqueous extract of the plant Scoparia dulcis has also been reported (Latha, Pari, Sitasawad & Bhonde, 2004). Although antidiabetic activity of this plant is well known, the active principle and mechanism of action has not investigated earlier (Arulselvana, Ghofar, Karthivashana, Halima, Ghafar & Fakurazi, 2014). Further, the relationship between diabetes and pancreatic cancer has also not been explored regarding the anticancer activity of plants. The pancreatic cancer may ultimately lead to diabetes in pancreatic cancer suffering patient. However, the primary rationale of selecting the plant is that the plant may be the potent source of compounds against pancreatic cancer which ultimately controls diabetes.

All fifty collected plants samples were shade dried, grounded by mechanical grinder and soaked in methanol for 72 hours in conical flasks. The soaked samples were filtered with the help of Whatmann 40 filter paper. The filtrate thus obtained was concentrated with the help of rotatory evaporator. After complete extraction the extract was dried, percentage yield was calculated and performed phytochemical analysis.

3.2 General experimental conditions 3.2.1 Physical constants

Melting points of the compounds were determined on a Yanaco MP-53 micro melting point apparatus. Optical rotations were measured on a JASCO digital polarimeter (Model DIP-3600) in chloroform and methanol.

3.2.2 Spectroscopic technique

UV spectra were recorded in methanol on Hitachi UV 3200 spectrophotometer. IR spectra were recorded in CHCl3 on a JASCO A-302 IR spectrophotometer. The mass spectra were measured on double focusing (Varian MAT 311 A) and Jeol HX 110

59 mass spectrometer. The 1H-NMR spectra were recorded on Bruker AC-300, AM-400 and Amx-500 MHz instruments, while 13C-NMR spectra were recorded at 75, 100, 125 and 150 MHz. Multiplicities of carbon signals were determined by using DEPT 90o and 135o experiments. Homonuclear 1H-1H connectivities were determined by using COSY 45o experiment. One-bond 1H-13C connectivities were determined by HMQC experiment. Two-and three-bond 1H-13C connectivities were determined by HMBC experiment. 1H-NMR chemical shifts are reported in δ (ppm) and coupling constant (J) were measured in Hz.

3.2.3 Chromatography and staining

Column chromatography was performed on Merck silica gel 60 (70-230 and 240-300 mesh sizes, E. Merck) Merck alumina (70-230 Mesh ASTM). Precoated silica gel TLC plates (E. Merck F254) were used for checking the purity of compounds. TLC plates were viewed under the ultraviolet light at 254 nm for fluorescence quenching spots and at 366 nm for fluorescence spots.

3.2.4 Equipments

Mixture grinder, mortar and pestle, digital weighing machine (GT 210), hot air oven (Griffin-Grundy), rotatory evaporator (Buchi RE 111 with Buchi 461 water bath), Column Chromatography 600 mm (Fortuna WGCO, Optifit, Germany), UV Chamber (JSGW), magnetic stirrer hot plate (Stuart scientific, UK), water bath (Clifton), iodine chamber, burette, Pipettes, micropipettes (Erba BIHOT), thermometer, condenser, melting point apparatus (Griffin and George Company Limited, UK), PD-303 UV spectrophotometer (APEL), rectangular water bath (Physilab Scientific Industries, Ambala Cantt, India), plastic cuvettes, quartz cuvette, incubator, IR prestige-21 FTIR spectrometer (SHIMADZU).

3.2.5 Chemicals

Most of the solvents and chemicals were of laboratory grade. Methanol and chloroform (Thermo Fischer Scientific India Pvt. Ltd., Mumbai), ethyl acetate and hexane (Merck Limited, Mumbai) were purchased. Silica gel used for column chromatography was of mesh 60-120 size from Hi-Media. Similarly, silica gel was used for thin layer chromatography and silica gel Rf values were recorded by using precoated TLC plates of Merck company. Folin-Ciocalteu reagent and double distilled

60 water was also purchased from the local vendor. Chemicals and reagents like DPPH, ascorbic acid, gallic acid, quercetin required for antioxidant test, total phenolic content test and total flavonoid content test were purchased from the local vendor.

3.2.6 Phytochemical screening

The methanolic extracts of different plant samples were analysed for the presence of secondary metabolites such as polyphenols, alkaloids, flavonoids, tannin, carotenoids, saponins, reducing sugars, cardiac glycosides, steroids, terpenoids, glycoside and anthraquinone according to the standard procedures of analysis (Wadood, Ghufran, Jamal, Naeem, Khan, Ghaffar & Asand, 2013). 3.2.6.1 Alkaloids: 0.2 g of the crude methanolic extract of each plant sample was warmed with 2% H2SO4 for 2 min. After filtration of the reaction mixture a few drops of Dragendroff’s reagent were added. Orange red precipitate was observed which indicates the presence of alkaloids. 3.2.6.2 Flavonoids: About 0.2 g of each plant extract was dissolved in diluted NaOH and HCl was added. A yellow solution turned into colorless which indicates the presence of flavonoids. 3.2.6.3 Steroids: 2 mL of acetic anhydride was added to 0.5 mL methanolic extract followed by adding 2 mL of H2SO4. The colour changed from violet to green or blue which is the indication of the presence of steroids. 3.2.6.4 Terpenoids: About 0.2 g of the plant extract was mixed with 2 mL of chloroform first and then 3 mL of concentrated H2SO4 was added to each mixture. There was formation of reddish brown color at the interface which indicates the presence of terpenoids. 3.2.6.5 Reducing sugars: Each samples were shaken with distilled water first then filtered. To the filtrate few drops of Fehling solution A and B were added and boiled for few minutes. The appearance of an orange red precipitate confirmed the presence of reducing sugars. 3.2.6.6 Glycosides: Methanolic extract was acidified with dil.HCl and then neutralized with NaOH solution to this few drops of Fehlings solution A and B were added to the mixture. Formation of red ppt. was observed which indicates the presence of glycosides.

3.2.6.7 Polyphenols: The methanolic extract was mixed with water to this solution 1percent (w/v) ferric chloride solution (3 drops) was added. A greenish colour was developed indicating the presence polyphenols. 3.2.6.8 Tannins: About 0.5 g of the extract was boiled in 10 mL of water in a test tube and then filtered. A few drops of 0.1 percent ferric chloride was added and observed for brownish green or blue black colouration. 3.2.6.9 Cardiac glycoside: To 0.5 g of extract diluted to 5 mL in water, 2 mL of glacial acetic acid containing one drop of ferric chloride solution was added. This was

underplayed with 1 mL of conc. H2SO4. A brown ring at the interface was observed that indicated the presence of a deoxysugar.

3.2.6.10 Anthraquinone: 0.5 g of the extract was boiled with 10 mL of H2SO4 and

filtered while hot. The filtrate was shaken with 5 mL of CHCl3. The chloroform layer

was pipette into another test tube and 1 mL of dil. NH3 was added. The resulting solution was observed for colour change. 3.2.6.11 Saponins: To 0.5 g of extract was added 5 mL of distilled water in a test tube. The solution was shaken vigorously and observed for a stable persistent froth. 3.2.6.12 Carotenoids: About 1 g of sample was extracted with 10 mL of chloroform in a test tube with vigorous shaking. The resulting mixture was filtered and 85 percent

H2SO4 was added. A blue colour at the interface showed the presence of carotenoids. 3.2.7 Antioxidant activity (DPPH radical scavenging assay)

The free radical scavenging activity was measured by using DPPH assay. Different concentration of test samples (5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 µg/mL) were prepared while the concentration of DPPH was 0.2 mM in the reaction mixture. These reaction mixtures were taken in Eppendorf tubes and incubation at 37 oC for 30 min. Discolorations were measured at 517 nm using a UV-Visible spectrophotometer. Percent radical scavenging activity by sample treatment was determined by comparison with methanol treated control group ascorbic acid was used as positive control. Measurement was performed at least in triplicate. The percentage scavenging of the DPPH free radical was calculated using the following equation:

Absorbance of the control – Absorbance of the test sample % Scavenging Activity = X 100 Absorbance of the control

The inhibition curve was plotted for the triplicate experiments and represented as percentage of mean inhibition ± standard deviation and the IC50 values were obtained.

Standard graph was plotted taking the concentration on the X-axis and percentage scavenging activity on the Y-axis. Based on this graph, IC50 value of each sample was calculated with the formula IC50= EXP (LN (Conc.>50%) – ((pi>50% - 50)/(pi>50% - pi<50%)*LN(conc.>50% /conc.< 50%))) following the (Saha, Hasana, Aktera,

Hossaina, Alarm & Mazumderc, 2008). The IC50 value of the different species was compared with standard ascorbic acid and species having the lowest IC50 is considered to have the best antioxidant property.

3.2.8 Total polyphenol content determination

The total phenolic content in the fifteen active medicinal plant extracts screened for DPPH radical scavenging assay was determined by using the Folin–Ciocalteu phenol reagent. According to the protocol (Eghdami & Sadeghi, 2010) 0.1 mL of each extract (2.5 mg/mL) was separately mixed with the 1 mL of Folin–Ciocalteu phenol reagent and 0.8 mL of aqueous 1 M Na2CO3 solution. The reaction mixture was allowed to stand for about 15 minutes and the absorbance of the reactants was measured at 765 nm using the UV- visible spectrophotometer. The calibration curve was obtained using the solution of gallic acid as standard in methanol using the concentration ranging from 25-250 μg/mL. Based on this standard graph, concentrations of the individual samples were calculated. The total polyphenol content was expressed in terms of the milligrams of the gallic acid equivalent per gram of the dry mass (mg GAE g-1).

The total phenolic content is calculated in all the plant extracts separately using the following formula

C = cV/m

Where,

C = Total content of phenolic compounds in mg/g, in gallic acid equivalent (GAE) c = Concentration of gallic acid established from the calibration curve in mg/mL

V = Volume of extract in mL m = Weight of plant extract

Data were recorded as mean of three determinations of absorbance for each concentration, from which linear correlation coefficient (R2) value can be calculated. The regression equation is given as.

Y = mx + c

Where,

Y = Absorbance of extract m = Slope from the calibration curve x = Concentration of extract c = Intercept

Using this regression equation concentration of extracts can be calculated. Thus, with the calculated value of concentration of each extract, the phenolic content can be calculated.

3.2.9 Total flavonoid content determination

The total flavonoid content in the fifteen active plants extracts screening for DPPH radical scavenging assay was estimated by using the aluminium chloride (AlCl3) colorimetric method. 0.25 mL of extract (10 mg/mL) was separately mixed with the 0.75 mL of methanol, 0.05 mL of the 10 percent aluminum chloride, 0.05 mL of the 1

M potassium acetate (CH3COOK) and 1.4 mL of the distilled water. The reaction mixture was allowed to stand for about 30 minutes in room temperature. The absorbance of the mixture was measured at 415 nm using the UV visible spectrophotometer. The calibration curve was constructed with the help of standard quercetin solutions in methanol with the concentration ranging from the 10-100 μg /mL. The total flavonoid content was expressed in terms of the milligram of quercetin equivalent per gram of the dry mass (mg QE/gm).

