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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

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Page 1: SCREENING OF SOME SELECTED MEDICINAL PLANTS OF NEPAL …

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

Table 8: 1H and

13C- NMR and chemical shift value of glutinol (CDCl3 ppm,

500 MHz) 98

Table 9: 1H- and

13C-NMR chemical shift value of glutinone (CDCl3, ppm, 500 MHz

100

Table 10: 1H- and

13C-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

.

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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.

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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

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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.

Angiogenesis

Metabolism Change Hypoxia Response

Tolerance to

nutrient starvation

Survival

Get nutrient supply

Glycolysis

Angiogenesis

Cancer

Ischemia

1 2 Austerity

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

Anti-Austerity Strategy

Compounds

Ordinary medium

+ Toxicity

Conventional drugs Nutrient Deprived Medium

- Toxicity

+ Toxicity

New strategy Invalid

- Toxicity

Targeting Cancer Cells Tolerance to Nutrient Starvation

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

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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

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(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.

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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 anti-

bacterial, 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.

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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.

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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-trihydr-

oxy-4-methoxyflavon-7-O-β-D-glucopyranoside (Sayani, Hemanta, Soumik, Soma,

Sanjukta, Santinath & Pubali, 2013).

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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).

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

Betulin Ethyl gallate

3-acetyloleanolic acid

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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

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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

2-hydroxy-4-methoxy-1,2,3,4-tetrahy-

dronaphthalen-1-yl benzene-1,2-diol

3,4-dihydroxyphenyl-1,2,3,4-

tetrahydronaphthalene-1,3-diol

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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).

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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).

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

Kaempferol Luteolin

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et al., 2013). The whole plant affords carbohydrates, alkaloids, flavonoids,

phytosterols, β-sitosterol, glycosides, proteins and triterpenes.

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.

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.

Apigenin

Luteolin

Vitexin Orientin

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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).

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

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

1,4-cyclohexylphenyl ethanone

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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).

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

Strychnine Brucine

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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

α-amyrin

Dihydroxyisoflavone

β-sitosterol

Leucoanthocyanidin

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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).

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

5-hydroxy-2-[2-(4-hydroxyphenyl)

acetyl]-3-methoxylbenzoic acid

(2S,3S)-3,7,8,3',4'-pentahydroxyflavane

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

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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).

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.

Caffeic acid

p-Coumaric acid

Maslinic acid

Eriodictyol

Astilbin Oleanolic acid

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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

chroma- tography revealed the presence of glycosides, steroids, flavonoids, coumarins

and alkaloids (Singh, Vikas & Bhandari, 2014). Subramanian, Manikandaraja and

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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).

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

Isobornyl acetate Camphene

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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).

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).

Syringic acid

Ferulic acid Chlorogenic acid

4-Hydroxy-3-methoxy benzoic acid

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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).

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).

Quercetin Gallic acid Vitamin c

Vanillic acid Gallic acid

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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-gl-

ucopyranoside, 3,4,8,9,10-pentahydroxydibenzopyran-6-one, 3,4,3'-tri-O-methylella-

gic acid, lupeol, lup-20,29-en-3β,3-O-diol, betulin and gallic acid (Sripanidkulchai &

Junlatat, 2014).

2.20 Berberis aristata

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, anti-

hyperglycemic, 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).

3,4,8,9,10-pentahydroxydibenzo

pyran-6-one

3,4,3'-tri-O-methylellagic acid

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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).

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).

Berberine

4-methylheptadec-6-enoic acid ethyl ester

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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

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

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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.

Cuscutin

Coumarin

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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-glucopyra-

nosyl 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-allopy-

ranoside 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

β- sitosterol Oleanolic acid

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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).

2.26 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).

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-hydroxy-4-methoxy-benzaldehyde

Dextropropoxyphene

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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-arabinopyran-

osyl, (1,6)-β-D-glucopyranoside, and (25S)-5β-furost-3β,22,26-triol-3-O-β-D-xylo-

pyranosyl, (1,4)α-L-arabinopyranosyl, (1,6)-β-D-glucopyranoside-26-O-β-D-gluco-

pyranoside (Cong, Ye & Che, 2000).

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

Glucuronic acid

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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).

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

trans-N-p-coumaroyltyramine Boldine

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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-pentamethoxy-

flavone, 7,5-dime- thoxy-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).

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

Betulin 4-hydroxybenzoic acid 5- hydroxy-3',4',6,7,8-

Pentamethoxy flavone

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(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-di-

hydroxy-5-methoxy-6,8-dimethylflavanone, 4,2',4'-trihydroxy-6'-methoxy-3',5'-dime-

thylchalcone, 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone, 7-hydroxy-5-met-

hoxy-6,8-dimethylfavanone, 2',4'-dihydroxy-3'-methyl-6'-methoxychalcone, 6-form-

yl-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).

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-glu-

copyranoside 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).

7-hydroxy-5-methoxy-6,8

dimethylisoflavone 6-formyl-8-methyl-7-O-

methylpinocembrin

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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).

5,7-dihydroxy-6,8-dimethylflavone 7-hydroxy-5-methoxy-6,8 dimethyl

flavanone

Naringegenin

Kaempferol-3-O-L-rhamnopyranoside

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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

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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).

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.

Lupeol 3-O-acetoxyoleanolic acid

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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).

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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).

Catechin Gallic acid Bergenin

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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).

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

Quercetin

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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-xylopyra-

noside, O-catechin-7-O-β-xylopyranoside, (+)-isolarisiresinol-9-O-β-glucopyranosi-

de 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

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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).

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).

di-(2-ethylhexyl) phthalate

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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).

Kaurenoic acid Iiriodenine Norushinsunine

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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).

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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, anti-

inflammatory 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 phytoecd-

ysteroids (Zhong-Yu Zhou, Yong Cao, Wei-Min Zhang & Jian-Wen Tan, 2012).

Ferulic acid Caffeic acid Vanillin

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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 anti-

hyperglycemic 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.

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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).

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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

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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).

α-amyrin Oleanolic acid

Maslinic acid Sigmasterol

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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-acetyl-

rhamnopyranosyl-(1-6)-β-D-glucopyranoside and apigenin-7-O-α-L-2,3-di-O-acetyl-

rhamnopyranosyl(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,

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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).

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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).

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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

Coixol Betulinic acid

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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:

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Scopadulcis acid A Scopadulcis acid B

Scoparic acid A Scoparic acid B

Apigenin Scoparic acid C

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Cirsimarin Hymenoxin

Betulinic acid Benzoxazolone

Alpha amyrin Acacetin

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Cynaroside Coumaric acid

Cirsitakaoside Gentisic acid

Iffaionic acid Glutinol

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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.

β-sitosterol Dulcitol

Vitexin Benzoxazin

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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

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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

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mass spectrometer. The 1H-NMR spectra were recorded on Bruker AC-300, AM-400

and Amx-500 MHz instruments, while 13

C-NMR spectra were recorded at 75, 100,

125 and 150 MHz. Multiplicities of carbon signals were determined by using DEPT

90o and 135

o 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

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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.

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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

X 100

% Scavenging Activity =

Absorbance of the control

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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

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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)

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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

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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

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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

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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

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

Aspirate medium and

wash with PBS

96-well plate

10% Cell Counting kit 8

in DMEM (100 μl /well)

3 h incubation

24 h incubation

for attachment

Test samples at different

concentration in DMEM

or NDM (100 µL / well)

24 h incubation

at 37 ooC

5% CO2

Absorbance measured at

450 nm

20000 cells/well in

DMEM (100 µL / well)

Aspirate

medium

wash with

PBS

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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

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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).

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

Scoparia dulcis (1.7 kg)

Crude extract (72.0 g)

Water soluble

n-hexane fraction (15.0 g) Water soluble

Extracted with 80% MeOH-H2O

Dissolved in distilled water (1.5 L)

Extracted with n-hexane

CH2Cl2 neutral fraction (2.0 g) Water soluble

Water soluble fraction (10.0 g)

g)

Ethyl acetate fraction (20.0 g)

Extracted with (CH2Cl2)

Extracted with ethyl acetate

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

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

Friedelin 4

(35.0 mg)

Glutinone 3

(40.0 mg)

Glutinol 2

(50.0 mg)

Sigmastanone 8

(10.0 mg)

Betulinic acid 5

(50.0 mg)

Tetratriacontan 1-ol 6

(10.0 mg)

β-sitosterol 7

(60.0 mg)

Hexane fraction

(15.0 g)

Fr-1 to Fr-27

Pure hexane

(800.0 mg)

Fr- 28 to Fr- 40

5% ethyl acetate

(750.0 mg)

Fr-41 to Fr 62 10%

ethyl acetate

(650.0 mg)

Fr-63 to Fr-85

15% ethyl acetate

(850.0 mg)

Fr-86 to Fr-120

20% ethyl acetate

(900.0 mg)

Column chromatography (Silica gel, 100g)

Eluted with n-hexane and ethyl acetate

(0-100%)

Fr- 1 to Fr-12

Pure hexane

(100.0 mg)

Fr-13 to Fr-27

5% to 10% (1 L)

(150.0 mg)

Fr-28 to Fr-48 10%

to 20% (2 L)

(300.0 mg)

Fr-49 to Fr-78 30%

to 40% (2.5 L)

(200.0 mg)

Compound 1 (45.0 mg)

Dichloromethane fraction

(2.0 g)

Column chromatography using silica gel

(70-230, mesh, 200 g) eluted with ethyl

acetate and n-hexane

Column chromatography using silica

(10% ethyl acetate in hexane)

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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

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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).

