In Vitro Pharmacological Investigations of Aqueous Fraction of Coccinia Cordifolia Leaves

IN V
A Disser

VITRO PH
FRA
rtation subm
fulfillmen
HARMACO
ACTION O
mitted to th
nt of the req
D
OLOGICA
OF COCCI
he Departme
quirements f
Sub
Samiya K
ID: 20
Departme
East W

AL INVES
CINIA COR

ent of Pharm
for the degr

bmitted B
Khondaker
010-3-70-0

ent Of Pha
West Univer
STIGATI
RDIFOLIA
macy, East
ree of Bache

By
r Rinta
048
armacy
rsity
IONS OF A
IALEAVE
West Unive
elor of Phar
AQUEOU
ES
ersity, in pa
rmacy.
i
US
artial

Dedication
This Research paper is dedicated to
My beloved parents,
Who are my biggest Inspirations
ii
DECLARATION BY THE CANDIDATE

I, Samiya Khondaker Rinta, hereby declare that this dissertation, entitled In vitro
pharmacological investigations of aqueous fraction of Coccinia cordifolia leaves submitted
to the Department of Pharmacy, East West University, in the partial fulfillment of the
requirement for the degree of Bachelor of Pharmacy (Honors) is a genuine & authentic research
work carried out by me under the guidance of Nigar Sultana Tithi, Lecturer and Mahbubul
Hoque Shihan, Senior Lecturer, Department of Pharmacy, East West University, Dhaka. The
contents of this dissertation, in full or in parts, have not been submitted to any other Institute or
University for the award of any Degree or Diploma of Fellowship.

----------------------------------
Samiya Khondaker Rinta
ID: 2010-3-70-048
Department of Pharmacy
East West University
Aftabnagar, Dhaka

iii
CERTIFICATION BY THE SUPERVISOR

This is to certify that the dissertion, entitled In vitro pharmacological investigations of
aqueous fraction of Coccinia cordifolia leaves is a bonafide research work done, under our
guidance and supervision by Samiya Khondaker Rinta (ID: 2010-3-70-048), in partial fulfillment
of the requirement for the degree of Bachelor of Pharmacy.

------------------------------------- -------------------------------------
Nigar Sultana TithiMahbubul Hoque Shihan
Lecturer & SupervisorSenior Lecturer & Co-supervisor
Department of Pharmacy Department of Pharmacy
East West UniversityEast West University
Aftabnagar, Dhaka Aftabnagar, Dhaka

iv
ENDORSEMENT BY THE CHAIRPERSON

This is to certify that the dissertation, entitled In vitro pharmacological investigations of
aqueous fraction of Coccinia cordifolia leaves is a bonafide research work done by Samiya
Khondaker Rinta (ID: 2010-3-70-048), in partial fulfillment of the requirements for the degree of
Bachelor of Pharmacy.

---------------------------------------
Dr. Chowdhury Faiz Hossain
Professor & Chairperson
Department of Pharmacy
East West University
Aftabnagar, Dhaka
ACKNOWLEDGEMENTS