The total flavonoid content in all plant extracts can be calculated separately using the formula,

C = cV/m

Where,

C = Total content of flavonoid compounds in mg/g, in quercetin equivalent (QE)

64 c = Concentration of quercetin established from the calibration curve in mg/mL

V = Volume of extract in mL m= Weight of plant extract

Data were recorded as a mean of these determinations of absorbance for each concentration, from which linear coefficient (R2) value can be calculated. The regression equation is given as,

Y = mx + c

Where,

Y = Absorbance of plant extracts x = Concentration of plant extracts m = Slope from the calibration curve c = Intercept

Using this regression equation concentration of extracts can be calculated. Thus, with the calculated value of concentration of each extract, the flavonoid content can be calculated.

3.2.10 In-Vitro antimicrobial activity

3.2.10.1 Preparation of culture media

3.2.10.2 Nutrient agar (NA)

About twenty eight gram of the powder (Hi Media Laboratories Pvt. Ltd, Mumbai, India) was carefully weighed and poured in distilled water. The contents were dissolved on the water completely and the final volume was maintained to 1000 mL followed by boiling for uniform mixing. This media was sterilized on an autoclave at 15l bs pressure at 121 °C for 15 minutes. The autoclave tape was used as an indicator for the completeness of sterilization. After this the media was taken out of the autoclave and cooled to about 45-50 °C and poured on sterilized and properly labeled petridishes. About 20 mL of the media was poured on each petridishes of 9 cm diameter. After this plates were left for the solidification. The pouring process was carried out on the sterile cabinet. For the preparation of the slant media screw tight bottles were filled with the media followed by autoclaving in the condition as

65 mentioned above and placing in an inclined position. These bottles were left for solidification.

3.2.10.3 Preparation of mueller hinton agar (MHA)

Thirty eight grams (Hi Media Laboratories Pvt. Ltd, Mumbai, India) of the powder was weighed and suspended in distilled water. The final volume was maintained 1000 mL. The content was heated to boiling to dissolve the medium completely. The media was sterilized by autoclaving at 15 lbs pressure and 121 °C for 15 minutes. The media was mixed carefully before pouring. The media was poured on sterile petridishes under aseptic conditions for further proposes (Maharjan, Mainali & Baral, 2011).

3.2.10.4 Preparation of standard culture inoculums

The individual pure culture of bacteria Bacillus subtilic, Escherichia coli, Salmonella typhi and Staphylococcus aureus were streaked on the different nutrient agar plates. Those plates were incubated on the incubator at 37 °C for about 24 hours and pure and isolated colonies were obtained. Each distant colony was aseptically transferred to the Luria Bertani (LB broth) for the suspension culture with the help of the sterilized inoculating loop. The inoculated bottles were kept on the shaking incubator at 37 °C and 120 rpm for overnight. These inoculums were used for the swapping of the plates to test the antimicrobial affects of the plant extracts.

3.2.10.5 Transfer of the bacteria on the petriplates

The test plates for the antimicrobial activity were first labeled with date, name of bacteria, and name of the plant species and the concentration of the plant extract to be added. The MHA plates were inoculated with the appropriate bacterial culture by a sterile cotton swab. One swab was used for one bacterium. The culture plates were allowed to dry for about five minutes.

3.2.10.6 Antibacterial test

The antimicrobial test was performed by modified agar diffusion method prescribed by (Nino, Narvaez, Mosquera & Correa, 2006) with slight modification. On the above prepared MHA plates five wells were prepared on the solid MHA media with the help of the sterile cork borer of 6 mm diameter. Three different concentrations (10 mg/mL, 15 mg /mL and 20 mg /mL) of the plant sample were prepared on the methanol. With the help of the sterile pipette the 50 µL of the each individual plant extract were

66 poured in the above prepared well. The methanol was taken as negative control while the penicillin at the concentration of the 50 µg/mL was taken as the positive control for the Gram-positive bacteria Bacillus subtilic and Staphylococcus aureus while tetracycline a broad spectrum antibiotic was taken as the positive control for Gram- negative bacteria Escherichia coli and Salmonella typhi. The plates were incubated on the microbial incubator overnight at 37 °C and the zone of inhibition was observed and noted for individual plant extract of individual bacteria for different concentration for further analysis.

3.2.10.7 Antimicrobial screening

Nutrient agar was added in distilled water in the ratio of 28 g/litre in appropriate size of conical flask and boiled with continuous shaking and autoclaved at 121 oC for 30 minutes. Sterilized media was allowed to cool about 50 oC. They were distributed in the sterile Petri-plates of size of 90 mm diameter in the ratio 25 mL per plate aseptically and labeled properly. Plates were left as such for solidification. The antibacterial of crude extracts of medicinal plants were screened against the test organisms by agar well diffusion method (Balouiri, Sadiki & Ibnsouda, 2016). Sterile Muller Hinton Agar (MHA) plates of approximately 4 mm thickness were prepared. Before using the plates, they were dried under hot air oven at appropriated temperature to remove excess of moisture from the surface of the media. The fresh inoculums comparable with turbidity standard were prepared. Then a sterile cotton swab was taken out and was dipped into the prepared inoculums. The excess of inoculums was removed by pressing and rotating against the upper inside side wall of the tube above the liquid level and then swabbed carefully all over the plate. The plate was rotated through the angle of 60o after each swabbing. Finally, the swab was passed round the edges of the agar surface. The inoculated plates were left to dry for few minutes at room temperature with the lid closed. Then with the help of sterile cork borer no 5, wells were made in the inoculated media plates and labeled properly. So, the diameter of a well was 6 mm. Then 50 µL of the test compound was introduced into respective well. In one well pure methanol was filled as control. The plates were then left for half an hour with the lid closed so that the extract diffused into media. The plate was incubated overnight at 37 oC. After proper incubation (18-24 hours) the plates were observed for the zone of inhibition around, well. The triplicate assay was performed in

67 the case of presence of zone of inhibition. The ZOI were measured using scale and mean was recorded.

3.3 Preferential cytotoxicity against PANC-1 cancer cell line

Testing cytotoxicity against PANC-1 cancer cell line was done according to the protocol adopted (Izuishi, Kato, Ogura, Kinoshita & Esumi, 2002) at University of Toyama Japan. In this method PANC-1 cancer cells were seeded in different plates and incubated in fresh Dulbecco’s Modified Eagle’s Medium at 37 oC under 5 percent

CO2 and 95 percent air for 24 hrs. The cell was then washed With PBS. After then the medium was changed to either DMEM or Nutrient Deprived Medium (NDM), absence of glucose, amino acid and serum, followed by immediate addition of serial dilution of the test samples. After 24 hour incubation, the cells were washed with PBS. Then 100 µL of DMEM with 10 percent WST-8 cell counting kit solution was added to the sample and kept for 2 hrs. Then the absorbance of the wells at 450 nm was measured.

Protocol for PANC-1 cytotoxicity assay

Test samples at different 20000 cells/well in concentration in DMEM DMEM (100 µL / well) Aspirate medium and or NDM (100 µL / well) wash with PBS

24 h incubation for attachment

96-well plate 24 h incubation

at 37 oC

5% CO2

3 h incubation

Aspirate 10% Cell Counting kit 8 medium Absorbance measured at wash with in DMEM (100 μl /well) 450 nm PBS

Figure 3: Preferential cytotoxic activity test against PANC-1 cell lines

Thus, going through the preferential cytotoxicity against PANC-1 cell line and DPPH bioassay screening methods only two plants; Scoparia dulcis and Bridelia retusa, were found as potent sources of active compounds against pancreatic cancer and antioxidant activity which ultimately prevents from diabetes. Some other plants were also found as potent sources of active compounds but they were not considered as antidiabetic plants because neither the literatures reviewed simply hinted their potentiality as source of antidiabetic constituents nor they are regarded as medicinal plants in curing diabetes by local people including traditional healers. Hence two plant samples; Scoparia dulcis and Bridelia retusa as sources of anticancer and antioxidants were selected for further study.

3.4 Isolation of pure compounds from Scoparia dulcis Linn.

3.4.1 Collection of plant samples

The plant Scoparia dulcis Linn was collected from Chitwan district of Nepal in July 2013. The plant was identified by Rita Chhetry, Research Officer, National Herbarium and Plant Resources, Ministry of Forest and Soil Conservation, Godawari, Nepal. A voucher specimen SD 2812 has been submitted to the same department in order to identify the plant.

3.4.2 Extraction and isolation of pure compounds

The shade dried whole plant of Scoparia dulcis (1.7 kg) was extracted with 80 percent methanol water (4.0 L) for three times. The concentrated methanolic extract (72.0 g) after evaporation of solvent was then dissolved in distilled water (1.5 L). The aqueous layer was then subjected to solvent-solvent extraction. In the beginning, the aqueous layer was extracted with n-hexanes (each 1.5 L volume of aqueous layer three times with 1.5 L of n-hexanes). After evaporation of n-hexanes, 15.0 g of the crude hexane fraction was obtained. The aqueous layer was then extracted with CH2Cl2 and 2.0 g of crude CH2Cl2 fraction was obtained. The aqueous layer was then extracted with ethyl acetate and 10.0 g of crude ethyl acetate fraction was obtained. The dichloromethane (DCM) fraction (2.0 g) was subjected to column chromatography in order of increasing polarity of ethyl acetate in hexanes, which yielded many sub-fractions. Out of them sub-fraction B (800 mg) obtained by 10 percent EtOAc/hexanes was further subjected to silica gel column chromatography using 10 percent EtOAc/hexanes as an eluting agent which yielded compound 1 (45.0 mg). The hexanes fraction (15.0 g) was

69 eluted in column chromatography using silica gel with n- hexanes and ethyl acetate. In order of increasing polarity of ethyl acetate in n-hexane yielded five sub-fractions. The sub-fraction A (800 mg), obtained by pure hexanes, was further subjected to column chromatography using pure hexanes as eluting agent to obtain the pure compound 3 (40.0 mg). The sub-fraction B (750.0 mg), obtained by 5 percent EtOAc/hexanes, was subjected for column chromatography on silica gel using 5 percent EtOAc/hexanes as eluting agent to obtain compounds 4 (35.0 mg), and compound 2 (50.0 mg). The sub- fraction E (900.0 mg), obtained from 30 percent EtOAc/hexanes, was further subjected to column chromatography using 20 percent EtOAc/hexanes to obtain compound 5 (50.0 mg), and compound 6 (10.0 mg). From the sub fraction A (800 mg) obtained by pure hexane, was further subjected to column chromatography using pure hexane as eluting agent to obtain the pure compound 7 (60.0 mg) and from sub fraction D (850 mg) on obtained by 15 percent ethyl acetate in hexane, was further subjected to column chromatography using 10 percent ethyl acetate as eluting agent to obtain the pure compound 8 (10.0 mg).