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

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

Extracted with ethyl acetate

Extracted with (CH2Cl2)

Crude extract (500 g)

Water soluble

Insoluble in n-hexane Water soluble

Extracted with 80% MeOH-H2O

Dissolved in distilled water (5 L)

Extracted with n-hexane

Insoluble in CH2Cl2 Water soluble

Water soluble fraction (460 g)

Ethyl acetate soluble fraction (15.0 g)

Bridelia retusa bark (8.5 kg)

Column chromatography using silica

gel (70-230, mesh, 200 g) eluted with

ethyl acetate and n-hexane

Fr- 1 to Fr-12

1% to 5% (750 mL)

(100 mg)

Fr-13 to Fr-27

5% to 10% (1 L)

(300 mg)

Fr-28 to Fr-48

10% to 25% (2 L)

(200 mg)

Fr-49 to Fr 78

30% to 40% (2.5 L)

(200 mg)

Compound (2),

β-sitosterol (65.0 mg)

Ethyl acetate fraction

(15.0 g)

Compound (1),

Tambulin (20.0 mg)

Compound (3) β-sitosterol

glucoside (10.0 mg)

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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).

3.5.3.2 Glutinol (2)

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.

3.5.3.3 Glutinone (3)

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

Physical state: white crystalline

Molecular weight: 165.04

Molecular formula: C8H7NO3

Yield: 45.0 mg 0.0625%

M.P: 151-156 oC

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

Specific rotation:

Coixol (1)

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%)

Elemental Analysis: C, 84.44; H,

11.81; O, 3.75

Glutinol (2)

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

chromatography.

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.

Glutinone (3)

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

(5.3%)

Elemental Analysis: C, 84.84; H, 11.39;

O, 3.77

Friedelin (4)

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

(100.0%), 427.39 (33.1%), 428.39 (5.3%)

Elemental Analysis: C, 84.44; H, 11.81; O,

3.75

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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.

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.

Betulinic acid (5)

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

(100.0%), 457.36 (32.6%), 458.37 (5.3%)

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

10.51

Physical state: white crystalline

Molecular weight: 494.54

Molecular formula: C34H70O

Yield: 10.0 mg 0.0138%

M.P: 135 oC

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

Tetratriacontan-1-ol (6)

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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.

3.5.3.8 Sigmastanone (8)

The hexane fraction (15.0 gm) of Scoparia dulcis 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.

β-sitosterol (7)

Physical state: white crystalline

Molecular weight: 414.39

Molecular formula: C29H50O

Yield: 60.0 mg 0.0833%

M.P: 132 oC

EIMs m/z (Relative intensity %):

414.39 (100.0%), 415.39 (32.0%),

416.39 (5.0%)

Elemental Analysis: C, 83.99; H,

12.15; O, 3.86

Sigmastanone (8)

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%),

416.39 (5.0%)

Elemental Analysis: C, 83.99; H,

12.15; O, 3.86

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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.

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.

Tambulin (9)

Physical state: yellow crystalline

Molecular weight: 344.32

Molecular formula: C18H16O7

Yield: 20.0 mg 0.013%

M.P: 205 oC

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

β-sitosterol glucoside (10)

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%)

Elemental Analysis: C, 72.87; H,

10.48 ; O, 16.64

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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.

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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,

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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

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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.

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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

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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

Figure 9b: Yield percentage of plant extracts

Figure 9a: Yield percentage of plant extracts

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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.

Figure 10: Calibration curve of standard ascorbic acid

A

bso

rban

ce

Concentration against absorbance

Concentration of ascorbic acid (µg/mL)

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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).

% S

cavem

gim

g

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

Per

cen

t sc

aven

gin

g

Concentration µg/mL

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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.

Figure 13: Calibration curve of standard gallic acid

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

Ab

sorb

ance

Concentration of gallic acid (µg/mL)

Absorbance against gallic acid concentration

concentration

Per

cen

t sc

aven

gin

g

Concentration µg/mL

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Table 1: Total phenolic content in potent antioxidant plant extracts

Plant extracts Abs.

I

Abs.

II

Conc.

I

Conc.

II

mg GAE/g

I

mg GAE/g

II

mg GAE/g

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).

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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.

Figure 14: Calibration curve of standard quercetin

Concentration of quercetin µg/mL

Absorbance against quercetin concentration

Abso

rban

ce

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Table 2: Total flavonoid content in potent antioxidant plant extracts

Plant extracts Abs.

I

Abs.

II

Conc.

I

Conc.

II

mg

QE/g I

mg

QE/g II

mg QE/g

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

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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).

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Table 3: Preferential cytotoxicity against pancreatic cancer cell lines

Plant extracts Preferential cytotoxic concentration (PC50)

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,

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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

Plant extracts Control

(mm)

Stayphylococcus

aureus

Bacillus

subtilic

Salmonella

typhi E.coli

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 - -

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

Plant extracts Control

(mm)

Stayphylococcus

aureus

Bacillus

subtilic

Salmonella

typhi E.coli

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

Plant extracts Control

(mm) Stayphylococcus aureus

Bacillus

subtilic Salmonella typhi E.coli

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 - - - -

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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

Plant extracts Control

(mm)

Concentration

(mg/mL)

Staphylococcus

aureus

Salmonella

typhi E. coli

Bacillus

subtilic

Astilbe

revularis

6 10 13 11 6 6

6 15 13 11 6 7

6 20 14 12 8 8

Bergenia

ciliata

6 10 12 10 11 16

6 15 12 12 12 16

6 20 13 12 13 18

Bauhinia

variegata

6 10 13 10 10 11

6 15 14 10 11 12

6 20 14 11 11 14

Curcuma

angustifolia

6 10 13 13 11 14

6 15 14 15 14 15

6 20 16 16 16 16

Betula

alnoides

6 10 11 9 10 13

6 15 11 10 12 14

6 20 12 11 13 16

Shorea

robusta

6 10 12 13 10 11

6 15 12 15 10 11

6 20 13 16 11 12

Ziziphus

mauritiana

6 10 9 12 9 15

6 15 9 13 9 16

6 20 10 12 9 18

Litsea

cubeba

6 10 8 6 6 16

6 15 9 6 6 17

6 20 11 6 6 18

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

Plant

extracts

Control

(mm)

Concentration

(mg/mL)

Staphylococcus

aureus

Salmonella

typhi

E.

coli

Bacillus

subtilic

Bridelia

retusa

6 10 13 12 16 11

6 15 14 13 17 16

6 20 19 16 19 18

Cleistocalyx

operculatus

6 10 18 13 16 18

6 15 19 15 18 19

6 20 20 16 19 19

Drymaria

diandra

6 10 10 12 9 9

6 15 11 10 10 10

6 20 11 11 10 12

Acacia

catechu

6 10 8 15 12 10

6 15 10 14 14 13

6 20 12 15 15 15

Phyllanthus

emblica

6 10 13 10 8 11

6 15 14 11 9 13

6 20 18 12 11 14

Annona

reticulata

6 10 6 8 17 12

6 15 6 9 16 11

6 20 6 10 15 12

Bombax ceiba

6 10 8 6 6 15

6 15 9 6 6 16

6 20 10 6 6 16

Callicarpa sp.

6 10 9 11 10 12

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

Drugs Conc.

(µg)/disc

Stayphylococcus

aureus

Bacillus

subtilic

Salmonella

typhi E. coli

Ciprofloxacin 5 32

Erythromycin 15 20

Nalidixic acid 30 24

Chloramphenicol 30 25

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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

formula C8H7NO3 was deduced from EI-MS and 13

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 13

C-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

No.

1H-NMR (δ ppm)

13C-NMR (δ ppm)

observed (MeOD)

13C-NMR (δ ppm)

reported (DMSO)

Multiplicity

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,

J5,7 = 2.5

110.60 109.10 CH

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

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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)

Table 8: 1H- and

13C-NMR chemical shift value of glutinol (CDCl3, ppm, 500 MHz)

Carbon

No.

1H-NMR (δ ppm)

observed

13C-NMR (δ ppm)

observed

13C-NMR (δ ppm)

reported Multiplicity

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

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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

m/z 424.3721 (calcd. for C30H40O = 424.3705) and the 13

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

Figure 16: Structure of glutinol (2)

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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).

Table 9: 1H- and

13C-NMR chemical shift values of glutinone (CDCl3, ppm, 500 MHz)

Carbon

No.

1H-NMR (δ ppm)

observed

13C-NMR (δ ppm)

observed

13C-NMR (δ ppm)

reported Multiplicity

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)

Table 10: 1H- and

13C-NMR chemical shift values of friedelin (CDCl3 ppm, 500 MHz)

Carbon

No.