All praise is for Almighty for all the bounties granted to me and only with His guidance and help
this achievement has become possible.
It is my pleasure and proud privilege to express my heartiest regards and gratitude to my
respected teacher and supervisor Nigar Sultana Tithi, Lecturer, Department of Pharmacy, East
West University, for her expert supervision, constructive criticism, valuable advice, optimistic
counseling, constant support and continuous backup and encouragement throughout every phase
of the project as well as to prepare this dissertation.
I am thankful to my honorable teacher and co-supervisor, Mahbubul Hoque Shihan, Senior
Lecturer, Department of Pharmacy, East West University, for his amiability to provide me with
untiring guidance, whole hearted cooperation and for his extensive knowledge in research that
helped me in all the spheres to perform the research work.
My special thanks to Nishat Nasrin, Senior Lecturer, Department of Pharmacy, East West
University and for giving me her valuable time, constant advising, encouragement and for her
kind words during my troubling moments.
I would also like to put forward my most sincere regards and profound gratitude to Dr.
Chowdhury Faiz Hossain, Professor and Chairperson, Department of Pharmacy, East West
University, for giving me the opportunity to conduct such an interesting project and for
facilitating a smooth conduction of my study
I would also like to extend my thanks to all the research students in the lab, lab officers and other
staffs of the Department of Pharmacy for their help and assistance, friendly behavior and earnest
co-operation which enabled me to work in a very congenial and comfortable ambience.
I owe special thanks to my fellow research group members for their immense support and
contribution in my research work.
Last but not the least, I would like to thank my family, and friends for their care and
encouragement during my research work.
vi
CONTENTS
1. Introduction 1-21
1.1 Medicinal plants 1
1.2 Medicinal plants of Bangladesh 2
1.3 Vernacular names of Coccinia cordifolia 7
1.4 Taxonomy of C. cordifolia 7
1.5 Cucurbitaceae family 8
1.5.1 Species of Cucurbitaceae available in Bangladesh 8
1.6 Coccinia cordifolia 11
1.7 Synonyms of Coccinia cordifolia 13
1.8 Distribution of Coccinia cordifolia 13
1.9 Habitant of Coccinia cordifolia 14
1.10 Morphology of plant 15
1.10.1 Stem of C. cordifolia 15
1.10.2 Leaf of C. cordifolia 15
1.10.3 Flower of C. cordifolia 16
1.10.4 Fruit of C. cordifolia 17
1.10.5 Seed of C. cordifolia 17
1.10.6 Root of C. cordifolia 18
vii
1.11 Reproduction and dispersal of Coccinia cordifolia 18

1.12 Vegetative propagation of Coccinia cordifolia 18
1.13 Nutritional value of Coccinia cordifolia 19
1.14 Local uses of C. cordifolia in Bangladesh 19
1.15 Toxicology of C. cordifolia 21
2. Literature Review 22-38
2.1 Phytochemical constituents 22
2.2 Pharmacological properties 22
2.2.1 Anti-diabetic and hypoglycemic activity 22
2.2.1.1 In vivo study on human model 22
2.2.1.2 In vivo study on animal model 24
2.2.2 Hypolipidemic and antidyslipidemic activity 25
2.2.3 Anti-inflammatory activity 27
2.2.4 Analgesic and antipyretic activity 28
2.2.5 Anti-bacterial and anti-fungal activity 28
2.2.6 Anthelmintic activity 30
2.2.7 Cytotoxic, antitumer and pesticidal activity 30
2.2.8 Antioxidant activity 31
2.2.9 Chemoprotective activity 32
2.2.10 Antiulcer and cytoprotective activity 32
viii
2.2.11 Anti-hepatotoxic activity 33

2.2.12 Fertility inducing activity 35
2.2.13 Larvicidal, repellent and egg hatching inhibition property 35
2.2.14 Antilithiatic activity 36
Rationale and objective of the work 38
3. Methods and Materials 39-57
3.1 Collection and preparation of plant material 39
3.2 Extraction of the plant material 39
3.3 Preparation of mother solution 40
3.4 Partition of mother solution 40
3.4.1 Partition with n-hexane 40
3.4.2 Partition with carbon tetrachloride 40
3.4.3 Partition with chloroform 40
3.4.4 Partition with ethyl acetate 40
3.4.5 Collection of aqueous Fraction 42
3.5 Antioxidant activity 42
3.5.1 DPPH (1,1-diphenyl-2-picrylhydrazyl) radical test 42
3.5.1.1 Principle 42
3.5.1.2 Apparatus and reagents 43
3.5.1.3 Procedure 43-44
ix
3.5.2 Total phenolic content 44

3.5.2.3 Procedure 45-46
3.5.3 Total flavonoid content 46
3.5.3.2 Procedure 47-49
3.6 Brine shrimp lethality bioassay 49
3.6.1 Principle 49
3.6.2 Apparatus and reagents 50
3.6.3 Procedure 50-52
3.6.3.1 Preparation of sea water 50
3.6.3.2 Hatching of brine shrimp 50
3.6.3.3 Preparation of test solutions 51
3.6.3.5 Counting of nauplii 52
3.7 Antimicrobial activity by disc diffusion method 52
3.7.1 Principle 52
3.7.2 Apparatus and reagents 52
3.7.2.1 Materials 52
x
3.7.2.2 Test sample of Coccinia cordifolia 53