Scoparia dulcis (1.7 kg)

Extracted with 80% MeOH-H2O

Crude extract (72.0 g) Dissolved in distilled water (1.5 L)

Water soluble Extracted with n-hexane

n-hexane fraction (15.0 g) Water soluble Extracted with (CH2Cl2)

CH2Cl2 neutral fraction (2.0 g) Water soluble Extracted with ethyl acetate

Water soluble fraction (10.0 g) Ethyl acetate fraction (20.0 g) g) Figure 4: Fractionation of crude methanolic extract of S. dulcis

Dichloromethane fraction (2.0 g)

Column chromatography using silica gel (70-230, mesh, 200 g) eluted with ethyl acetate and n-hexane

Fr- 1 to Fr-12 Fr-13 to Fr-27 Fr-28 to Fr-48 10% Fr-49 to Fr-78 30% Pure hexane 5% to 10% (1 L) to 20% (2 L) to 40% (2.5 L) (100.0 mg) (150.0 mg) (300.0 mg) (200.0 mg)

Column chromatography using silica (10% ethyl acetate in hexane)

Compound 1 (45.0 mg)

Figure 5: Isolation of coixol (1) from dichloromethane fraction of Scoparia dulcis

Hexane fraction (15.0 g)

Column chromatography (Silica gel, 100g) Eluted with n-hexane and ethyl acetate (0-100%)

Fr-1 to Fr-27 Fr- 28 to Fr- 40 Fr-41 to Fr 62 10% Fr-63 to Fr-85 Pure hexane Fr-86 to Fr-120 5% ethyl acetate ethyl acetate 15% ethyl acetate 20% ethyl acetate (800.0 mg) (750.0 mg) (650.0 mg) (850.0 mg) (900.0 mg)

Friedelin 4 Glutinol 2 Sigmastanone 8 (35.0 mg) (50.0 mg) (10.0 mg)

Glutinone 3 β-sitosterol 7 Tetratriacontan 1-ol 6 Betulinic acid 5 (40.0 mg) (60.0 mg) (10.0 mg) (50.0 mg)

Figure 6: Isolation of compounds 2, 3, 4, 5, 6, 7 and 8 from hexane fraction of Scoparia dulcis

3.5 Isolation of pure compounds from Bridelia retusa

3.5.1 Plant materials The bark of Bridelia retusa (Euphorbiaceae) was collected in the January 2, 2013 from Syangja district, Chinnebash 6. The plant was identified by Rita Chhetry, Research Officer, National Herbarium and Plant Resources, Ministry of Forests and Soil Conservation, Godawari, Nepal. A voucher specimen 3424 has been submitted to the same department. 3.5.2 Extraction The plant sample was shade dried at room temperature and powdered material was then weighed (8.5 kg), soaked in methanol water (80 percent) for 72 h and filtered. The filtrate obtained was concentrated under reduced pressure in a rotatory evaporator to obtain the crude extract (500 g).The crude extract was dissolved in distilled water (4.0 L) and extracted by solvent-solvent extraction with increasing order of polarity. The crude extract was not soluble in hexane and dichloromethane due to which it was further extracted with ethyl acetate. 3.5.3 Isolation of pure compounds from bark extract of Bridelia retusa

The shade dried bark of B. retusa (8.5 kg) was extracted with 80 percent methanol water (30.0 L) for three times. The concentrated methanolic extract (500.0 g) after evaporation of solvent was then dissolved distilled water (5.0 L). The aqueous layer was then subjected to solvent-solvent extraction. In the beginning, the aqueous layer was extracted with n-hexanes (each 2.0 L volume of aqueous layer three times with

2.0 L of n-hexanes) and the aqueous layer was then extracted with CH2Cl2. The extract was insoluble in both the hexanes and dichloromethane solvents. The aqueous layer was then extracted with ethyl acetate and 15.0 g of crude ethyl acetate fraction was obtained. The ethyl acetate fraction (15.0 g) was subjected to column chromatography in order of increasing polarity of ethyl acetate in hexane, which yielded many sub fractions. Out of them sub fraction B (3.0 g) obtained by 25 percent EtOAc/hexane was further subjected to silica gel column chromatography using 15 percent EtOAc/hexane as an eluting agent which yielded compound 1 (10.0 mg). The sub fraction A (1.5 g) obtained by 10 percent EtOAc/hexanes was further subjected to silica gel column chromatography using 5 percent EtOAc/hexanes as an eluting agent which yielded compound 2 (65.0 mg). The sub fraction C (2.0 g) obtained by 40

percent EtOAc/hexanes was further subjected to silica gel column chromatography using 30% EtOAc/hexanes as an eluting agent which yielded compound 3 (10.0 mg).

Bridelia retusa bark (8.5 kg)

Extracted with 80% MeOH-H2O Crude extract (500 g)

Dissolved in distilled water (5 L)

Water soluble Extracted with n-hexane

Insoluble in n-hexane Water soluble

Extracted with (CH2Cl2)

Insoluble in CH2Cl2 Water soluble

Extracted with ethyl acetate

Ethyl acetate soluble fraction ( 15 .0 g) Water soluble fraction (460 g)

Figure 7: Fractionation of crude methanolic extract of Bridelia retusa bark

Ethyl acetate fraction (15.0 g)

Column chromatography using silica gel (70-230, mesh, 200 g) eluted with ethyl acetate and n-hexane

Fr- 1 to Fr-12 Fr-13 to Fr-27 Fr-28 to Fr-48 Fr-49 to Fr 78 1% to 5% (750 mL) 5% to 10% (1 L) 10% to 25% (2 L) 30% to 40% (2.5 L) (100 mg) (300 mg) (200 mg) (200 mg)

Compound (2), Compound (1), Compound (3) β-sitosterol

β-sitosterol (65.0 mg) Tambulin (20.0 mg) glucoside (10.0 mg)

Figure 8: Isolation of compounds 1, 2 and 3 from ethyl acetate fraction of Bridelia retusa bark

3.5.3.1 Coixol (1)

The dichloromethane fraction (2.0 gm) of Scoparia dulcis was subjected to silica gel column chromatography and eluted with ethyl acetate and hexane in increasing order of polarity resulting in the isolation of needle shaped crystalline coixol (1).

Physical state: white crystalline

Molecular weight: 165.04

Molecular formula: C8H7NO3

Yield: 45.0 mg 0.0625%

M.P: 151-156 oC Coixol (1) EIMs m/z (Relative intensity %): 165.04 (100.0%), 166.05 (8.8%)

Elemental Analysis: C, 58.18; H, 4.27; N, 8.48; O, 29.06

3.5.3.2 Glutinol (2) Specific rotation: The hexane fraction (15.0 gm) of Scoparia dulcis was subjected for column chromatography by using silica gel, fraction 41 - 62 in 10 percent ethyl acetate and hexane were collected in the vial. The glutinol (2) was obtained by further silica gel column chromatography.

Physical state: white crystalline

Molecular weight: 426.7174

Molecular formula: C30H50O

Yield: 50.0 mg 0.0694% M.P: 206-208 oC EIMs m/z (Relative intensity %): 426.39 (100.0%), 427.39 (33.1%),

428.39 (5.3%) Glutinol (2) Elemental Analysis: C, 84.44; H, 3.5.3.3 Glutinone (3) 11.81; O, 3.75

The hexane fraction (15.0 gm) of Scoparia dulcis was subjected for column chromatography by using silica gel, fraction 1-27 (800.0 mg) in pure hexane was

74 collected in the vial. The glutinone (3) was obtained by further silica gel column chromatography.

Physical state: white crystalline Molecular weight: 424.37

Molecular formula: C30H50O Yield: 40.0 mg 0.0555% M.P: 206-208 oC EIMs m/z (Relative intensity %): 424.37

(100.0%), 425.37 (32.5%), 426.38 Glutinone (3) (5.3%) Elemental Analysis: C, 84.84; H, 11.39; O, 3.77

3.5.3.4 Friedelin (4)

The hexane fraction (15.0 gm) of Scoparia dulcis was subjected for column chromatography by using silica gel, fraction 28-40 (750.0 mg) in 5 percent ethyl acetate and hexane was collected in the vial. The friedelin (4) was obtained by further silica gel column chromatography.

Physical state: white crystalline

Molecular weight: 426.39

Molecular formula: C30H50O

Yield: 35.0 mg 0.0486%

M.P: 262 oC

EIMs m/z (Relative intensity %): 426.39 Friedelin (4) (100.0%), 427.39 (33.1%), 428.39 (5.3%) Elemental Analysis: C, 84.44; H, 11.81; O,

3.75

3.5.3.5 Betulinic acid (5)

The hexane fraction (15.0 gm) of Scoparia dulcis was subjected for column chromatography by using silica gel, fraction 86-120 (900 mg) in 20 percent ethyl acetate and hexane was collected in the vial. The betulinic acid (5) was obtained by further silica gel column chromatography. Physical state: white crystalline

Molecular weight: 456.36

Molecular formula: C30H48O3

Yield: 50.0 mg 0.0694%

M.P: 316 oC

EIMs m/z (Relative intensity %): 456.36 Betulinic acid (5) (100.0%), 457.36 (32.6%), 458.37 (5.3%)

Elemental Analysis: C, 78.90; H, 10.59; O, 10.51

3.5.3.6 Tetratriacontan-1-ol (6)

The hexane fraction (15.0 gm) of Scoparia dulcis was subjected for column chromatography by using silica gel, fraction 86-120 (900.0 mg) in 20 percent ethyl acetate and hexane was collected in the vial. The tetratriacontan-1-ol (6) was obtained by further silica gel column chromatography.

Physical state: white crystalline

Molecular weight: 494.54

Molecular formula: C34H70O Yield: 10.0 mg 0.0138% o Tetratriacontan-1-ol (6) M.P: 135 C EIMs m/z (Relative intensity %):

494.54 (100.0%), 495.55 (37.6%), 496.55 (7.1%)

Elemental Analysis: C, 82.51; H, 14.26; O, 3.23

3.5.3.7 β-sitosterol (7)

The hexane fraction (15.0 gm) of Scoparia dulcis was subjected for column chromatography by using silica gel, fraction 1-27 (800.0 mg) in pure hexane was collected in the vial. The β-sitosterol (7) was obtained by further silica gel column chromatography. Physical state: white crystalline

Molecular weight: 414.39

Molecular formula: C29H50O

Yield: 60.0 mg 0.0833%

M.P: 132 oC

β-sitosterol (7) EIMs m/z (Relative intensity %): 414.39 (100.0%), 415.39 (32.0%),

416.39 (5.0%) 3.5.3.8 Sigmastanone (8) Elemental Analysis: C, 83.99; H, The hexane fraction (15.0 gm) of Scoparia12.15; dulcisO, 3.86 was subjected for column chromatography by using silica gel, fraction 63-85 (850.0 mg) in 15 percent ethyl acetate in hexane was collected in the vial. The sigmastanone (8) was obtained by further silica gel column chromatography.

Physical state: white crystalline

Molecular weight: 414.39

Molecular formula: C29H50O

Yield: 10.0 mg 0.0138%

EIMs m/z (Relative intensity %): 414.39 (100.0%), 415.39 (32.0%), Sigmastanone (8) 416.39 (5.0%)

Elemental Analysis: C, 83.99; H, 12.15; O, 3.86

3.5.3.9 Tambulin (9)

The ethyl acetate fraction (15.0 gm) of Bridelia retusa bark was subjected for column chromatography by using silica gel, fraction 28-48 (3.0 gm) in 20 percent ethyl acetate and hexane was collected in the vial. The tambulin (9) was obtained by further silica gel column chromatography. Physical state: yellow crystalline

Molecular weight: 344.32

Molecular formula: C18H16O7

Yield: 20.0 mg 0.013%

M.P: 205 oC

Tambulin (9) EIMs m/z (Relative intensity %): 344.09 (100.0%), 345.09 (19.7%), 346.10 (1.9%), 346.09 (1.4%)

Elemental Analysis: C, 62.79; H, 4.68; O, 32.53

3.5.3.10 β-sitosterol glucoside (10)

The ethyl acetate fraction (15.0 gm) of Bridelia retusa bark was subjected for column chromatography by using silica gel, fraction 49-78 (2.0 gm) in 30 percent ethyl acetate and hexane was collected in the vial. The β-sitosterol glucoside (10) was obtained by further silica gel column chromatography.