1H-NMR (δ ppm)

observed

13C-NMR (δ ppm)

observed

13C-NMR (δ ppm)

reported Multiplicity

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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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.

Figure 18: Structure of friedelin (4)

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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.

Figure 19: Structure of betulinic acid (5)

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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.

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

Figure 21: Structure of sigmastanone (8)

Figure 20: Structure of β-sitosterol (7)

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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=

6.55 was assigned to C3- OH proton. The 13

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).

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

The molecular mass of compound glucopyranosyl-β-sitosterol glucoside is 576.85

corresponding to the formula C35H60O6. The 1H-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,

Figure 22: Structure of tambulin (9)

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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.

4.12 Biological activity of isolated pure compounds

4.12.1 Insulin secretory activity of coixol (1)

Compounds isolated from Scoparia dulcis were evaluated for their insulin secretory

activity in isolated islets. Among them, glutinone (3), friedelin (4), betulinic acid (5),

and tetratriacontan-1-ol (6) showed little to no effect on glucose stimulated insulin

secretion (Fig. 24B). Glutinol (2) showed a moderate insulin secretion activity (137.25

± 7.63 percent), as compared to insulin secretion by 16.7 mM glucose (100 ±8.33

percent). Interestingly, coixol (1) showed a potent insulin secretory activity (230.35 ±

11.12 percent) in isolated islets (Fig. 24A). At 200 µM dose, coixol (1) stimulated

insulin secretion higher than tolbutamide (212.01 ± 16.76 percent), a known insulin

secretagogue (Sharma et al., 2015).

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

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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.

Figure 24: Effect of compounds 1-6 (A), and dose response of compound 1(B) on glucose stimulated

insulin secretion from isolated mice islets

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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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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

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

βTC-6

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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).

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

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.

Compound

NO %

inhibition

TNF-α %

inhibition

IL-1β %

inhibition

Oxidative Burst (IC50 µg/mL)

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

Compounds

Positive

control

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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.

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

Concentration (ppm)

Cell survival and concentration of compounds

Cel

l su

rviv

al p

erce

nt

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

pancreatic cancer (PANC-1) cell line

Concentration µM Concentration µM

Concentration µM

Glutinone (3)

Betulinic acid (5)

Concentration µM

Cel

l su

rviv

al (

%)

Cel

l su

rviv

al (

%)

β-sitosterol (7) Glutinol (2)

Cel

l su

rviv

al (

%)

Cel

l su

rviv

al (

%)

Tetratriacontan-1-ol (6)

Cel

l su

rviv

al (

%)

Cel

l su

rviv

al (

%)

Coixol (1)

Concentration µM

Concentration µM

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116

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

pancreatic cancer (PSN-1) cell lines

Concentration µM

Concentration µM

Concentration µM

Concentration µM

Concentration µM

Concentration µM

Cel

l su

rviv

al (

%)

Cel

l su

rviv

al (

%)

Cel

l su

rviv

al (

%)

Cel

l su

rviv

al (

%)

Cel

l su

rviv

al (

%)

Cel

l su

rviv

al (

%)

Coixol (1)

Tetratriacontan-1-ol (6)

β- sitosterol (7)

Glutinol (2)

Glutinone (3) Betulinic acid (5)

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Figure 30: Preferential cytotoxicity of betulinic acid (5) against pancreatic cancer (PANC-1) cell line in

dose dependent manner

The result on preferential cytoxicity against PANC-1 and PSN-1 shows that the

betulinic acid (5) is active against PANC-1 and PSN-1. It indicates that the betulinic

acid (5) may be used in controlling pancreatic cancer. Control in pancreatic cancer

could be preventive measure to check diabetes as the literature by Permert (1993; as

cited in Gullo, 1994) mentions that diabetes occurs more frequently in patients with

pancreatic cancer than in the general population, because pancreatic cancer may cause

diabetes by destroying islet cells of pancreas or by causing peripheral resistance to

insulin. Thus betulinic acid (5) may be used as active constituent that prevents diabetes

through preventing pancreatic cancer.

Fluorescent Phase contrast Overlay

L = Live D = Dead

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118

4.12.7 Antioxidant activity of tambulin (9)

The radical scavenging activity showed that the tambulin (9) has potent antioxidant

activity with IC50 166.15±1.92 SEM [µM] with radical scavenging activity 86.03

percent where as the standard butylated hydroxytoluene has IC50 128.83±2.1 SEM

[μM] and 85.87 percent radical scavenging activity. This indicates that tambulin (9) is

an active antioxidant compound act against oxidative damage of the cell which causes

several chronic diseases like cancer and Parkinsons (Sharma, Adhikari, Choudhary,

Awale & Kalauni, 2015).

Figure 31: DPPH radical scavenging activity of tambulin and standard BHT

4.12.8 Urease activity of tambulin (9)

Tambulin (9) showed potent urease inhibiting agent of IC50 41.82 ± 1.60 SEM [µM] as

compared to the standard thiourea IC50 21.00 ± 0.11 SEM [µM]. Urease activity of

natural compound tambulin from Bridelia retusa was compared to some results of

previous research work. From the previous research work it was reported that

methanolic extract of Melilotus indicus Linn. and its sub-fraction in different solvents

showed remarkable urease inhibitory activities with IC50 values 0.95 µg/mL, 0.89

µg/mL, 1.53 µg/mL, 0.98 µg/mL and 4.96 µg/mL in methanolic, chloroform, ethyl

acetate, n-butanol and water fraction respectively (Ahmed, Younas, & Mughal, 2014).

86.06

85.87

Tambulin Butylated hydroxytoluene Butylated hydroxy toluene Tambulin

128.83±2.1

166.15±1.92

Rad

ical

sca

ven

gin

g p

erce

nta

ge

Ssc

aven

gin

g I

C50 S

EM

M]

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119

Figure 32: Urease Inhibitory concentration and percentage inhibition of tambulin and standard thiourea

4.12.9 Immunomodulatory activity of tambulin (9)

Oxidative burst studies using chemiluminescence technique showed that tambulin (9)

has immunomodulatory activity on whole blood from healthy human volunteers and

on isolated neutrophils using luminol as a probe, and zymosan as an activator. The

compound showed IC50 = <1 µg/mL as compared to the standard drug ibuprofen in

whole blood (IC50 = 11.2± 1.9 µg/mL).

Table 13: Immunomodulatory activity of tambulin on ROS with respect to Ibuprofen

Compounds Amount used (mg) Conc. mg/mL) IC50±SD

Tambulin 0.5 (100, 10, 1 μg/mL <1

Ibuprofen (standard) drug 0.5 (100, 10, 1 μg/mL 11.2±1.9

Tambulin (9) showed potent immunomodulatory activity (IC50 = <1 μg/mL) on ROS

as compared to the standard Ibuprofen IC50 = 11.2±1.9 µg/mL.

Tambulin Thiourea Thiourea Tambulin

41.82±1.60

21±0.11

88

98

Per

cen

tag

e in

hib

itio

n

Inh

ibit

ory

con

cen

trat

ion

M]

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CHAPTER 5

CONCLUSIONS AND RECOMMENDATION

5.1 Conclusions

Screening of fifty selected medicinal plants collected from different regions of Nepal

showed that most of them are potent sources of secondary metabolites as an

antioxidant and preferential cytotoxic against pancreatic cancer cell lines (PANC-1).

Among them plant extracts of Acacia catechu, Bauhinia variegata, Shorea robusta,

Bridelia retusa and Phyllanthus emblica are found potent sources of antioxidant

compounds with the strongest DPPH radical scavenging activity. Most of the plant

extracts are potent against Gram positive and Gram negative bacteria which are

valuable source of antimicrobial constituents.

Compounds isolated from Scoparia dulcis were evaluated for their insulin secretory

activity in isolated islets. Among them, glutinone (3), friedelin (4), betulinic acid (5),

and tetratriacontan-1-ol (6) showed no effect on glucose stimulated insulin secretion.

Glutinol (2) showed a moderate insulin secretion activity (137.25 ± 7.63 %) when

compared with the insulin secretion as 16.7 mM glucose (100 ±8.33 %). Interestingly,

coixol (1) showed a potent insulin secretory activity (230.35 ± 11.12 %) in isolated

islets. At 200 µM dose, compound (1) stimulated insulin secretion higher than

tolbutamide (212.01 ± 16.76 %), a known insulin secretagogue. Among all five tested

natural compounds isolated from S. dulcis, glutinone (3) exerted potent inhibition of

oxidative burst from whole blood cells. Whereas remaining compounds; friedelin,

glutinol, sigmastanone and β-sitosterol showed no effect on insulin secretory activity,

immunomodulatory and oxidative burst. Glutinone (3) showed potent inhibition of

intracellular reactive oxygen species (ROS), when tested on zymosan activated

isolated human PMNS using luminol as probe. Glutinone (3) showed moderate level

of inhibition on the production of proinflammatory cytokines TNF-α and IL-1β and

weak inhibition was observed when it was tested for IL-1β. Glutinone (3) showed low

level of inhibition for its effect on nitric oxide production. Current study demonstrated

the antiinflammatory potential of glutinone (3) and it can be the lead compound for

further drug discovery process.