3.7.2.3 Test organisms 53
3.7.3 Procedure 53-57
3.7.3.1 Preparation of the medium 53
3.7.3.2 Sterilization procedure 54
3.7.3.3 Preparation of test plate 55
3.7.3.4 Preparation of discs 55
3.7.3.5 Preparation of test sample 56
3.7.3.6 Application of test samples 56
3.7.3.7 Diffusion and incubation 56
3.7.3.8 Determination of antimicrobial activity by measuring
the zone of inhibition
57
4. Results and Discussion 58-71
4.1 Antioxidant test results 58
4.1.1 DPPH test results 58
4.1.1.1 Preparation of standard curve 59
4.1.1.2 Preparation of aqueous fraction curve 60
4.1.1.3 Discussion 61
4.1.2 Total phenol content test results 62
xi
4.1.2.2 Total phenolic content present in aqueous fraction 63

4.1.3 Total flavonoid content result 64
4.1.3.2 Total flavonoid content present in aqueous fraction 65
4.2 Brine shrimp lethality bio-assay result 66
4.2.1 Preparation of standard curve 67
4.2.2 Preparation of aqueous fraction curve 68
4.2.3 Discussion 69
4.3 Antibacterial test results 70
4.3.1 Zone of inhibition of standard and aqueous fraction 70
4.3.2 Discussion 71
5. Conclusion 72
5.1 Conclusion 72
6. Reference 73-79

xii
LIST OF FIGURES
Figure 1.1: Coccinia cordifolia plant 12
Figure 1.2: Coccinia cordifolia, A: Twig with flower; B: Male flower; C:
Female flower; D: L.S. female flower; E: Male flower with
corolla split open; F: T.S. Ovary 13
Figure 1.3: Regional distribution of C. cordifolia (Synonym: Coccinia
indica) and other most frequently mentioned plants 14
Figure 1.4: C. cordifolia leaves, fruits and flowers. 15
Figure 1.5: Leaves of C. cordifolia 16
Figure 1.6: Flower of C. cordifolia 16
Figure 1.7: Fruits of C. cordifolia- (a) unripe fruit; (b) ripe fruit; (c) cross-
section of unripe fruit 17
Figure 1.8: Seeds of C. cordifolia 17
Figure 1.9: Roots of C. cordifolia 18
Figure 3.1: Drying of extract using rotary evaporator 39
Figure 3.2: Schematic representation of the partitioning of methanolic
crude extract of Coccinia cardifolia leaves 41
Figure 3.3: Mechanism of free radical scavenging activity 42
Figure 3.4: Schematic diagram of DPPH test 44
Figure 3.5: Schematic diagram of flavonoid content test 49
Figure 3.6: Artemia salina 24 hours old 51
Figure 3.7: Autoclave machine 54
Figure 3.8: Laminar hood 55
Figure 3.9: Incubator 57
Figure 4.1: Regression line and R
2
value of ascorbic acid 59
Figure 4.2: Regression line and R
2
value of aqueous fraction of Coccinia
xiii
cordifolia 60
Figure 4.3: Comparison between IC
50
values of standard and extract 61
Figure 4.4: Graphical representation of assay of phenolic content of
ascorbic acid 62
Figure 4.5: Comparison between absorbances of standard and extract over
different concentrations 63
Figure 4.6: Graphical representation of assay of flavonoid content of
ascorbic acid 65
Figure 4.7: Plot of % mortality and predicted regression line of Tamoxifen
(standard) 67
Figure 4.8: Plot of % mortality and predicted regression line of aqueous
fraction (extract) 68
Figure 4.9: Comparison between LC
50
values of standard and extract 69
Figure 4.10: Comparison of antimicrobial activity between Azithromycin
and aqueous fraction 71