Physical state: white crystalline Molecular weight: 576.85

Molecular formula: C35H60O6 Yield: 10.0 mg, 0.002% EIMs m/z (Relative intensity %) m/z: 576.44 (100.0%), 577.44 (38.1%), 578.45 (7.3%) β-sitosterol glucoside (10) Elemental Analysis: C, 72.87; H, 10.48 ; O, 16.64

3.6 Biological assay of isolated pure compounds

3.6.1 Antidiabetic activity of coixol (1)

Coixol (1) isolated from dichloromethane soluble fraction of Scoparia dulcis was subjected for antidiabetic activity in mouse insulinoma pancreatic beta cells (MIN-6). It is because some of the compounds found in Scoparia dulcis were likely to be active against pancreatic cancer that protects diabetes as well.

3.6.1.1 Islets isolation and insulin secretion assay

Isolation of islets and insulin secretion assay was carried out as described previously (Siddiqui, Hasana, Mairaj, Mehmood, Hafizur, Hameed & Khan, 2014). In brief, batches of three size-matched islets were incubated for 60 min in KRB buffer solution with 3 mM (basal) or 16.7 mM (stimulatory) glucose, supplemented with test compound. At the end of incubation, 100 μL aliquots were removed from each tubes and secreted insulin was measured using an ultra sensitive mouse insulin ELISA kit. Insulin concentrations were normalized for the number of islets.

3.6.1.2 MIN-6 cell culture and insulin secretion assay

Mouse insulinoma pancreatic beta cells (MIN-6) were kindly provided by Dr. Jun-Ichi Miyazaki (Osaka University, Japan) and cultured as described previously (Miyazaki, Araki,Yamato, Ikegami, Asano, Shibasaki, Oka & Yamamura, 1990). Briefly, MIN-6 cells were cultured in Dulbecco’s Modified Eagle’s Medium containing 25 mM glucose supplemented with 12 percent fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and 5 µL β-mercaptoethanol at 37 ºC in a humidified atmosphere of 5 percent CO2 and 95 percent air. Cells of passage number 20-27 were used for insulin secretion assay.

MIN-6 Cells were seeded onto 24-well plates at a density of 5 x 105 cells per well. After 24 hours of plating, cells were washed twice and pre-incubated with Krebs-

Ringer HEPES buffer containing 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM

MgSO4, 1.2 mM KH2PO4, 10 mM HEPES (pH 7.4), 2 mM glucose, 0.1 percent BSA for 60 min at 37 ºC. These cells were then incubated in Krebs-Ringer HEPES buffer containing 16.7 mM glucose with or without test compound for 60 minutes at 37 ºC. After incubation, 100 µL supernatant was collected, centrifuged at 1000 rpm, and stored at -40 ºC until insulin assay. Insulin was measured using an ultra sensitive mouse insulin ELISA kit. 79

3.6.1.3 Toxicity assay

Cytotoxicity of coixol (1) was evaluated 3T3 cell line in 96-well flat bottom micro- titer plates by using the standard MTT (3-[4,5-dimethylthiazole-2-yl]-2,5- diphenyltetrazolium bromide) colorimetric assay. In this assay coixol (1) showed no toxic effect (IC50 = 200 μM).

It has been reported that, a single intraperitoneal dose of 500 mg/kg coixol in mice resulted in transient sedation but no mortality (Zhu, 1998). A single intravenous dose of 100 mg/kg of coixol caused no death or any abnormal manifestations in mice. There was no toxic reaction after oral administration of 20,100, or 500 mg/kg coixol for 30 days (Wang, 1983). We also did toxicity study with coixol in mice. Coixol was orally administered to mice at a dose of 100 mg/kg and mortality and general behaviors of the mice were observed continuously for the initial 4h and intermittently for the next 6h and then again at 24, 48 and 72h following coixol administration. After 72h of coixol treatment autopsy was performed for checking any abnormalities in the liver, kidney, gastrointestinal tract, spleen and heart. Serum creatinine, ALT and AST, were also done to check the toxicity to kidney and liver, respectively. The acute and sub- acute toxicity results showed no mortality or any abnormal manifestation in mice at a dose of 100 mg/kg body weight and no significant change was observed in serum creatinine, ALT and AST levels.

3.6.2 Immunomodulatory activity of glutinone (3)

Glutinone (3) isolated from hexane soluble fraction of Scoparia dulcis was subjected for immunomodulatory activity for determination of ROS by Chemiluminescence assay, nitric oxide (NO) assay and cytokine assay.

3.6.2.1 Determination of ROS by chemiluminescence assay

Oxidative burst studies using chemiluminescence technique was performed as described by (Helfand, Werkmeister & Roder, 1982). The assay was performed on whole blood from healthy human volunteers and on isolated neutrophils using luminol as a probe, and zymosan as an activator. Briefly 25 µL of whole blood (1:20 dilution in HBSS++) [Sigma, St. Louis, USA] or isolated neutrophils (1x106 cells/mL) were incubated with 25 µL of different concentrations of compounds (1, 10 and 100 µg/mL) each in triplicate in white half area 96 well plates [Costar, NY, USA]. The plate was incubated at 37 ºC for 15 min in the thermostat chamber of luminometer [Labsystems,

Helsinki, Finland]. 25 µL of (7 x 10-5 M) luminol [Research Organics, Cleveland, OH, USA], and 25 µL serum opsonized zymosan (SOZ) (2 mg/mL) [Fluka, Buchs, Switzerland] was then added into the wells. The plate was then read in luminometer for 50 min, and results were recorded as total integral readings as relative light units (RLU).

3.6.2.2 Nitric oxide (NO) assay

The assay was performed as previously described by (Andrade, Siles-Lucas, Arellano, Barreto, Valladares et al., 2005). Briefly (1x106 cells/mL) macrophages from J774.2 cell line (European Collection of Cell Cultures, UK) were plated in 24-well tissue culture plates. Cells were activated by adding 30 µg/mL of E. coli lipopolysaccharide (LPS) (DIFCO Laboratories michigon, USA) and treated with test compound at a concentration 25 µg/mL. The supernatant was collected after 48 hours for analysis. Nitrites (a stable product of nitric oxide) concentration in supernatant was measured using the griess reagent.

3.6.2.3 Cytokine assay Effect of compound on the release of proinflammatory cytokines TNF-α and IL-1β was performed by method previously described by (Singh, Tabibian, Venuqopal, Devraj & Jialal, 2005). Briefly THP-1 cells (European Collection of Cell Cultures, UK) were differentiated by adding 20 ng/mL of phorbol myristate acetate (PMA),

(SERVA, Heidelberg, Germany) for 24 hours at 37 ºC in 5 percent CO2 and then stimulated with 50 ng/mL E. coli Lipopolysaccharide B (DIFCO Laboratories, michigon, USA). 25 µg/mL of test compound was then added for four hours, and plate was incubated at 37 ºC in 5 percent CO2. Cytokines quantification in supernatants was performed using the human TNF-α and IL-1β Duo Set (R&D Systems, Minneapolis, USA), according to manufacturer’s instructions.

3.6.3 Cytotoxicity against MCF-7 (breast cancer) cell lines 3.6.3.1 Cell culture

Two human breast cancer cell lines (MCF-7, ER positive breast cancer cells (ATCC, HTB-22TM); MDA-MB-231, triple negative breast cancer cells (ATCC, HTB-26TM) and MCF-10A, normal mammary epithelial cells (ATCC, CRL-10317TM) were cultured in ATCC recommended medium containing 10 percent (v/v) fetal bovine serum (ATCC, 30-2020), streptomycin (S 9137-Sigma, 0.1 mg/mL), penicillin (P

3032-Sigma, 100 U/mL) and insulin (I 6634-Sigma, 0.01 mg/mL). MCF-7 and MCF-

10A cells were maintained in a humidified incubator at 37 ºC with 5 percent CO2.

MDA-MB-231 cells were cultured without CO2.

3.6.3.2 Cytotoxic assay

Two human cancer cell lines (MCF-7 and MDA-MB-231) and one non-carcinoma cell lines (MCF-10A) were used to assess cytotoxic activity of isolated compounds. Cancer cells, on 80 percent confluency, were trypsinized and plated in cell culture treated 96 well plates (5 x 103 cells/ well) and incubated for 24h. After incubation, cancer cells and normal cells were treated with different concentrations (5, 10, 15, 20 and 25 µg/mL) of isolated compounds. After the incubation period (24h) cells were fixed with 50 μL of ice-cold 50 percent trichloroacetic acid solution. The plates were then stored for 60 min at 4 ºC and wells were then rinsed five times with tap water and stained with 0.4 percent SRB solution (100 μL stain/well) for 15 min. Unbound SRB dye was removed by washing five times with1 percent acetic acid solution. Unbuffered Tris- basesolution (200 μL/well) was added to solubilize unbound SRB dye and plates were placed on a plate shaker for 1 h at room temperature. Absorbance was read at OD 540 nm using a microplate reader and the results expressed as a percentage of control values (Nohara, Wang & Spiegel, 1998).

3.6.4 Urease inhibition assay

Reaction mixtures comprising 25 μL of enzyme (jack bean urease) solution and 55 μL of buffers containing 100 mM urea were incubated with 5 μL of test compounds (0.5 mM concentration) at 30 °C for 15 min in 96-well plates. Urease activity was determined by measuring ammonia production using the indophenol method as described by Weather burn. Briefly, 45 μL each phenol reagent (1 percent w/v phenol and 0.005 percent w/v sodium nitroprusside) and 70 μL of alkali reagent (0.5 percent w/v NaOH and 0.1 percent active chloride NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50 min, using a micro plate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200 μL. The results (change in absorbance per min) were processed by using soft Max Pro software (molecular Device, USA). The entire assays were performed at pH 6.8. Percentage inhibitions were calculated from the formula. 100 -

(ODtest well /ODcontrol) *100. Thiourea was used as the standard urease inhibitor.

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Results and discussion

4.2 The yield percentage of plant extracts

The yield percentage of fifty selected medicinal plant extracts collected from different regions of Nepal showed varying level of extracts (Fig. 9, 9a & 9b). The result showed that some of the plants; Pterocarpus santalinus and Curcuma angustifolia, were good sources of secondary metabolites and some others; Oxalis corniculata and Coccinia grandis, were very poor sources of secondary metabolites. The plants with rich secondary metabolites likely to have large number of compounds and can be used for isolation of pure compounds.

Figure 9: Yield percentage of plant extracts

Figure 9a: Yield percentage of plant extracts

Figure 9b: Yield percentage of plant extracts

4.3 Phytochemical screening of plant extracts

All the methanolic plant extracts were found rich in secondary metabolites such as alkaloids, flavonoids, steroids, terpenoids, reducing sugars, glycosides, polyphenols, tannins, cardiac glycosides, anthraquinone, saponins and carotenoids (Sharma, Kalauni, Awale & Pokharel, 2015).