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Betunilic acid (5) isolated from Scoparia dulcis is active against pancreatic cancer

PANC-1 and PSN-1. Controlling pancreatic cancer means preventing the patient from

diabetes as well. One of the scholars (Permert, 1994 as cited in Gullo, 1999) mentions

that diabetes occurs more frequently in patients with pancreatic cancer than in normal

people, because pancreatic cancer may cause diabetes by destroying islet cells of

pancreas or by causing peripheral resistance to insulin. Thus, the betulinic acid (5)

may be used against pancreatic cancer which ultimately prevents diabetes. Coixol (1)

thus isolated from Scoparia dulcis is found active against diabetes. It may therefore be

used as antidiabetic compound that increases the rate of insulin secretion in diabetic

patients. As found by Donghui, Yeung, Konopleva and Abbruzzese (2009) diabetes is

thought to be both a potential cause and effect of pancreatic cancer. Tambulin (9)

firstly isolated from ethylacetate soluble fraction of Bridelia retusa bark showed

potent antioxidant activity. The compound tambulin also showed the potent activity

against urease inhibition and immunomodulatory activity.

The isolated active compounds; coixol (1), glutinone (3), betunilic acid (5) and

tambulin (9) are found potent in insulin secretion, immunomodulation, against

pancreatic cancer and antioxidant activity respectively. Thus as hypothesized in the

beginning of this research medicinal plants, Scoparia dulcis and Bridelia retusa

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 and anticancer which can be used in drug discovery

process against disease like pancreatic cancer that may ultimately leads to diabetes.

Therefore, these compounds may be recommended for the further process of

discovering the drugs against diabetes, pancreatic cancer and antioxidant.

5.2 Recommendations

Recommendation for drug discovery and policy making

The active compounds; coixol (1), glutinone (3), betunilic acid (5) and tambulin (9)

are found potent in insulin secretion, immunomodulation, against pancreatic cancer

and antioxidant activity respectively. On the basis of these findings the following

recommendations are made:

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a) Synthesizing the active compound coixol (1) in required quantity and testing it in

animal models for its insulin secreting activity someone can go for drug discovery

process,

b) Synthesizing the active compound glutinone (3) in required quantity and testing it in

animal models for its immunomodulatory activity someone can go for drug discovery

process,

c) Synthesizing the active compound betunilic acid (5) in required quantity and test it

in animal models for its activity against pancreatic cancer cell (PANC-1) someone can

go for the process of discovery of anticancer drug,

d) Synthesizing the active compound tambulin in required quantity and testing it in

animal models for its antioxidant activity someone can go for drug discovery process,

e) The plant Scoparia dulcis (Chhini jhar) has been used by different communities of

Nepal since long year for management of diabetes. Hence, the isolated compound

coixol (1) is safe from all side effects and may be the potent antidiabetic drug of

Nepalese origin.

Recommendations for researcher

Based on the experience and activities performed during this research work, following

recommendations have been made:

Screening of many medicinal plants available in different regions of Nepal has to be

done by future scholars particularly focusing on their medicinal values. The focus of

screening could be on their biological activities through different biological assays in

order to isolate an active antioxidant, antimicrobial and anticancer compounds.

In addition, some plant extracts were only screened for their preliminary in-vitro

activities, so the advance in vivo and clinical trial of them deserves to be further

investigated. These plants can be used to investigate phytochemical discovery and

subsequent screening are needed for opening new opportunities to develop

pharmaceuticals based on plant constituents. Therefore, isolation of bioactive chemical

compounds from plants, identification and structural elucidation of isolated pure

compounds, as well as synthesis of compounds must be the main aim of incoming

researcher. It is important to find out the biochemical importance of such chemicals

for the human welfare and conservation and cultivation of such plant species.

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CHAPTER 6

SUMMARY

People of Nepal have been using many plants as medicine in curing various diseases

throughout the history. However, whether various plants in different ecological belts

of Nepal possess medicinal compounds, can be extracted and used as medicine within

the area of natural product chemistry, focusing particularly in the diseases like

pancreatic cancer and diabetes, is the key research problem of this study. Regarding

this research problem the objectives of this study were to screen some selected

medicinal plants, to extract secondary metabolites, to test anti-bacterial, anti-oxidant

and preferential cytotoxicity against pancreatic cancer cell line (PANC-1) of the

extract and to determine total phenolics and flavonoid contents of the potent

antioxidant extracts and finally to isolate the active compounds against pancreatic

cancer that also prevents diabetes in patients with pancreatic cancer. The hypothesis of

this study was therefore formulated as medicinal plants collected from different

regions of Nepal have bioactivity such as antibacterial, antidiabetic, antioxidant,

immunomodulatory and anticancer.

In order to achieve the objectives or to test hypothesis of this study fifty different

medicinal plants were collected from different districts and regions of Nepal.

Methanolic extracts of fifty selected medicinal plants collected from different regions

of Nepal were screened for DPPH radical scavenging assay, microbial activity and

preferential cytotoxicity against pancreatic cancer PANC-1 cell lines under nutrient

deprived condition. The potent antioxidant samples were further subjected for

determination of total phenolic and flavonoid content. For the purpose of isolation of

active compounds from the screened plant samples chromatographic techniques like

column and HPLC were applied. Structure of isolated compounds were determined by

modern spectroscopic techniques; 1H-NMR, 2D-NMR, mass, UV and IR. Compounds

were further tested for antidiabetic, antioxidant, immunomodulatory and anticancer

activity.

Screening of fifty selected medicinal plants based on antioxidant and preferential

cytotoxicity activity against pancreatic cancer showed that the selected plants were

rich sources of secondary metabolites. The plant extract of Scoparia dulcis was found

potent against PANC-1 cancer cell lines with PC50 10.00 µg/mL. Hence, these two

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plants Bridelia retusa as antioxidant active and Scoparia dulcis as cytotoxic against

pancreatic cancer cell lines were selected to isolate active pure compounds.

Betulinic acid (5) was found as potent cytotoxic against breast cancer cell line MCF-7

and MDA-MB-231 with IC50 value 13.65 ppm. 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. Betulinic acid (5) was also

found active against pancreatic cancer cell under NDM condition. 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). Whereas, betulinic acid (5) is

found to be highly active against PANC-1 cancer cell line which 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). Therefore, betulinic acid (5) may be used against parncreatic

cancer which may ultimately controls diabetes.

The evaluation of insulin secretory activity of coixol (1) showed a dose dependent

insulin secretory activity. 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.

Tambulin (9) showed significant radical scavenging activity with IC50 166.15±1.92

SEM [µM] with 86.03 percent radical scavenging activity whereas the standard

butylated hydroxytoluene (BHT) has IC50 value 128.83±2.1 SEM (µM) with the

radical scavenging 85.87 percent. The total phenolic content in ethyl acetate fraction of

Bridelia retusa bark was found to be 147.20±1.5 mg GAE/gm and the total flavonoid

content was found to be 16.64±0.00 mg QE/gm. Among the three tested compounds

isolated from Bridelia retusa, tambulin (9) exhibited good urease enzyme inhibition

with IC50 41.82 ± 1.60 SEM [µM] and 88 percent which is comparable to the standard

inhibitor, thiourea (IC50 21.00 ± 0.11 SEM [µM] and which shows 98 percent

inhibition. Tambulin (9) showed a significant inhibitory effect on the release of

reactive oxygen species (ROS) from zymosan activated cells from whole blood (IC50 =

<1µg/mL) as compared to standard drug ibuprofen in whole blood (IC50 = 11.2± 1.9

µg/mL) whereas, compounds 7 and 10 showed no effect (IC50 = >100) on the release

of ROS from zymosan activated cells.

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Betulinic acid (5) is active against pancreatic cancer which ultimately indirectly

influences the condition of diabetes. The reason is that controlling pancreatic cancer

means preventing the patient from diabetes as well. One of the scholars (Permert, 1994

as cited in Gullo, 1999) mentions that diabetes occurs more frequently in patients with

pancreatic cancer than in normal people, because pancreatic cancer can cause diabetes

by destroying islet cells of pancreas or by causing peripheral resistance to insulin.

Thus, there is association between pancreatic cancer and diabetes. The results indicate

that the compounds rich in antioxidant capacity may help to reduce the inflammatory

response.

Coixol (1) thus isolated from Scoparia dulcis is found active against diabetes. It may

therefore be used as antidiabetic compound that 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.

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APPENDICES

Appendix 1a: Research paper published in International Journals

1. Potent Insulin Secretagogue from Scoparia dulcis Linn of Nepalese Origin.

Khaga Raj Sharma, Achyut Adhikari, Rahman M. Hafizur, Abdul Hameed, Sayed

Ali Raza, Surya Kant Kalauni, Jun-Ichi Miyazaki, M. Iqbal Choudhary, (2015);

Wiley online library. Phytotherapy Research, vol. 29, issue 10, pages 1672-1675.

