i
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
ii
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
iii
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
iv
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
v
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
vi
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
vii
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.
viii
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.
ix
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
x
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
xi
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
xii
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
xiii
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
xiv
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
xv
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
xvi
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
xvii
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
xviii
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
xix
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
xx
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
.
1
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.
2
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
3
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)
4
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
5
(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.
6
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.
7
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.
8
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).
9
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
10
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
11
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
12
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).
13
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
14
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
15
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
16
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
17
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
18
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
19
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
20
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
21
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
22
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
23
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
24
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
25
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
26
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
27
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
28
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
29
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
30
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
31
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
32
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
33
(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
34
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
35
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
36
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
37
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).
38
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
39
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
40
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
41
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
42
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
43
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).
44
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
45
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.
46
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).
47
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
48
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
49
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,
50
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).
51
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).
52
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
53
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:
54
Scopadulcis acid A Scopadulcis acid B
Scoparic acid A Scoparic acid B
Apigenin Scoparic acid C
55
Cirsimarin Hymenoxin
Betulinic acid Benzoxazolone
Alpha amyrin Acacetin
56
Cynaroside Coumaric acid
Cirsitakaoside Gentisic acid
Iffaionic acid Glutinol
57
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
58
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
59
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
60
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
61
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.
62
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
63
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
64
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)
65
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
66
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
67
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
68
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
69
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
70
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
71
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)
72
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
73
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)
74
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)
75
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
76
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)
77
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
78
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
79
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.
80
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,
81
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
82
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.
83
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
84
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
85
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)
86
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
87
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
88
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).
89
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
90
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
91
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).
92
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,
93
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 - -
94
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 - - - -
95
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
96
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
97
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
98
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
99
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)
100
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.
101
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
102
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)
103
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)
104
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)
105
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)
106
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)
107
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
108
*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).
109
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
110
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.
111
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.
112
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
113
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
114
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).
115
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
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)
117
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
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
[µ
M]
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
[µ
M]
120
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.
121
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:
122
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.
123
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
124
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.
125
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.
126
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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.
147
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.
148
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
149
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.
150
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.
151
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.
152
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.
153
Appendix 2b: Letter of invitation as fellow researcher at H. E. J. Research
Institute of Chemistry, ICCBS, University of Karachi, Pakistan
154
Appendix 2c: Letter of recommendation/participation in different academic
activities in H. E. J. research institute of chemistry ICCBS,
University of Karachi, Pakistan.
155
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
156
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)
157
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
158
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
159
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
160
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
161
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
162
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
163
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
164
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
165
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
166
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
167
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
168
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
169
Appendix 9: List of spectra of some isolated pure compounds
1H-NMR spectrum of friedelin (4)
170
Mass spectrum of glutinol (2)
171
DEPT 135 and 1H-NMR spectrum of glutinone (3)
172
1H-NMR and DEPT 135 spectrum of glutinone (3)
173
1H-NMR of coixol (1)
174
Broad band and DEPT 90 spectrum of coixol (1)
175
UV and IR spectrum of coixol (1)
176
1H-NMR of betulinic acid (5)