4.4 Antioxidant activity (DPPH radical scavenging assay)

Free radical scavenging activity was determined by using 2,2-diphenyl-1-picryl hydrazyl radical (DPPH), which is very stable free radical having purple color. When

84 free radical scavengers were added, DPPH was reduced and its color was changed to yellow based on the efficacy of antioxidants. Scavenging of DPPH free radical determines the free radical scavenging capacity or antioxidant potential of test sample which showed its effectiveness, prevention, interception and repair mechanism against injury in a biological system.

DPPH assay was conducted for each plant extract by using ascorbic acid as standard. In this assay, different concentrations of different extract solutions and ascorbic acid solution were incubated at room temperature and their absorbance was recorded by spectrophotometer. The IC50 values of each extract were calculated.

Antioxidant activity shows the reducing power of the different plant extracts expressed as ascorbic acid equivalents. Generally, the reducing power of the different extracts was found to vary significantly and antioxidant properties were evaluated to find a new natural source of antioxidant.

Concentration against absorbance

Absorbance

Concentration of ascorbic acid (µg/mL)

Figure 10: Calibration curve of standard ascorbic acid

Figure 11: Free radical scavenging activity of active plant extracts

Out of fifty selected medicinal plants, fifteen plant extracts showed potent antioxidant activity against DPPH radical scavenging assay. Plant extracts such as Acacia catechu, Bauhinia variegata, Shorea robusta and Berberis aristata showed high

antioxidant activity with low inhibitory concentration IC50 (Sharma et al., 2015).

scavenging

Percent Percent Scavemgimg %

Concentration µg/mL

Figure 12: Free radical scavenging activity and the concentration of plant extracts

ging Percent scaven Percent

Concentration µg/mL

Figure 12a: Free radical scavenging activity and concentration of plant extracts

4.5 Total phenolic content

The absorbance values of different concentration of each extract were recorded at 760 nm. The total phenolic content in different extracts was calculated with the help of calibration curve using regression equation y = 0.004x, R2=0.972 followed by the formula C= cV/m and expressed as mg GAE/gm of extract in dry weight (mg/g). The total phenolic content calculated in different methanolic extract of different plant material is given in Table 1.

Absorbance against gallic acid concentration

concentration

Absorbance

Concentration of gallic acid (µg/mL)

Figure 13: Calibration curve of standard gallic acid

Table 1: Total phenolic content in potent antioxidant plant extracts

Plant extracts Abs. Abs. Conc. Conc. mg GAE/g mg GAE/g mg GAE/g I II I II I II SEM

Drymaria diandra 1.215 1.234 303.75 308.50 121.50 123.40 122.45±0.96

Euphorbia hirta 1.430 1.332 357.50 333.00 143.00 133.20 138.10±4.90

Shorea robusta 1.408 1.508 352.00 377.00 140.80 150.80 145.80±5.00

Acacia catechu 1.696 1.691 424.00 422.75 169.60 169.10 169.35±0.25

Lyonia ovalifolia 1.362 1.393 340.50 348.25 136.20 139.30 137.75±1.55

Phyllanthus emblica 1.533 1.550 383.25 387.50 153.30 155.00 154.15±0.85

Berberis aristata 1.458 1.457 364.50 364.25 145.80 145.70 145.75±0.05

Bridelia retusa 1.457 1.485 364.25 371.25 145.70 148.50 147.20±1.50

Cleistocalyx operculatus 1.519 1.576 379.75 394.00 151.90 157.60 154.75±2.85

Bauhinia variegata 1.560 1.566 390.00 391.50 156.00 156.60 156.30±0.30

Bergenia ciliata 1.457 1.460 364.25 365.00 145.70 146.00 145.85±0.15

Bombax ceiba 1.460 1.483 365.00 370.75 146.60 148.30 147.45±0.85

Callicarpa sp. 1.267 1.285 316.75 321.25 126.70 128.50 127.60±0.90

Ziziphus mauritiana 0.929 0.994 232.25 248.50 92.90 99.40 95.80±3.60

Scoparia dulcis 1.458 1.457 364.50 364.25 145.80 145.70 145.75±0.05

Abs. = Absorbance Conc. = Concentration

The result showed that the total phenolic content in Acacia catechu has highest among the fifteen screened medicinal plants. Plant extracts of Bauhinia variegata,

Cleistocalyx operculatus, Berberis aristata and Phyllanthus emblica also showed relatively high total phenolic content. More importantly, the plant Bridelia retusa selected for further isolation of antioxidant was also found rich in total phenolic content (Sharma et al., 2015).

4.6 Total flavonoid content

The absorbance values for different concentration of each extract were recorded at 510 nm. The total flavonoid content in different extracts was calculated from the standard calibration curve using regression equation Y = 0.010x, R2=0.980 followed by the formula C= cV/m and expressed as mg QE/gm of extract in dry weight (mg/g). The total flavonoid contents calculated in different methanolic extracts of plant materials are given in table 2.

Absorbance against quercetin concentration

Absorbance

Concentration of quercetin µg/mL

Figure 14: Calibration curve of standard quercetin

Table 2: Total flavonoid content in potent antioxidant plant extracts

Plant extracts Abs. Abs. Conc. Conc. mg mg mg QE/g I II I II QE/g I QE/g II SEM

Drymaria diandra 1.185 1.117 118.50 111.70 11.85 11.17 11.51±0.30

Euphorbia hirta 1.146 1.162 114.60 116.20 11.46 11.62 11.54±0.00

Shorea robusta 1.568 1.408 156.80 140.80 15.68 14.08 14.88±0.80

Acacia catechu 1.831 1.896 183.10 189.60 18.31 18.96 18.63±0.30

Lyonia ovalifolia 1.251 1.262 125.10 126.20 12.51 12.62 12.56±0.00

Phyllanthus emblica 1.587 1.533 158.70 153.30 15.87 15.33 15.60±0.20

Berberis aristata 1.589 2.076 158.90 207.60 15.89 20.76 18.32±2.40

Bridelia retusa 1.671 1.657 167.10 165.70 16.71 16.57 16.64±0.00

Cleistocalyx operculatus 1.447 1.319 144.70 131.90 14.47 13.19 13.83±0.60

Bauhinia variegata 1.457 1.751 145.70 175.10 14.57 17.51 16.04±1.40

Bergenia ciliata 1.586 1.557 158.60 155.70 15.86 15.57 15.71±0.10

Bombax ceiba 1.042 1.066 104.20 106.60 10.42 10.66 12.54±0.10

Callicarpa sp. 1.061 1.079 106.10 107.90 10.61 10.79 10.70±0.09

Ziziphus mauritiana 1.116 1.116 111.60 111.60 11.16 11.16 11.16±3.60

Scoparia dulcis 1.042 1.066 104.30 107.02 10.50 10.70 12.54±0.10

Abs. = Absorbance Conc. = Concentration

The total phenolic content was found to be 95.80±3.6 mg GAE/gm in Ziziphus mauritiana and 169.35±0.2 mg GAE/mg in Acacia catechu. The total flavonoid content was found to be 10.70±0.0 mg QE/gm in Callicarpa sp. and 18.6±0.3 mg QE/gm in Acacia catechu. The extracts of Acacia catechu, Bauhinia variegata, Cleistocalyx operculatus, Phyllanthus emblica, Shorea robusta, Berberis aristata have high value of phenolic and flavonoid content exhibited the greatest antioxidant activity. The correlation between the total flavonoid and phenolic content with free

90 radical scavenging (IC50) values showed that higher the phenolic and flavonoid content lower the IC50 values and higher the antioxidant activity (Sharma et al., 2015).

The total phenolic content of these plant extracts are compared to the plant extracts of some previously studied plants. Total phenolics of some previously studied plant extracts was found as Origanum dictamnus (8.2±0.3 mg GA/gm), Eucalyptus globules

(10.5±0.3), Sideritis cretica (8.6±0.2), Thymus vulgaris (8.0±0.1), Satureja thymbra

(9.2±0.1), Lavandula vera (4.9±0.1), Lippatri phylla (7.7±0.1) and Matricaria chamomilla (6.1±0.1) (Zorica, Hatida, Albina, Majda & Mirzeta, 2009). The result showed that the plant extracts studied in this work are found as the potent sources of secondary metabolites and could be used as the sources to isolate the active ingredient.

4.7 Preferential cytotoxicity against pancreatic cancer cell lines (PANC-1)

Fifty selected medicinal plant extracts were submitted to the University of Toyama

Japan for screening of plant samples with preferential cytotoxicity against pancreatic cancer cell lines. The result showed that the plant extracts of Scoparia dulcis,

Euphorbia hirta and Bridelia retusa were found potent against pancreatic cancer cell lines under nutrient deprived condition.

Among fifty medicinal plants collected from different regions of Nepal, the plant extract of Scoparia dulcis showed 100 percent preferential cytotoxicity at the concentration of 10.00 µg/mL in nutrient deprived medium. Peoples have been using the plant since many years for treatment of diabetes and the plant is popularly known as antidiabetic plant in Nepalese community. The disease pancreatic cancer and diabetes has very unique relationship.The activity shown against pancreatic cancer implies that the plant is useful in preventing diabetes among pancreatic cancer patients as explained (Donghui, Yeung, Hassan, Konopleva & Abbruzzese, 2009).

Table 3: Preferential cytotoxicity against pancreatic cancer cell lines

Preferential cytotoxic concentration (PC50) Plant extracts in NDM (µg/mL) Scoparia dulcis 10.00 Euphorbia hirta 24.64 Pterocarpus santilinus 21.54 Oxalis corniculata 30.06 Curcuma angustifolia 27.87 Betula alnoides 39.88 Mahonia napaulensis 49.84 Ziziphus mauritiana 50.50 Desmostachya bipinnata 50.84 Bridelia retusa 59.23 Bergenia ciliata 53.99 Bauhinia variegata 52.11 Melia azadarach 48.71 Cissampelos pareira 52.35 Litsea cubeba 49.48 Pogostemon amarantoides 70.85 Astilbe rivularis 50.36 Piper mullesua 70.92 Bombax ceiba 72.90 Callicarpa sp. 72.90 Arctigenin (Standard) 2.38

4.8 Antimicrobial activity

The methanolic extract of Cleistocalyx operculatus ZOI value 19 mm/disc showed greater sensitivity for Bacillus subtilic, the plant extracts of Euphorbia hirta, Ziziphus mauritiana, Bergenia ciliata showed zone of inhibition against Bacillus subtilic 18 mm/disc. Aegle marmelos showed good sensitivity for Bacillus subtilic but has no sensitivity for the organism E. coli, Staphyllococcus aureus and Salmonella typhi. Some plant extracts like Bridelia retusa, Cleistocalyx operculatus, Acacia catechu, Justicia adhatoda, Bauhinia variegata, Berberis aristata, Curcuma angustifolia,

Betula alnoides, Shorea robusta, Ziziphus mauritiana, Euphorbia hirta showed sensitivity toward both Gram positive and Gram negative organism. Hence, these plant extracts are active sources of antimicrobial compound.

4.9 Anti-microbial screening of plant extracts

All plant extracts were tested against Gram positive and negative organisms. The result of antimicrobial screening is given in table 4, 4.1 and 4.2. Some antibiotics given in table 6 are used as positive control and methanol is used as negative control.