DOI: 10.1002/ptr.5412, Submitted: 16 Dec. 2014, Revised: 12 May 2014,

Accepted: 15 June, 2015, Article first published online: 14 Jul 2015.

2. In Vitro Free Radical Scavenging Activity of Methanol Extracts of Some

Selected Medicinal Plants of Nepal.

Khaga Raj Sharma, Surya Kant Kalauni, Suresh Awale and Yuba Raj Pokharel,

(2015). Austin Journal of Biotechnology and Bioengineering, 2 (1), 1035 (ISSN:

2378-3036), Received: January19, 2015; Accepted: February11, 2015; Published:

February 24, 2015.

3. Immunomodulatory Studies on Triterpenoids from Scoparia dulcis Linn

Khaga Raj Sharma, Achyut Adhikari, Almas Jabeen, Nida Dastagir, Surya Kant

Kalauni, M. Iqbal Choudhary, Yuba Raj Pokharel, (2015). Journal of

Biochemistry and Pharmacology (Los Angel) vol. 4, issue 4, DOI: 10.4173/2167-

0501.100182. Received: July 22, 2015; Accepted: August 13, 2015; Published:

August 17, 2015.

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Appendix 1b: Paper Published in National Journals

1. Bioassay Guided Isolation of Free Radical Scavenging Agent from the Bark of

Bridelia retusa.

Khaga Raj Sharma, Achyut Adhikari, M. Iqbal Choudhary, Suresh Awale, Surya

Kant Kalauni. Journal of Institute of Science and Technology, 20(1), 2015, 108-

113.

2. Antioxidant, Phytotoxic and Antimicrobial Activities of Methanolic Extract of

Bauhinia variegata Barks.

Khaga Raj Sharma, Surya Kant Kalauni and Suresh Awale. Journal of Institute of

Science and Technology, 20 (2), 2015, 37-41.

3. Cytotoxic and Antioxidant Activities of Extract of the Leaves of Annona

reticulata.

Khaga Raj Sharma, Surya Kant Kalauni and Suresh Awale. The Journal of

University Grants Commission, 4(1), 2015, 10-19.

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Appendix 1c: Paper presented in national and international seminar/workshop

1. Khaga Raj Sharma, Rajani Malla

and Surya Kant Kalauni, In Vitro Free Radical

Scavenging Activity of Methanol Extracts of Selected Medicinal Plants of Nepal,

International Conference on Emerging Trends in Science and Technology,

organised by Research Council of Science and Technology (RCOST), Biratnagar,

Nepal, on March 22-23, 2014.

2. Khaga Raj Sharma, Surya Kant Kalauni, Phytochemical Analysis, Antioxidant,

Phytotoxic and Antimicrobial Activity of Methanol Extract of the Barks of

Bauhinia variegata, International Conference on Advance Materials and

Nanotechnology for Sustainable Development, organized by Nepal Chemical

Society In Co-operation with Central Department of Chemistry, on November 4-6,

2014.

3. Khaga Raj Sharma, Achyut Adhikari, M. Iqbal Choudhary, Surya Kant Kalauni,

Isolation of free radical scavenging agent from the Bridelia retusa of Nepal,

National Seminar on Recent Advances in Material Research (RAMR-2015),

organised by Department of Chemistry, Marwar Business School, Gorakhpur, India,

on February 10 -11, 2015.

4. Khaga Raj Sharma, Achyut Adhikari, M. Iqbal Choudhary, Surya Kant Kalauni,

Isolation and Structure Elucidation of Chemical Constituents of Scoparia dulcis

from Chitwan district of Nepal, 16th

International Symposium on Eco-Materials

Processing and Design (ISEPD-2015), Kathmandu, Nepal, on January 12-15, 2015.

5. Khaga Raj Sharma, Achyut Adhikari, M. Iqbal Choudhary, Suresh Awale, Surya

Kant Kalauni, Isolation of urease inhibitory agent from the bark of Bridelia retusa

of Syangja district of Nepal, International Workshop on Science, Environment and

Education (IWOSEE)-2015 Organised by Action Research Consultancy Nepal with

collaboration of Prithivi Narayan Campus, Pokhara, on April, 2015.

6. Khaga Raj Sharma, Achyut Adhikari, M. Iqbal Choudhary, Suresh Awale, Surya

Kant Kalauni, Isolation of radical scavenging agent from the Bridelia retusa, Nepal

Chemical Society Chemical Symposium 2015.

7. Khaga Raj Sharma, Achyut Adhikari, Almas Jabeen, Nida Dastagir, Surya Kant

Kalauni, M. Iqbal Choudhary, Immunomodulatory studies on triterpenoids from

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Scoparia dulcis Linn. 5th

International Science Congress, ISC-2015 organised by

International Science Congress Association at Tribhuvan University, Kathmandu,

Nepal, on December 8-9, 2015.

8. Khaga Raj Sharma, Achyut Adhikari, Rahman M. Hafizur, Abdul Hameed , Sayed

Ali Raza, Surya Kant Kalauni, Jun-Ichi Miyazaki, M. Iqbal Choudhary,

Antidiabetic Study of Coixol Isolated from Scoparia dulcis Linn of Nepalese

Origin. Ist National Conference on Chemical Sciences, organised by Nepal

Chemical Society Regional Committee, Butwal In-Co-operation with Department

of Chemistry, Butwal Multiple Campus, Tribhuvan University, on January 9, 2016.

9. Khaga Raj Sharma, Achyut Adhikari, Rahman M. Hafizur, Abdul Hameed, Sayed

Ali Raza, Jun-Ichi Miyazaki, M. Iqbal Choudhary, Surya Kant Kalauni, Insulin

Secreting Activity of Compounds Isolated from Scoparia dulcis Linn of Nepalese

Origin, 7th

National Conference on Science and Technology, organised by Nepal

Academy of Science and Technology (NAST), on March 29-31, 2016.

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Appendix 1d: Poster Presented in national and international seminar/workshop

1. Khaga Raj Sharma , Achyut Adhikari, M. Iqbal Choudhary, Surya Kant Kalauni,

Isolation and biological activity of chemical constituents of Scoparia dulcis from

Chitwan district of Nepal, organised by Nanotechnology and Material Processing

(NSNMP), Kathmandu University, Dhulikhel, on January 18, 2015.

2. Khaga Raj Sharma, Achyut Adhikari, M. Iqbal Choudhary, Suresh Awale, Surya

Kant Kalauni, Isolation of Free Radical Scavenging Agent from Bridelia retusa of

Nepal, International Workshop on Science, Environment and Education

(IWOSEE)-2015, organised by Action Research Consultancy Nepal with

collaboration of Prithivi Narayan Campus, Pokhara, on April 18, 2015.

3. Khaga Raj Sharma, Achyut Adhikari, M. Iqbal Choudhary, Suresh Awale, Surya

Kant Kalauni, Isolation of Free Radical Scavenging Agent from Bridelia retusa of

Nepal, National Seminar on Recent Advances in Material Research (RAMR-2015),

organised by Department of Chemistry, Marwar Business School, Gorakhpur,

India, on February 10-11, 2015.

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Appendix 2a: Seminar attended

1. Actively participated in an International conference on Emerging Trends in

Science and Technology, organised by Research council of Science and

Technology (RCOST), Biratnagar, Nepal on March 22-23, 2014.

2. Actively participated in 16th

International Symposium on Eco-materials

processing and Design (ISEPD-2015), Kathmandu, Nepal on January 12-15,

2015.

3. Actively participated in National Symposium on Nanotechnology and Material

Processing (NSNMP), organised by Kathmandu University, on January 18, 2015.

4. Actively Participated in Mini Symposium on Envision-Life Science and

Medicine, organised by Kathmandu Center for Genomics and Research Laboratory

and Biotechnology Society of Nepal on June 24, 2014.

5. Actively Participated in National Workshop on Chemical and Biological Safety,

organized by H.E.J. Research Institute of Chemistry, International Center for

Chemical and Biological Sciences, University of Karachi, Pakistan on September

23-24, 2014.

6. Participated in 2nd

Symposium on Bioequivalence and Bioavailability Studies,

organised by International Center for Chemical and Biological Sciences, University

of Karachi, Pakistan, on November 18-19, 2014.

7. Actively Participated in NASIC Workshop on Modern Spectroscopic

Techniques and Their Application in Structure Determination, Jointly

organised by NASIC, H.E.J Research Institute of Chemistry, ICCBS University of

Karachi Pakistan held on December 1-3, 2014.

8. Actively participated in National Seminar on Recent Advances in Material

Research (RAMR-2015), organised by Department of Chemistry, Marwar

Business School, Gorakhpur, India, on February 10-11, 2015.

9. Actively participated in the Chemical Symposium organized by Nepal Chemical

Society on April 11, 2015.

10. Actively participated in an International Workshop on Science, Environment

and Education (IWOSEE)-2015, organised by Action Research Consultancy

Nepal with collaboration of Prithivi Narayan Campus, Pokhara, on April 18, 2015.