xiv
LIST OF TABLES
Table 1.1: Name and medicinal uses of some common plants in Bangladesh 2
Table 1.2: Showing the vernacular names of Coccinia cordifolia 7
Table 1.3: Some plants of Cucurbitaceae family available in Bangladesh 9
Table 1.4: Nutritional value of per 100gm of edible portion of C. cordifolia 19
Table 1.5: Use of C. cordifolia in different parts of Bangladesh 19
Table 3.1: Composition of 100mg Folin-Ciocalteu Reagent 44
Table 3.2: Different concentrations of ascorbic acid solution preparation 48
Table 3.3: List of micro-organisms 53
Table 4.1: % inhibition and IC
50
values of ascorbic acid 59
Table 4.2: % inhibition and IC
50
values of aqueous fraction of Coccinia
cordifolia 60
Table 4.3: Free radical scavenging capacity of ascorbic acid and aqueous
fraction of Coccinia cordifolia leaves 61
Table 4.4: Total phenol content of ascorbic acid 62
Table 4.5: Total phenolic content of aqueous fraction of leaves of Coccinia
cordifolia 63
Table 4.6: Total flavonoid content of ascorbic acid 64
Table 4.7: Total flavonoid content of aqueous fraction of leaves of
Coccinia cordifolia 65
Table 4.8: Results of the bioassay of Tamoxifen (standard) 67
Table 4.9: Results of the bioassay of aqueous fraction (extract) 68
Table 4.10: Cytotoxic activity of Tamoxifen and aqueous fraction of
Coccinia cordifolia leaves 69
Table 4.11: Antimicrobial activity of standard sample (Azithromycin) and
aqueous fraction 74
xv
LIST OF ABBREVIATIONS
g microgram
l microliter
AAE Ascorbic Acid Equivalent
ALK.P alkaline phosphatase
ALP alkaline phosphatase
ALT alkaline aminotransferase
AST aspartate aminotransferase
DMSO dimethyl sulfoxide
DPPH 1,1-diphenyl-2-picrylhydrazyl
CI Coccinia indica
CLEt Coccinia indica leaf extract
CP cyclophosphamide
FCR Folin-Ciocalteu Reagent
g gram
GSH glutathione
hr hour
IC
50
The concentration of a drug which is required for 50% of inhibition of a
specific test.
LC
50
The lethal concentration required to kill 50% of the sample population of
a specific test.
LPO Lipid peroxidation
L.S. Longitudinal Section
MDA malondialdehyde
mg milligram
ml milliliter
xvi
PI protease inhibitor
SGOT Serum glutamic oxaloacetic transaminase
SGPT Serum glutamic pyruvic transaminase
SOD Superoxide dismutase
STZ Streptozotocin
TB Total billirubin
TP Total proteins
T.S. Transverse Section
USDA United States Department of Agriculture
UV Ultraviolet
WHO World Health Organization

xvii
ABSTRACT
The study was designed for pharmacological investigation of aqueous fraction of methanol
extract of the leaves of Coccinia cordifolia (Family: Cucurbitaceae). The powdered leaves of
Coccinia cordifolia were extracted with methanol and then partitioned with n-hexane, carbon
tetrachloride, chloroform and ethyl acetate consecutively. The aqueous fraction remaining at the
end was investigated for free radical scavenging activity (DPPH Test), total flavonoid content,
total phenol content, brine shrimp lethality test and antimicrobial test. From DPPH test the IC
50

values obtained were 42.82g/ml and 25.19g/ml for standard (ascorbic acid) and aqueous
fraction, respectively. The fraction contained 64.38mg AAE/g of total phenolic content and
212mg AAE/g of total flavaniod content. Screening for cytotoxic properties using brine shrimp
lethality bioassay with tamoxifen (LC
50
value of 13.38g/ml) as positive control showed that the
fraction have considerable cytotoxic potency exhibiting LC
50
value 12.39g/ml. In antimicrobial
activity investigation, the aqueous fraction showed low antibacterial and antifungal activity
against the tested organisms compared to azithromycin (30g/disc) that was used as positive
control.The aqueous fraction showed strong cytotoxic activity, moderate antioxidant activity and
slight antimicrobial activity. Further investigations are needed for the proper identification and
isolation of these bioactive compounds to produce safer drugs for treatment of harmful diseases.
Key words: Coccinia cordifolia, Brine shrimp lethality bio-assay, DPPH test, phenolic content,
flavonoid content,antimicrobial activity.