Table 4: Microbial screening of plant extracts zone of inhibition (ZOI) mm

Control Stayphylococcus Bacillus Salmonella Plant extracts E.coli (mm) aureus subtilic typhi Oxalis corniculata 6 - - - - Drymaria diandra 6 10 9 12 9 Melia azedarach 6 9 8 10 8 Cyperus rotundus 6 - 9 - 7 Cissampelos pareira 6 - - - - Coccinia grandis 6 - - - - Euphorbia hirta 6 9 11 9 9 Cynodon dactylon 6 9 9 7 9 Ageratum houstonianum 6 - 9 9 8 Curcuma angustifolia 6 13 12 11 11 Shorea robusta 6 12 11 13 10 Acacia catechu 6 14 11 14 13 Lyonia ovalifolia 6 - 9 7 - Pterocarpus santalinus 6 - - - - Demostachya bipinnata 6 - 7 7 - Cinnamomum tenuipile 6 7 - - - Justicia adhatoda 6 8 16 - -

Table 4.1: Microbial screening of plant extracts zone of inhibition (ZOI) mm

Control Stayphylococcus Bacillus Salmonella Plant extracts E.coli (mm) aureus subtilic typhi Aegle marmelos 6 7 15 - - Mahonia napaulensis 6 - - - - Phyllanthus emblica 6 9 13 10 10 Berberis aristata 6 11 - 11 - Tinospora cordifolia 6 - - - - Cuscuta reflexa 6 - 7 7 8 Leucas cephalotes 6 - 10 8 9 Drynaria propinqua 6 - - - - Tinospora sinensis 6 - - - - Centella asiatica 6 - - - - Asparagus filicinus 6 7 7 7 10 Achyranthes bidentata 6 - - - - Bridelia retusa 6 13 11 10 12 Litsea cubeba 6 10 13 10 10 Oxalis corniculata 6 - - - - Justicia adhatoda 6 - 9 - - Cleistocalyx operculatus 6 14 15 12 14

Table 4.2: Microbial screening of plant extracts zone of inhibition (ZOI) mm

Control Bacillus Plant extracts Stayphylococcus aureus Salmonella typhi E.coli (mm) subtilic Bauhinia Variegata 6 11 10 13 10 Pogostemon amaranthoides 6 - 16 - - Betula alnoides 6 9 11 10 10 Scoparia dulcis 6 - - - - Bergenia ciliata 6 9 16 10 11 Periploca calophylla 6 - - - - Astilbe rivularis 6 13 9 14 9 Piper mullesua 6 - - - - Bombax ceiba 6 10 6 9 8 Calotropis gigantea 6 12 - 7 - Annona reticulata 6 6 12 8 19 Callicarpa sp. 6 9 12 11 10 Mimosa pudica 6 - - - - Ziziphus mauritiana 6 9 15 13 9 Cascabela thevetia 6 - - - -

4.10 Antimicrobial activity of screened plant extracts

Out of fifty medicinal plants collected from different regions of Nepal, sixteen plant samples were found active against Gram positive and negative bacteria. These active plant extracts were further tested against these organisms in dose dependent manner. The list of active plants against these organisms is given in Table 5 and 5.1.

Table 5: Antimicrobial activity of screened plant extracts zone of inhibition (ZOI) mm

Control Concentration Staphylococcus Salmonella Bacillus Plant extracts E. coli (mm) (mg/mL) aureus typhi subtilic 6 10 13 11 6 6 Astilbe 6 15 13 11 6 7 revularis 6 20 14 12 8 8 6 10 12 10 11 16 Bergenia 6 15 12 12 12 16 ciliata 6 20 13 12 13 18 6 10 13 10 10 11 Bauhinia 6 15 14 10 11 12 variegata 6 20 14 11 11 14 6 10 13 13 11 14 Curcuma 6 15 14 15 14 15 angustifolia 6 20 16 16 16 16 6 10 11 9 10 13 Betula 6 15 11 10 12 14 alnoides 6 20 12 11 13 16 6 10 12 13 10 11 Shorea 6 15 12 15 10 11 robusta 6 20 13 16 11 12 6 10 9 12 9 15 Ziziphus 6 15 9 13 9 16 mauritiana 6 20 10 12 9 18 6 10 8 6 6 16 Litsea 6 15 9 6 6 17 cubeba 6 20 11 6 6 18

Table 5.1: Antimicrobial activity of screened plant extracts zone of inhibition (ZOI) mm

Plant Control Concentration Staphylococcus Salmonella E. Bacillus extracts (mm) (mg/mL) aureus typhi coli subtilic 6 10 13 12 16 11 Bridelia 6 15 14 13 17 16 retusa 6 20 19 16 19 18 6 10 18 13 16 18 Cleistocalyx 6 15 19 15 18 19 operculatus 6 20 20 16 19 19 6 10 10 12 9 9 Drymaria 6 15 11 10 10 10 diandra 6 20 11 11 10 12 6 10 8 15 12 10 Acacia 6 15 10 14 14 13 catechu 6 20 12 15 15 15 6 10 13 10 8 11 Phyllanthus 6 15 14 11 9 13 emblica 6 20 18 12 11 14 6 10 6 8 17 12 Annona 6 15 6 9 16 11 reticulata 6 20 6 10 15 12 6 10 8 6 6 15 Bombax ceiba 6 15 9 6 6 16 6 20 10 6 6 16 6 10 9 11 10 12 Callicarpa sp. 6 15 9 12 11 12 6 20 10 12 11 13

Table 6: Antimicrobial activity of drugs (positive control) against the organisms, ZOI mm

Conc. Stayphylococcus Bacillus Salmonella Drugs E. coli (µg)/disc aureus subtilic typhi Ciprofloxacin 5 32 Erythromycin 15 20 Nalidixic acid 30 24 Chloramphenicol 30 25

4.11 Structure elucidation of isolated pure compounds

4.11.1 Coixol (1)

Compound (1) was obtained as yellowish needles from DCM fraction of the methanolic extract of Scoparia dulcis. The EI-MS displayed the molecular ion [M+] at m/z 165. The UV spectrum showed absorptions at 291 and 230 nm. Its molecular 13 formula C8H7NO3 was deduced from EI-MS and C-NMR (BB and DEPT) spectra.

Figure15: Structure of coixol (1)

The 1H-NMR spectrum (in MeOD) of compound (1) displayed resonances for a methoxy group at δ 3.76 (s, H3-OMe), and three downfield signals at δ 6.71 (dd, J5, 4=

8.5 Hz, J5,7 =2.5 Hz; H-5); 6.86 (d, J7,5 = 2.5 Hz, H-7); and 6.94 (d, J4,5 = 8.5 Hz, H-4). The 13C-NMR spectrum (in MeOD) displayed resonances at δ 56.4 (C-OMe), 98.1 (C- 7), 110.6 (C-5), 110.9 (C-4), 124.9 (C-8), 146.1 (C-9), 157.6 (C-2), and 157.6 (C-6). Structure of the compound was further confirmed from 2D-NMR spectra (COSY, HSQC, HMBC and NOESY). All the spectral data of compound (1) was identical to the reported compound coixol (Nagao, Otsuka, Kohda, Sato & Yamasaki, 1985). Structures of compounds 2-6 were also identified by comparing their spectral data with the literature data (Mahmood et al., 1995; Chauhan et al., 2002; Chandramu et al., 2003; Xing et al., 2012).

Table 7: 1H- and 13C-NMR chemical shift value of coixol (MeOD, ppm, 500 MHz)

Carbon 1H-NMR (δ ppm) 13C-NMR (δ ppm) 13C-NMR (δ ppm) Multiplicity No. observed (MeOD) reported (DMSO) 2 157.60 155.10 C 4 6.94 (d, J4,5 = 8.5) 110.90 109.90 CH

5 6.71 (dd,J5,4 = 8.5, 110.60 109.10 CH

J5,7 = 2.5

6 H3OMe- 3.76 s 157.60 155.40 C

7 6.86 ( d, J7,5 = 2.5) 98.10 97.10 CH 8 124.90 123.90 C 9 146.10 144.30 C 56.40 55.80 OMe

All chemical assignment was made on the basis of 1H-NMR, COSY, HMBC and DEPT NMR technique (Sharma, Adhikari, Hafizur, Hameed, Raza, Kalauni, Miyazaki & Choudhary, 2015).

4.11.2 Glutinol (2)

1 13 Table 8: H- and C-NMR chemical shift value of glutinol (CDCl3, ppm, 500 MHz)

Carbon 1H-NMR (δ ppm) 13C-NMR (δ ppm) 13C-NMR (δ ppm) No. Multiplicity observed observed reported

1 1.42, 1.14 19.00 18.20 CH2

2 1.56, 1.32 27.70 27.80 CH2 3 3.44, 5.60(-OH) 76.30 76.40 CH 4 - 40.80 40.80 C 5 - 141.50 141.60 C 6 5.61 122.10 122.10 CH

7 2.00, 1.79 23.60 23.60 CH2 8 1.43 47.30 47.40 CH 9 - 34.60 34.90 C 10 1.93 49.60 49.70 CH

11 1.56, 1.32 34.60 34.60 CH2

12 1.56, 1.32 30.30 30.40 CH2 13 - 39.00 39.30 C 14 - 39.30 39.30 C

15 1.56, 1.32 33.00 33.10 CH2

16 1.56, 1.32 36.00 36.00 CH2 17 - 30.00 30.10 C 18 1.38 43.00 43.10 CH

19 1.45, 1.20 35.00 35.10 CH2 20 - 28.00 28.30 C

21 1.56, 1.31 32.00 32.10 CH2

22 1.56, 1.31 39.00 39.00 CH2

23 1.24 25.40 25.50 CH3

24 1.24 29.00 29.00 CH3

25 1.25 16.20 16.20 CH3

26 1.24 19.50 19.60 CH3

27 1.02 19.00 19.00 CH3

28 1.02 32.00 32.00 CH3

29 0.98 34.40 34.50 CH3

30 0.98 32.40 32.40 CH3

Figure 16: Structure of glutinol (2)

4.11.3 Glutinone (3)

Compound (3) was isolated from hexane fraction of methanloic extract of Scoparia dulcis Linn. The EI-MS showed a [M+.] at m/z 424 and a base peak at m/z 274. The +. molecular formula C30H40O, was deduced from the HREI-MS which showed a [M ] at 13 m/z 424.3721 (calcd. for C30H40O = 424.3705) and the C-NMR spectra (BB and DEPT). The IR spectrum indicated the presence of carbonyl (1706 cm-1), and olefinic group (1631 cm-1).

Figure 17: Structure of glutinone (3)

The 1H-NMR spectrum of compound (3) displayed singlets for protons of eight methyl groups at δ 0.79 (H-25), 0.93 (H-29), 0.96 (H-30), 1.01 (H-26), 1.07 (H-27), 1.14 (H- 28), 1.20 (H-23), and 1.22 (H-24). A downfield signal at δ 5.6 br m was assigned to olefinic H-6. Position of double bond between C-5/C-6 was inferred from the base peak at m/z 274 (EI-MS) due to the retro Diels -Alder cleavage of the B ring, which was further supported by the HMBC spectrum, in which H-23 and H-24 showed HMBC correlation with carbonyl C-3 (215.6) and olefinic C-5 (142.4). Structure of the compound was further confirmed from 2D-NMR spectra (COSY, HSQC, HMBC, and

NOESY). Structure of coixol (1), friedelin (4), glutinol (2), and betulinic acid (5) were identified by comparing their spectral data with reported data in literature (Nagao et al., 1985; Mahmood et al., 1995; Chauhan et al., 2002; Chandramu et al., 2003).