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11. Actively participated in 5th

International Science Congress, ISC-2015,

organised by International Science Congress Association at Tribhuvan University,

Kathmandu, Nepal, on December 8-9, 2015.

12. Actively participated in Ist National Conference on Chemical Sciences,

organised by Nepal Chemical Society Regional Committee, Butwal In-Co-

operation with Department of Chemistry, Butwal Multiple Campus, Tribhuvan

University, on January 9, 2016.

13. Actively participated in 7th

National Conference on Science and Technology,

organised by Nepal Academy of Science and Technology (NAST), on March 29-

31, 2016.

14. Actively participated in the short course on Natural Products: Identification,

Characterization and Utilization, organised by RECAST, Tribhuvan University

held in Kathmandu, Nepal, on April 26-27, 2017.

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Appendix 2b: Letter of invitation as fellow researcher at H. E. J. Research

Institute of Chemistry, ICCBS, University of Karachi, Pakistan

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Appendix 2c: Letter of recommendation/participation in different academic

activities in H. E. J. research institute of chemistry ICCBS,

University of Karachi, Pakistan.

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Appendix 3: List of studied plants with their family, local name, English name,

yield percentage, parts used and therapeutic uses

Scientific Name Family English Name Local Name Locality % yield Parts used

Oxalis corniculata Oxalidaceae Wood sorrel Chari amilo Chitwan 5.5 Whole plant

Drymaria diandra Caryophyllaceae Chickweed Abhijhalo Chitwan 6.8 Whole plant

Melia azadarach Meliaceae China berry Bakaino Chitwan 8.6 Leaf

Cyperus rotundus Cyperaceae Nut grass Mothe jhar Chitwan 6.4 Whole plant

Cissampelos pareira Menisermaceae Abuta Batulpate Chitwan 6.4 Aerial parts

Coccinia grandis Cucurbitaceae Ivy gourd Kunruk Chitwan 6.0 Aerial parts

Euphorbia hirta Euphorbiaceae Snake weed Dudhe jhar Chitwan 10.8 Whole plant

Cynodon dactylon Poacceae Balama grass Dubo Chitwan 9.0 Whole plant

Ageratum houstonianum Asteraceae Garden Ageratum Gandhe jhar Chitwan 15.2 Whole plant

Curcuma angustifolia Zingiberaceae Turmeric Beshar Daman 27.2 Rhizomes

Strychnos nux-vomica loganiaceae Strychine Tree Kuchila Daman 16.0 Seed

Shorea robusta Dipterocarpaceae Saltree Sal Chitwan 14.6 Bark

Acacia catechu Fabaceae Cutch tree Khayar Chitwan 30.0 Bark

Lyonia avalifolia Ericaceae Pieris elliptica Aanger Syangja 12.0 Leaf

Pterocarpus santalinus Fabaceae Red sandalwood Rakta chandan Chitwan 37.4 Leaf

Desmostachya bipinnata Poacceae Halfa grass Kush Chitwan 13.4 Aerial parts

Cinnamomum tenupile Lauraceae Sugandhakokila Sugandhakokila Chitwan 32.0 Leaf

Justicia adhatoda Acanthaceae Malabar nut Asuro Syangja 22.4 Leaf

Aegle marmelos Rutaceae Golden apple Bel Chitwan 24.8 Leaf

Mahonia napaulensis Berberidaceae Mahonia napaulensis Jamane mandro Kathmandu 14.4 Leaf

Phyllanthus emblica Phyllanthaceae Emblic Amala Chitwan 36.0 Leaf

Berberis aristata Berberidaceae Tree turmeric Chutro Kathmandu 28.0 Leaf

Tinospora sinensis Menisermaceae Tinospora Gurjo ko lahara Syangja 6.6 Aerial parts

Cuscuta reflexa Convolvulaceae Giant dodder Aakash belli Syangja 28.0 Aerial parts

Leucas cephalotes Ranunculaceae Bara Halkusha Bish mara Syangja 24.0 Leaf

Drynaria propinqua Polypodiaceae Broken bone repairing Commeri Syangja 10.4 Bark

Tinospora cordifolia Menisermaceae Valvet leaf Gurjo gano Syangja 14.4 Node

Strengthen bones, headache

Gastrointestinal, antivirus

Diabetes

Antifungal, antibacterial

Anticancer, antidiabetic

Antibacterial, carminative

Analgesics, antibacterial

Antidiuretics, coolant

Skin diseases, Antiparasite

Skin care, cooling agent

Diarrhea, indigestion, asthma

Therapeutic uses

Insecticide

Antidiabetic, dysyntery

Antihelmintic, antiinflammatory

Antioxidant, antibacterial

Anti HIV, sinusitis

Antitumor, cytotoxic

Jaundice, leprosy

Cardiac tonic, antioxidant

Toxic

Antibacterial

Imparting odour

Dyeing

Poisonous

Cough, cold, asthma

Antioxidant, antibacterial

To kill feral mammals, rodents

Centella asiatica Mackinlayaceae Centella Ghottapre Kaski 46.6 Aerial parts

Asparagus filicinus Asparagaceae Asparagus Kurilo Syangja 20 Aerial parts

Justicia adhatoda Acanthaceae Malabar nut Asuro Chitwan 15.5 Leaf

Bridelia retusa Phyllanthaceae Kantakoi, Kanta Gayo Syangja 42.2 Bark

Litsea cubeba Lauraceae Exotic verbena Sidharlo Syangja 24.0 Aerial parts

Oxalis corniculata Oxalidaceae Wood sorrel Chari amilo Syangja 10.4 Whole plant

Achyranthes bidentata Amaranthaceae Oxknee Datiwan Syangja 7.4 Aerial parts

Cleistocalyx operculatus Oxalidaceae Water Banyan Kyamuno Syangja 31.0 Bark

Bauhinia variegata Fabaceae Mountain ebony Koiralo Syangja 55.2 Bark

Pogostemon amaranthoides Labiatae Night jasmine Rudilo Syangja 34.6 Aerial parts

Betula alnoides Betulaceae Indian birch Sour Manang 38.6 Bark

Scoparia dulcis Scorphulariaceae Broomweed Chini jhar Chitwan 12.9 Whole plant

Bergenia ciliata Saxifragaceae Hairy bergenia Pakhanvedh Manang 37.2 Root

Periploca calophylla Asclepiadaceae Callophyllum wight Shikari lahara Manang 13.0 Aerial parts

Astilbe rivularis Saxifragaceae Thulo ausadhi Thulo ookhati Manang 24.2 Root

Piper mullesua Piperaceae wild pepper Pipla Syangja 6.0 Aerial parts

Bombax ceiba Bombacaceae Malabar semal Simal Chitwan 48.4 Bark

Calotropis gigantean Apocynaceae Crown flower Aak Chitwan 7.4 Leaf

Annona reticulata Annonaceae Custard apple Sarifa Chitwan 39.3 Leaf

Callicarpa sp. Labiatae Verbenaceae Dhaichamle Chitwan 10.4 Aerial parts

Mimosa pudica Fabaceae Humble plant Lajjawati Chitwan 6.3 Aerial parts

Ziziphus mauritiana Rhamnaceae Wild berry Bayar Chitwan 24.8 Leaf

Cascabela thevetia Apocynaceae Lucky nut tree Karbir Chitwan 16.2 Aerial parts Heart stimulant, poisonous

Toxic alkaloids, neurology

Gastrointestinal, antivirus

Dysentry, diarrhoea

Asthma and bronchitis, antitumor

Oxylosis activity

Paralysis, swelling

Toxic to intestinal worms

Rheumatism, stomach trouble

Cerebral fever

Cardiac depressant

Antirheumatic, antifungal

Antidepressant

Anti-inflammatory

Toothache, inflammatory

Muscular swelling

Asthma and ulcer

Cough, cold

Cure diabetes

Hypertension, bronchitis diabetes

Dissolving stone in the body

Antioxidant, antibacterial

Brain stimulating, healing

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Appendix 4: Antioxidant screening of plant extract (DPPH radical scavenging

assay)

1 2 3 1 2 3

5 0.645 0.623 0.606 37.127 39.865 41.506

10 0.569 0.616 0.568 45.077 40.541 45.174

20 0.345 0.318 0.320 66.699 69.305 69.112

30 0.094 0.053 0.047 90.927 94.884 95.463

40 0.045 0.046 0.051 95.656 95.560 95.077

50 0.066 0.053 0.042 93.629 94.884 95.946

60 0.061 0.047 0.043 94.112 95.463 95.849

70 0.057 0.054 0.060 94.498 94.788 94.208

80 0.053 0.049 0.052 94.884 95.270 94.981

90 0.049 0.046 0.050 95.270 95.560 95.174

100 0.057 0.056 0.053 94.498 94.595 94.884

15.869 16.357 15.746

Lyonia ovalifolia

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

15.99±0.13

1 2 3 1 2 3

5 0.823 0.788 0.835 36.497 39.198 35.571

10 0.717 0.689 0.797 44.676 46.836 38.503

20 0.716 0.684 0.677 44.753 47.222 47.762

30 0.494 0.495 0.497 61.883 61.806 61.651

40 0.425 0.435 0.432 67.207 66.435 66.667

50 0.422 0.425 0.426 67.438 67.207 67.130

60 0.403 0.426 0.478 68.904 67.130 63.117

70 0.407 0.421 0.426 68.596 67.515 67.130

80 0.403 0.403 0.424 68.904 68.904 67.284

90 0.446 0.445 0.407 65.586 65.664 68.596

100 0.322 0.328 0.344 75.154 74.691 73.457

26.662 26.142 26.011

26.27±0.19

Drymaria diandra

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

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1 2 3 1 2 3

5 0.54 0.52 0.56 47.876 49.807 45.946

10 0.177 0.161 0.172 82.915 84.459 83.398

20 0.084 0.077 0.071 91.892 92.568 93.147

30 0.077 0.065 0.064 92.568 93.726 93.822

40 0.078 0.073 0.065 92.471 92.954 93.726

50 0.061 0.077 0.067 94.112 92.568 93.533

60 0.068 0.066 0.062 93.436 93.629 94.015

70 0.072 0.069 0.05 93.050 93.340 95.174

80 0.076 0.077 0.04 92.664 92.568 96.139

90 0.066 0.07 0.033 93.629 93.243 96.815

100 0.073 0.07 0.035 92.954 93.243 96.622

6.488 6.326 6.632Inhibitory concentration(IC50)

6.48±0.08

Bauhinia variegata

Conc.μg/mLAbsorbance Percentage scavenging

1 2 3 1 2 3

5 0.893 0.863 0.883 15.675 18.508 16.619

10 0.495 0.457 0.464 53.258 56.846 56.185

20 0.178 0.164 0.110 83.192 84.514 89.613

30 0.085 0.069 0.086 91.974 93.484 91.879

40 0.063 0.077 0.077 94.051 92.729 92.729

50 0.083 0.066 0.062 92.162 93.768 94.145

60 0.083 0.064 0.071 92.162 93.957 93.296

70 0.098 0.08 0.078 90.746 92.446 92.635

80 0.077 0.068 0.071 92.729 93.579 93.296

90 0.072 0.072 0.089 93.201 93.201 91.596

100 0.073 0.073 0.084 93.107 93.107 92.068

9.609 9.211 9.305

Bombax ceiba

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

9.37±0.12

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1 2 3 1 2 3

5 0.854 0.887 0.800 22.757 19.641 27.856

10 0.780 0.789 0.733 29.745 28.895 34.183

20 0.723 0.671 0.652 35.127 40.038 41.832

30 0.554 0.542 0.543 51.086 52.219 52.125

40 0.523 0.524 0.591 54.013 53.919 47.592

50 0.400 0.400 0.396 65.628 65.628 66.006

60 0.267 0.221 0.277 78.187 82.531 77.243

70 0.279 0.272 0.233 77.054 77.715 81.398

80 0.272 0.285 0.223 77.715 76.487 82.342

90 0.118 0.105 0.122 92.257 93.484 91.879

100 0.115 0.184 0.114 92.540 86.025 92.635

29.655 29.085 28.965Inhibitory concentration(IC50)

29.23±0.21

Euphorbia hirta

Conc.μg/mLAbsorbance Percentage scavenging

1 2 3 1 2 3

5 0.81 0.843 0.875 23.513 20.397 17.375

10 0.424 0.455 0.427 59.962 57.035 59.679

20 0.127 0.114 0.144 88.008 89.235 86.402

30 0.085 0.077 0.065 91.974 92.729 93.862

40 0.073 0.079 0.093 93.107 92.540 91.218

50 0.050 0.068 0.075 95.279 93.579 92.918

60 0.085 0.07 0.085 91.974 93.390 91.974

70 0.076 0.076 0.073 92.823 92.823 93.107

80 0.069 0.064 0.071 93.484 93.957 93.296

90 0.068 0.067 0.07 93.579 93.673 93.390

100 0.07 0.063 0.099 93.390 94.051 90.652

8.817 9.154 9.000

Phyllanthus emblica

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

8.99±0.09

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1 2 3 1 2 3

5 0.576 0.565 0.598 41.523 42.640 39.289

10 0.289 0.234 0.245 70.660 76.244 75.127

20 0.074 0.076 0.063 92.487 92.284 93.604

30 0.073 0.071 0.070 92.589 92.792 92.893

40 0.070 0.073 0.070 92.893 92.589 92.893

50 0.071 0.079 0.072 92.792 91.980 92.690

60 0.073 0.081 0.073 92.589 91.777 92.589

70 0.091 0.073 0.069 90.761 92.589 92.995

80 0.071 0.075 0.069 92.792 92.386 92.995

90 0.076 0.077 0.076 92.284 92.183 92.284

100 0.072 0.075 0.062 92.690 92.386 93.706

7.214 6.979 7.241

7.14±0.08

Berberis aristata

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

1 2 3 1 2 3

5 0.663 0.628 0.653 22.727 26.807 23.893

10 0.436 0.449 0.454 49.184 47.669 47.086

20 0.19 0.184 0.16 77.855 78.555 81.352

30 0.088 0.08 0.084 89.744 90.676 90.210

40 0.078 0.072 0.078 90.909 91.608 90.909

50 0.076 0.073 0.073 91.142 91.492 91.492

60 0.074 0.077 0.107 91.375 91.026 87.529

70 0.081 0.075 0.088 90.559 91.259 89.744

80 0.078 0.086 0.082 90.909 89.977 90.443

90 0.083 0.085 0.076 90.326 90.093 91.142

100 0.081 0.079 0.085 90.559 90.793 90.093

14.950 15.162 15.205

Bergenia ciliata

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

15.10±0.07

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1 2 3 1 2 3

5 0.715 0.776 0.726 16.667 9.557 15.385

10 0.603 0.618 0.622 29.720 27.972 27.506

20 0.552 0.546 0.536 35.664 36.364 37.529

30 0.5 0.472 0.528 41.725 44.988 38.462

40 0.386 0.259 0.369 55.012 69.814 56.993

50 0.148 0.220 0.18 82.751 74.359 79.021

60 0.115 0.134 0.128 86.597 84.382 85.082

70 0.106 0.082 0.088 87.646 90.443 89.744

80 0.068 0.071 0.075 92.075 91.725 91.259

90 0.084 0.064 0.058 90.210 92.541 93.240

100 0.066 0.063 0.053 92.308 92.657 93.823

38.187 36.261 38.186

37.54±0.64

Callicarpa sp.