1 13 Table 9: H- and C-NMR chemical shift values of glutinone (CDCl3, ppm, 500 MHz)

Carbon 1H-NMR (δ ppm) 13C-NMR (δ ppm) 13C-NMR (δ ppm) Multiplicity No. observed observed reported

1 1.65, 1.45 21.50 21.80 CH2

2 2.50, 2.40 38.00 38.30 CH2 3 - 215.50 215.70 C 4 - 50.20 50.20 C 5 - 142.30 142.70 C 6 5.65 121.30 121.60 CH

7 2.00, 1.84 23.50 23.80 CH2 8 1.44 47.00 47.30 CH 9 - 35.90 35.50 C 10 1.95 51.00 50.90 CH

11 1.56, 1.36 34.00 34.30 CH2

12 1.56, 1.36 30.30 30.60 CH2 13 - 38.00 38.20 C 14 - 39.30 39.60 C

15 1.55, 1.36 35.90 35.30 CH2

16 1.57, 1.37 36.50 36.20 CH2 17 - 30.50 30.50 C 18 1.38 43.00 43.40 CH

19 1.46, 1.20 33.00 33.30 CH2 20 - 28.50 28.50 C

21 1.56, 1.36 32.00 32.20 CH2

22 1.56, 1.37 32.00 32.20 CH2

23 1.36 28.80 28.80 CH3

24 1.37 24.60 24.60 CH3

25 1.05 15.60 18.90 CH3

26 1.03 19.60 19.60 CH3

27 1.03 18.30 18.60 CH3

28 1.03 32.00 32.20 CH3

29 0.97 32.30 32.20 CH3

30 0.97 34.50 34.80 CH3

All chemical assignment was made on the basis of 1H-NMR, COSY, HMBC and DEPT NMR technique.

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4.11.4 Friedelin (4)

1 13 Table 10: H- and C-NMR chemical shift values of friedelin (CDCl3 ppm, 500 MHz)

1 13 13 Carbon H-NMR (δ ppm) C-NMR (δ ppm) C-NMR (δ ppm) No. Multiplicity observed observed reported

1 1.94,1.64 22.20 22.20 CH2

2 2.27, 2.29 41.50 41.40 CH2 3 - 213.30 213.10 C 4 2.38 58.20 58.20 C 5 - 42.50 42.10 C 6 1.55,1.31 41.50 41.10 CH

7 1.52,1.31 18.20 18.20 CH2 8 1.33 53.00 53.00 CH 9 - 37.40 37.40 C 10 1.37 59.40 59.50 CH

11 1.54,1.31 35.60 35.80 CH2

12 1.53,1.31 30.40 30.40 CH2 13 - 39.60 39.60 C 14 - 38.20 38.20 C

15 1.52,1.32 32.40 32.30 CH2

16 1.50,1.33 36.00 36.00 CH2 17 - 30.40 30.00 C 18 1.37 42.70 42.90 CH

19 1.46,1.15 35.00 35.00 CH2 20 - 28.10 28.10 C

21 1.54,1.31 30.40 30.80 CH2

22 1.53,1.31 39.20 39.20 CH2

23 1.15 6.70 6.70 CH3

24 1.02 14.60 14.60 CH3

25 0.85 18.00 17.80 CH3

26 0.93 20.00 20.20 CH3

27 0.98 18.60 18.60 CH3

28 0.97 32.00 32.00 CH3

29 0.97 31.80 31.80 CH3

30 0.98 33.00 33.10 CH3

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Figure 18: Structure of friedelin (4)

4.11.5 Betulinic acid (5)

The isolated compound was identified by spectroscopic analysis as well as by comparison of the spectral data with previously reported values. Betulinic acid (5) was isolated as white crystal (MeOH). IR spectrum exhibited hydroxyl [λ max: 3610, 1020 cm-1] and exomethylene [λ max: 3060, 1630, 880]. Its mass spectrum displayed an [M+ + ] peak at m/z 456 corresponding to C30H48O3, together with fragments at m/z 441 [M + + -15] and 438 [M -18] and a base peak at m/z 43 [C3H7 ].

The spectral data of compound (5) thus shows that the compound is betulinic acid. The observed spectral data of compound (5) are similar to the reported spectral data (Nagao et al., 1985; Mahmood et al., 1995; Chauhan et al., 2002; Chandramu et al., 2003). Comparison of reported and observed spectral data the compound (5) was confirmed as betulinic acid.

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Figure 19: Structure of betulinic acid (5)

4.11.6 β-sitosterol (7)

β-sitosterol (7) was isolated as white crystals from the hexane fraction of methanolic extract of Scoparia dulcis. The HREI MS, of the compound (7) showed M+ at m/z

414.3857 in agreement with the formula C29H50O (Calcd 414.3841), which corresponded to five degree of unsaturation. The EI MS of compound (7) showed the M+ at m/z 414. Other ions at m/z 400 and 396 were characteristics of β-sitosterol. The 1H- NMR spectrum of compound (7) showed two quaternary methyls which appeared as singlet at δ 0.66 and 1.00 and were ascribed to the C-18 and C-19 protons respectively. Three others methyl signals appeared as doublets at δ 0.81(J18,27 = 6.5

Hz) 0.83 (J18,26 = 6.8 Hz), 0.84 (J28,29 = 7.0 Hz) and 0.92 (J20,21 = 6.5 Hz), were assigned to the C-27, C-26, C-29 and C-21 methyl proton respectively. A down field proton at δ 3.53 (multiplet) was assigned to C-3 proton geminal to a hydroxyl group. A broad doublet at δ 5.33 (J6,7 =5.1 Hz) was assigned to the C-6 olefinic proton. Compound (7) was thus identified as β- sitosterol by comparison of its 1H-NMR data with the reported literature values.

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Figure 20: Structure of β-sitosterol (7)

4.11.7 Sigmastanone (8)

Sigmastanone (8) also known as stigmastan-3-one was isolated as white crystals from the hexane fraction of methanolic extract of Scoparia dulcis. The HREI MS, of the + compound (8) showed M at m/z 414.39 in agreement with the formula C29H50O (Calcd 414.39), which corresponded to five degree of unsaturation. The observed spectral data of compound (8) were unambiguously matched with the reported spectral data. Hence the structure of compound is given below.

Figure 21: Structure of sigmastanone (8)

4.11.8 Tambulin (9)

The compound was obtained as yellow powder. The EI-MS spectrum of compound showed molecular ion [M+] at m/z 344 and base peak at m/z 329 which was corresponding to molecular formula C18H16O7. The IR spectrum displayed absorptions

104 at 3327 (OH), 1651 (aromatic), and 1556 (olefinic) cm-1. The UV spectrum showed absorptions at 367, 325 and 273 nm.

The 1H-NMR spectrum exhibited resonances for three signals at δ 3.88 s, 3.92 s, and 3.94 s were attributed for protons of methoxy group attached to C-4, C-7 and C-8 respectively. A downfield singlet resonated at δ 6.43 was ascribed to H-6, similarly two downfield ortho coupled doublets at δ 7.02 d (J = 9.0 Hz) and 8.22 d (J = 9.0 Hz), were assigned to H-3/5 and H-2/6 respectively. A down field signal at δ 11.58 s was assigned to intramolecular hydrogen bonded C5-OH proton and a broad singlet at δ= 13 6.55 was assigned to C3- OH proton. The C-NMR spectra (Broad band and DEPT) displayed the resonances for all eighteen carbons including three methyl, five methine and ten quaternary carbons. Structure of compound was further confirmed from 2D- NMR spectra (COSY, HSQC, HMBC and NOESY). Position of hydroxyl and methoxy groups was assigned with the help of the HMBC correlation. The HMBC correlation between methoxy protons at δ 3.88 showed long range correlation with (C- 4) δ 160.5 and 3.92 showed correlation with (C-8) δ 129.4, 3.94 and 159.1 (C-7) clearly indicated the position of methoxy groups in compound.

All the spectral data of compound tambulin, isolated from Bridelia retusa in this research, were unambiguously matched with reported data of tambulin (Babu, Khurana, Sakhuja, Srivastava & Jain, 2007).

Figure 22: Structure of tambulin (9)

4.11.9 3-O-β-D-glucopyranosyl-β-sitosterol glucoside (10)

The molecular mass of compound glucopyranosyl-β-sitosterol glucoside is 576.85 1 corresponding to the formula C35H60O6. The H-NMR spectrum of compound (10) showed a broad singlet at δ 5.35, assigned to H-6. The H-3 appeared as a multiplet at δ 3.96. The methyl signals appeared at δ 0.66, 0.85, 0.88, 0.91, 0.93 and 0.99,

105 corresponding to the C-18, C-27, C-26, C-29, C-19 and C-21 methyl protons. The C-1 anomeric proton appeared at δ 5.04 as a doublet (J6,7 =7.6 Hz) indicating the presence of a β-sugar unit. The compound was identified as known compound, 3-O-β-D- glucopyranosyl-β- sitosterol glucoside is a common constituent in many plants.

Figure 23: Structure of 3-O-β-D-glucopyranosyl-β-sitosterol glucoside (10)

4.12 Biological activity of isolated pure compounds

4.12.1 Insulin secretory activity of coixol (1)

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Figure 24: Effect of compounds 1-6 (A), and dose response of compound 1(B) on glucose stimulated insulin secretion from isolated mice islets

Note: Groups of 3 size-matched islets from BALB/c mice were incubated for one hour at 37 ºC in KRB buffer, containing 3 mM or 16.7 mM glucose in the absence or presence of test compound. Values are mean ± SEM from 3 independent experiments.

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*p <0.05, ***p <0.001 compared with the value for 16.7 mM glucose alone (control). Compounds 1-6, were tested at a dose of 200 µM. TB, tolbutamide (200 µM).

Insulin secretory activity of coixol (1) was further evaluated with different doses in mice islets and pancreatic MIN-6 cells. In isolated islets, compound coixol (1) showed a dose dependent insulin secretory activity (Fig. 24B). The dose 10 or 50 µM could stimulate insulin secretion but did not reach to significant level. The dose 100 µM significantly (p < 0.05) stimulated the glucose induced insulin secretion. The dose 200 µM stimulated the insulin secretion more than the standard drug tolbutamide (227.25 ± 8.33 percent vs. 209.65 ± 16.65 percent). Similar pattern was observed when insulin secretory activity of coixol (1) was evaluated in MIN-6 pancreatic β-cell line. Coixol (1) stimulated the insulin secretion 2.1fold and 3.2 fold at 100 µM and 200 µM doses respectively (Sharma et al., 2015).

4.12.2 Coixol (1) exerts an exclusive glucose dependent insulinotropic effect in βTC-6 cells

βTC-6 cells were incubated with coixol (1) at 2 mM (Fig. 25A) and 20 mM (Fig. 25B) and incubated at 37 ºC for 60 min in KRB buffer. After the incubation period, cells were immunostained for insulin by mouse anti-insulin/Alexa 594-donkey anti-mouse IgG and the image was visualized using a Nikon 90i microscope (Nikon, Japan) and the images were acquired with a Nikon DXM 1200C camera using NIS-Elements image analysis software AR 3.0.

At 2 mM glucose, insulin staining is dispersed throughout the cells suggest that coixol (1) has little to none effects on insulin secretion at low glucose concentration. In sharp contrast, decreased insulin staining was observed by coixol at 20 mM glucose suggest that coixol stimulated insulin secretion at high glucose concentration. Additionally, at 20 mM glucose, more insulin staining at the peripheries suggest that the insulin granules are on the way to be secreted. These data strongly suggest that coixol exerts an exclusive glucose-dependent insulinotropic effect in βTC-6 cells (Hafizur, Raza, Hameed, Adhikari & Sharma, 2015).

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Figure 25: Showing coixol exerts an exclusive glucose-dependent insulinotropic effect in βTC-6

The compound thus isolated from Scoparia dulcis is found active against diabetes. It may therefore be used as antidiabetic as the compound increases the rate of insulin secretion in diabetic patients which ultimately controls diabetes. As found by Donghui et al. (2009) diabetes is thought to be both a potential cause and effect of pancreatic cancer. This compound may thus further prevent diabetes patient from pancreatic cancer.

4.12.3 The clinical effect and safety

All the human studies have found safety profile of this plant. In animal study, oral administration of water or ethanol extracts of Scoparia dulcis up to 2 g/kg did not produce any toxicity of mice and rats (Freire, Torres, Roque, Souccar & Lapa, 1991). It is also found that oral administration of 2 g/kg methanol extracts of Scoparia dulcis has no acute toxic effects on Wistar rats. Though people from many countries are using this plant from long time and also 2 g/kg extract did not produce any toxicity in rats, it can be said that Scoparia dulcis is safe.

Cytotoxicity of coixol (1) was evaluated in 96-well flat-bottom micro-titer plates by using the standard MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay. In this assay coixol (1) showed no toxic effect (IC50 = 200 μM).

It has been reported that, a single intraperitoneal dose of 500 mg/kg, coixol (1) in mice resulted in transient sedation but no mortality (Zhu et al., 1998). A single intravenous dose of 100 mg/kg of coixol caused no death or any abnormal manifestations in mice. There was no toxic reaction after oral administration of 20,100, or 500 mg/kg coixol for 30 days (Wang et al., 1983). Coixol (1) was orally administered to mice at a dose

109 of 100 mg/kg and mortality and general behaviors of the mice were observed continuously for the initial 4-h and intermittently for the next 6h and then again at 24, 48 and 72h following coixol administration. After 72 h of coixol (1) treatment autopsy was performed for checking any abnormality in the liver, kidney, gastrointestinal tract, spleen and heart. Serum creatinine, ALT and AST, were also done to check the toxicity to kidney and liver, respectively. The acute and sub-acute toxicity results showed no mortality or any abnormal manifestation in mice at a dose of 100 mg/kg body weight and no significant change was observed in serum creatinine, ALT and AST levels (Sharma et al., 2015).

Coixol (1) was orally administered to mice at a dose of 100 mg/kg and mortality and general behaviors of the mice were observed continuously for the initial 4-h and intermittently for the next 6-h and then again at 24, 48 and 72h following coixol administration. After 72 h of coixol (1) treatment autopsy was performed for checking any abnormality in the liver, kidney, gastrointestinal tract, spleen and heart. Serum creatinine, ALT, AST, were also done to check the toxicity to kidney and liver, respectively. The acute and sub-acute tests of toxicity results showed no mortality or any abnormal manifestation in mice at a dose of 100 mg/kg body weight and no significant change was observed in serum creatinine, ALT, AST levels. It has also been reported that, a single intraperitoneal dose of 500 mg/kg coixol (1) in mice resulted in transient sedation but no mortality (Zhu et al. 1998). There was no toxic reaction after oral administration of 20,100, or 500 mg/kg coixol for 30 days (Zhu et al. 1998).

Additionally, in our all insulin secretory experimental, no significant insulin secretion was observed after incubation of islets or MIN-6 cells with at 3 mM glucose, further suggesting that exposure of coixol (1) did not cause any cytotoxic effect on isolated islets or MIN-6 cells to leak out the insulin (Sharma et al., 2015).

4.12.4 Immunomodulatory activity of glutinone (3)

Among all tested compounds, glutinone (3), (IC50 = 4.30 µg ⁄ mL) exerted potent inhibition of oxidative burst from whole blood cells. Whereas remaining compounds showed no effect (IC50 >100 µg ⁄ mL). Glutinone (3) was also found to be a potent inhibitor (IC50 = 5.0 µg/mL) of intracellular reactive oxygen species (ROS), when tested on zymosan activated isolated human PMNs using luminol as probe.

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Glutinone (3) was further studied for its effect on the production of proinflammatory cytokines TNF-α and IL-1β. In case of TNF-α, moderate level of inhibition was observed (19 percent) at a concentration of 25 µg/mL as compared to the pentoxifillin

(IC50 = 98.4 µg/mL), which was used as standard TNF-α inhibitor. Very weak inhibition was observed when it was tested for IL-1β, 3.3 percent (Sharma, Adhikari, Jabeen, Dastagir, Kalauni, Choudhary & Pokharel, 2015).

Glutinone (3) was also evaluated for its effect on nitric oxide production in cellular assay. Low level of inhibition was observed (13 percent), when compared to standard L-NMMA (65.5 percent) at concentration 25 µg/mL (Sharma et al., 2015).

All isolated compounds were evaluated for their toxicity on 3T3 fibroblasts cells, where they found to be nontoxic. Among all tested compounds, glutinone (3) was found to be potent inhibitor of ROS production. It also moderately inhibited TNF-α and showed weak inhibition on IL-1β and NO. The anti inflammatory effect of compound (3) was reported earlier. Previously it was reported to reduce inflammation in carrageenan induced rat paw oedema (Duwiejua, Zeitlin, Gray & Waterman, 1999) and found to inhibit protaglandin E2 (PGE2) in mouse peritoneal cells and thromboxane B2 (TXB2) in human platelets (Bermejo, Abad, Diaz, Villaescusa & Gonzalez, 2002). Present study describes the potent suppressive effect of this compound on myeloperoxidase dependent intracellular ROS production in human PMNs. Luminol is used as a probe in this assay, as being having a low molecular weight it can cross the cell membrane, and hence can detect both intra and extra cellular ROS, produced by the cells (Bryan, Abswin, Smart, Bayon & Wohlert, 2012). Previously reported anti-inflammatory activities of glutinone (3) are in agreement with in current results as a reciprocal relationship between COX and ROS. The mediators derived from ROS and prostanoids from COX, such as PGE2, are well known in promoting inflammation and enhancing pathogenesis of various inflammatory diseases including cardiovascular disorders and hypertension. The In vivo reduction in inflammation in rat paw oedema by this compound might be due to its strong suppressive activity on derivatives of COX and in this study on ROS. Suppression of other inflammatory markers, including proinflammatory cytokines TNF-α, IL-1β and on NO also accounts for reduction in inflammation. Furthermore this compound found to be non toxic when tested on mouse 3T3 fibroblast cells. Most inflammatory conditions are associated with oxidative stress and hyperactivation of COX mediators.

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Current studies showed a strong relationship between mediators of both pathways. Glutinone (3) that have ability to block the mediators of both pathways might be of therapeutic value against inflammatory diseases. However further studies to unreveal exact underlying molecular mechanism, as well as detailed in vivo studies and clinical trials are needed to evaluate the effects of this compound in reducing inflammation (Sharma et al., 2015).

Positive control Compounds

Figure 26: Effect of compounds on luminol enhanced oxidative burst using zymosan activated whole blood. Readings presented as mean ± SD of three determinations

Table 11: Effect of glutinone (3) on nitric oxide (NO), proinflammatory cytokines, TNF-α and IL-1β

NO % TNF-α % IL-1β % Oxidative Burst (IC50 µg/mL)

Compound inhibition inhibition inhibition Whole blood PMNs

Glutinone 3 13 ± 0.7 19 ± 1.0 3.34 ± 3.5 4.3 ± 0.6 5.0 ± 0.3

L-NMMA 65.5 ± 1.1 - - - -

Ibuprofen - - - 11.2 ± 1.9 2.5 ± 0.6

Note: Effect on luminol enhanced myeloperoxidase dependent oxidative burst using zymosan activated PMNs and mice peritoneal macrophages. Readings presented as mean ± SD of three determinations L-NMMA= NG Monomethyl L-Arginine Acetate: Standard Inhibitor for NO and PMNs = polymorphonuclear leukocytes.

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4.12.5 Cytotoxicity of betulinic acid (5) against breast cancer cell lines

Betulinic acid (5) was found potent cytotoxic against breast cancer cell line MCF-7 and MDA-MB-231 with IC50 value 13.65 ppm as compared to the standard paclitaxel

IC50 4.653±1.3 ppm at 24 h post incubation 3.53±0.32 ppm at 48 h incubation but non- cytotoxic against normal cell lines. Amongst the tested compounds, coixol (1) glutinol

(2), glutinone (3) and tetratriacontan-1-ol (6) has IC50 value 31.45 ppm, 47.10 ppm, 56.91 ppm and 78.69 ppm respectively showed weak cytotoxicity against breast cancer cell lines. Friedelin (4) has no cytotoxicity against breast cancer cell line with IC50 220.5 ppm.

Cell survival and concentration of compounds

Cell survival percent survival Cell

Concentration (ppm)

Figure 27: Cytotoxicity of compounds against MCF-7 (breast cancer) cell lines

Table 12: Cytotoxicity of compounds against MCF-7 (breast cancer) cell lines

S. N. Compounds IC50 ppm Log IC50 ppm

1 Betulinic acid (5) 13.56 1.132

2 Coixol (1) 31.45 1.498

3 Glutinol (2) 47.10 1.67

4 Glutinone (3) 56.91 1.755

5 Sigmastanone ( 8) 78.69 1.896

6 Friedelin (4) 220.50 2.343

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The result clearly shows that natural compound betulinic acid (5) isolated from

Scoparia dulcis has potent cytotoxicity effect against breast cancer cell lines with IC50 13.56 ppm.

4.12.6 Preferential cytotoxicity of pure compounds against pancreatic cancer cell line (PANC-1) and PSN-1.

All compounds isolated from Scoparia dulcis were subjected to test against pancreatic cancer cell (PANC-1) under nutrition rich (DMEM) and deprived condition (NDM). Betulinic acid (5) showed preferential cytotoxicity against pancreatic cancer cell PANC-1 in nutrition deprived condition (NDM).

Glutinol (2) and glutinone (3) showed mild activity against PANC-1 under nutrition deprived condition, coixol (1), tetratriacontan-1-ol (6), β-sitosterol (7) showed no activity against PANC-1. Whereas, betulinic acid (5) is found to be highly active against PANC-1 cancer cell line showed 100 percent preferential cytotoxicity under nutrition deprived condition (NDM) at a concentration of 31.60 μM. The result clearly showed that betulinic acid (5) is active against PANC-1 under NDM (Fig. 28).

All compounds were subjected to test against pancreatic cancer cell line PSN-1. Glutinol (2) showed mild activity against PSN-1. coixol (1), glutinone (3), tetratriacontan-1-ol (6), and β-sitosterol (7) showed no activity. Whereas betulinic acid (5) is potent and showed 100 percent preferential cytotoxicity at a concentration of 3.893 μM against PSN-1 cancer cell line (Fig 29).

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Coixol (1)

Betulinic acid (5)

Cell survival (%) survival Cell