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

1 2 3 1 2 3

5 0.850 0.848 0.846 17.954 18.147 18.340

10 0.850 0.846 0.851 17.954 18.340 17.857

20 0.764 0.766 0.803 26.255 26.062 22.490

30 0.674 0.760 0.582 34.942 26.641 43.822

40 0.578 0.567 0.536 44.208 45.270 48.263

50 0.463 0.473 0.489 55.309 54.344 52.799

60 0.448 0.591 0.402 56.757 42.954 61.197

70 0.437 0.437 0.477 57.819 57.819 53.958

80 0.402 0.406 0.429 61.197 60.811 58.591

90 0.477 0.594 0.554 53.958 42.664 46.525

100 0.368 0.357 0.393 64.479 65.541 62.066

47.715 47.713 47.072

Ziziphus mauritiana

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

47.57±0.21

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1 2 3 1 2 3

5 0.694 0.684 0.645 33.012 33.977 37.741

10 0.372 0.395 0.476 64.093 61.873 54.054

20 0.068 0.066 0.063 93.436 93.629 93.919

30 0.063 0.065 0.065 93.919 93.726 93.726

40 0.064 0.068 0.063 93.822 93.436 93.919

50 0.059 0.060 0.06 94.305 94.208 94.208

60 0.065 0.068 0.074 93.726 93.436 92.857

70 0.072 0.052 0.071 93.050 94.981 93.147

80 0.062 0.061 0.07 94.015 94.112 93.243

90 0.07 0.056 0.068 93.243 94.595 93.436

100 0.058 0.057 0.06 94.402 94.498 94.208

8.116 8.220 8.919

8.41±0.25

Cleistocalyx operculatus

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

1 2 3 1 2 3

5 0.820 0.81 0.805 20.849 21.815 22.297

10 0.549 0.532 0.529 47.008 48.649 48.938

20 0.402 0.311 0.282 61.197 69.981 72.780

30 0.192 0.114 0.123 81.467 88.996 88.127

40 0.107 0.053 0.096 89.672 94.884 90.734

50 0.069 0.061 0.068 93.340 94.112 93.436

60 0.07 0.062 0.074 93.243 94.015 92.857

70 0.073 0.076 0.068 92.954 92.664 93.436

80 0.063 0.063 0.070 93.919 93.919 93.243

90 0.068 0.056 0.065 93.436 94.595 93.726

100 0.064 0.068 0.079 93.822 93.436 92.375

15.107 15.107 15.022

Bridelia retusa

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

15.07±0.02

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1 2 3 1 2 3

5 0.520 0.515 0.540 49.807 45.650 47.876

10 0.264 0.328 0.234 74.517 68.340 77.413

20 0.065 0.062 0.062 93.726 94.015 94.015

30 0.065 0.069 0.056 93.726 93.340 94.595

40 0.056 0.055 0.065 94.595 94.691 93.726

50 0.071 0.070 0.074 93.147 93.243 92.857

60 0.077 0.071 0.064 92.568 93.147 93.822

70 0.076 0.077 0.065 92.664 92.568 93.726

80 0.098 0.088 0.084 90.541 91.506 91.892

90 0.084 0.078 0.079 91.892 92.471 92.375

100 0.087 0.08 0.082 91.602 92.278 92.085

6.332 6.892 6.522

6.58±0.16

Shorea robusta

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

1 2 3 1 2 3

5 0.671 0.666 0.686 35.232 35.714 33.784

10 0.069 0.085 0.075 93.340 91.795 92.761

20 0.063 0.072 0.07 93.919 93.050 93.243

30 0.065 0.076 0.071 93.726 92.664 93.147

40 0.062 0.084 0.071 94.015 91.892 93.147

50 0.052 0.069 0.071 94.981 93.340 93.147

60 0.06 0.064 0.067 94.208 93.822 93.533

70 0.075 0.073 0.063 92.761 92.954 93.919

80 0.1 0.071 0.062 90.347 93.147 94.015

90 0.079 0.066 0.06 92.375 93.629 94.208

100 0.081 0.055 0.057 92.181 94.691 94.498

7.093 7.095 7.161

Acacia catechu

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

7.11±0.02

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1 2 3 1 2 3

5 0.705 0.776 0.726 17.832 9.557 15.385

10 0.603 0.618 0.622 29.720 27.972 27.506

20 0.552 0.546 0.536 35.664 36.364 37.529

30 0.500 0.472 0.528 41.725 44.988 38.462

40 0.386 0.259 0.369 55.012 69.814 56.993

50 0.148 0.220 0.18 82.751 74.359 79.021

60 0.115 0.134 0.128 86.597 84.382 85.082

70 0.106 0.082 0.088 87.646 90.443 89.744

80 0.068 0.071 0.075 92.075 91.725 91.259

90 0.084 0.064 0.058 90.210 92.541 93.240

100 0.066 0.063 0.053 92.308 92.657 93.823

38.187 36.261 38.186

36.60±1.20

Scoparia dulcis

Conc.μg/mLAbsorbance Percentage scavenging

Inhibitory concentration(IC50)

Appendix: 5 Total phenolic, flavonoid content and free radical scavenging (IC50)

Plant extracts Free radical scavenging (IC50) Total phenolic mg GAE/gm Total flavonoid mg QE/gm

Drymaria diandra 26.27±0.19 122.45±0.96 11.51±0.30

Euphorbia hirta 29.23±0.21 138.10±4.90 11.54±0.00

Shorea robusta 6.58±0.16 145.80±5.00 14.88±0.80

Acacia catechu 7.11±0.02 169.35±0.25 18.63±0.30

Lyonia ovalifolia 15.99±0.13 137.75±1.55 12.56±0.00

Phyllanthus emblica 8.99±0.09 154.15±0.85 15.60±0.20

Berberis aristata 7.14±0.08 145.75±0.05 18.32±2.40

Bridelia retusa 15.07±0.02 147.20±1.50 16.64±0.00

Cleistocalyx operculatus 8.41±0.25 154.75±2.85 13.83±0.60

Bauhinia variegata 6.48±0.08 156.30±0.30 16.04±1.40

Bergenia ciliata 15.10±0.07 145.85±0.15 15.71±0.10

Bombax ceiba 9.37±0.12 147.45±0.85 12.54±0.10

Callicarpa sp. 37.54±0.64 127.60±0.90 10.70±0.09

Ziziphus mauritiana 47.50±0.21 95.80±3.60 11.16±3.60

Scoparia dulcis 36.60±1.20 145.75±0.05 12.54±0.10

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Appendix 6:Total phenolic content (standard calibration curve for gallic acid)

Concentration (μg/mL) Radical scavenging

25 0.133

50 0.197

75 0.371

100 0.507

125 0.600

150 0.705

175 0.884

200 0.906

225 0.912

250 1.120

Appendix 7: Cytotoxicity (breast cancer) of compounds glutinone, betulinic acid,

sigmastanone, friedelin and coixol.

Sigmastanone (6)

S. N. Concentration

(ppm)

Absorbance

I II II

1 1.56 0.404 0.400 0.435

2 3.12 0.457 0.455 0.383

3 6.25 0.413 0.430 0.400

4 12.50 0.428 0.420 0.350

5 25.00 0.404 0.430 0.056

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Glutinone (9)

S. N. Concentration

(ppm)

Absorbance

I II II

1 1.56 0.400 0.400 0.4

2 3.12 0.372 0.375 0.365

3 6.25 0.388 0.350 0.340

4 12.50 0.356 0.378 0.360

5 25.00 0.344 0.340 0.33

Friedelin (4)

S. N. Concentration

(ppm)

Absorbance

I II II

1 1.56 0.448 0.367 0.407

2 3.12 0.408 0.361 0.416

3 6.25 0.405 0.408 0.344

4 12.50 0.38 0.402 0.406

5 25.00 0.409 0.400 0.337

Coixol (1)

S. N. Concentration

(ppm)

Absorbance

I II II

1 1.56 0.378 0.394 0.407

2 3.12 0.344 0.43 0.416

3 6.25 0.367 0.411 0.344

4 12.50 0.325 0.303 0.406

5 25.00 0.261 0.242 0.337

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Betulinic acid (5)

S. N. Concentration

(ppm)

Absorbance

I II II

1 1.56 0.404 0.470 0.404

2 3.12 0.473 0.439 0.425

3 6.25 0.393 0.414 0.464

4 12.50 0.145 0.154 0.127

5 25.00 0.095 0.105 0.094

Appendix 8: List of identified plants used in the study

Code Scientific Name Family

KS1 Oxalis corniculata (From Syangja) Oxalidaceae

KS2 Drymaria diandra Caryophyllaceae

KS3 Melia azadarach Meliaceae

KS4 Cyperus rotundus Cyperaceae

KS5 Cissampelos pareira Menisermaceae

KS6 Coccinia grandis Cucurbitaceae

KS7 Euphorbia hirta Euphorbiaceae

KS8 Cynodon dactylon Poacceae

KS9 Ageratum houstonianum Asteraceae

KS10 Curcuma angustifolia Zingiberaceae

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KS11 Strychnos nux-vomica loganiaceae

KS12 Shorea robusta Dipterocarpaceae

KS13 Acacia catechu Fabaceae

KS14 Lyonia avalifolia Ericaceae

KS15 Pterocarpus santalinus Fabaceae

KS16 Desmostachya bipinnata Poacceae

KS17 Cinnamomum tenupile Lauraceae

KS18 Justicia adhatoda (From Chitwan) Acanthaceae

KS19 Aegle marmelos Rutaceae

KS20 Mahonia napaulensis Berberidaceae

KS21 Phyllanthus emblica Phyllanthaceae

KS22 Berberis aristata Berberidaceae

KS23 Tinospora cordifolia Menisermaceae

KS24 Cuscuta reflexa Convolvulaceae

KS25 Leucas cephalotes Ranunculaceae

KS26 Drynaria propinqua Polyodiaceae

KS27 Tinospora sinensis Menisermaceae

KS28 Centella asiatica Mackinlayaceae

KS29 Asparagus filicinus Asparagaceae

KS30 Achyranthes bidentata Amaranthaceae

KS31 Bridelia retusa (Voucher No. 3424) Phyllanthaceae

KS32 Litsea cubeba Lauraceae

KS33 Oxalis corniculata (From Chitwan) Oxalidaceae

KS34 Justicia adhatoda (From Syangja) Acanthaceae

KS35 Cleistocalyx operculatus Oxalidaceae

KS36 Bauhinia variegata Fabaceae

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KS37 Pogostemon amaranthoides Labiatae

KS38 Betula alnoides Betulaceae

KS39 Scoparia dulcis (Voucher No. 2812) Scorphulariaceae

KS40 Bergenia ciliata Saxifragaceae

KS41 Periploca calophylla Asclepiadaceae

KS42 Astilbe rivularis Saxifragaceae

KS43 Piper mullesua Piperaceae

KS44 Bombax ceiba Bombacaceae

KS45 Calotropis gigantea Apocynaceae

KS46 Annona reticulata Annonaceae

KS47 Callicarpa sp. Labiatae

KS48 Mimosa pudica Fabaceae

KS49 Ziziphus mauritiana Rhamnaceae

KS50 Cascabela thevetia Apocynaceae

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Appendix 9: List of spectra of some isolated pure compounds

1H-NMR spectrum of friedelin (4)

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Mass spectrum of glutinol (2)

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DEPT 135 and 1H-NMR spectrum of glutinone (3)

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1H-NMR and DEPT 135 spectrum of glutinone (3)

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1H-NMR of coixol (1)

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Broad band and DEPT 90 spectrum of coixol (1)

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UV and IR spectrum of coixol (1)

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1H-NMR of betulinic acid (5)