In vitro pharmacological investigations of aqueous fraction of Coccinia cordifolia leaves

Chapter One
INTRODUCTION
In vitro pharmacological investigations of aqueous fraction of Coccinia cordifolia leaves 1
1.1 Medicinal Plants
The use of natural products with therapeutic properties is as ancient as human civilization and,
for a long time, mineral, plant and animal products were the main sources of drugs. The
development of organic chemistry resulted in a preference for synthetic products for
pharmacological treatment. Plant derived medicines are used in selfmedication in all cultures.
Only a fraction of the worlds available plants have been studied. Discovery and use of synthetic
drugs have caused side effects or adverse reactions that were not for seen in preclinical and
clinical examinations. As a result, a resurgence of interest in the study and use of medicinal
plants has been taken place during the last two decades. As a result of modern isolation
techniques and pharmacological testing procedures, new plant drugs found their way into
modern medicine as purified substances rather than in the form of galenical preparations (Reddy
et al., 2010). Compounds such as muscarine, physostigmine, cannabinoids, yohimbine, forskolin,
colchicine and phorbol esters, all obtained from plants, are important tools used in
pharmacological, physiological and biochemical studies (Williamson et al., 1996).
Plants are valuable for modern medicine in four basic ways:
1) They are used as sources of direct therapeutic agents.
2) They serve as raw materials base for elaboration of more complex semi synthetic chemical
compounds.
3) The chemical structures derived from plant sources can be used as models for new synthetic
compounds.
4) Finally plants can be used as taxonomic markers for the discovery of new compounds
(Reddy et al., 2010).
The study of traditional human uses of plants is called Ethnobotany. It is a recognised way to
discover new effective medicines for future. In ancient Greece, plants were classified and
descriptions of them were given by scholars. It aids in the identification process. Researchers
identified in 2001 that 122 compounds that are used in modern medicine, were isolated and
identified from "ethnomedical" plant sources. The current use of the active elements of the plants
is 80% similar to those of ethnomedical use (Fabricant and Farnsworth, 2001).
In vitro pha
About 25
being in
Organisa
drugs ob
digoxin
vinblastin
codeine f
1.2 Med
Medicina
prescribe
used as h
country.
desire. T
stands at
as growin
Table 1.1
Scientifi
Family:
Name: K
Distribu
Plant pa
Medicin
worm kil
armacological in
5% of the d
current use
ation (WHO)
btained from
from Digita
ne from Ca
from Papave
dicinal Plan
al plants m
ed by practit
household re
From long a
The total num
t about 2000
ng or availab
1: Name and
c Name: An
Acanthacea
Kalomegh (lo
tion: Chittag
art used: wh
al uses: m
ller, dysenter
nvestigations of
drugs prescri
. Of the 252
), 11% are e
m natural pre
alis spp., q
atharanthus
er somniferu
nts of Bang
mainly used
ioners of tra
emedies by t
ago medicin
mbers of pl
0. About 450
ble in Bangla
d medicinal u
ndrographis p
ocal name), C
gong and Ch
hole plant
metabolic pr
ry, liver dise
f aqueous fracti
ibed worldw
2 drugs con
exclusively o
cursors. Exa
quinine and
roseus, atro
um (Shu, 199
gladesh
in the prep
aditional med
the common
nal plant assa
lants with m
0 to 500 nam
adesh (Sadi,
uses of some
paniculata
Creat (englis
hittagong Hi
roblem, gas
ease
ion of Coccinia
wide come fr
nsidered as b
of plant origi
amples of im
d quinidine
opine from
98).
parations of
dicine in dif
n people. Me
ail their leaf
medicinal pro
mes of such m
, 2012).
e common p
sh name)
ill Tracts
tric, fever,
cordifolia leave
from plants,
basic and es
in and a sign
mportant dru
from Cinch
Atropa bel
f Unani and
fferent parts
edical plant i
f, stem, root,
operties in t
medicinal p
plants in Ban
es
121 such a
ssential by th
nificant num
ugs obtained
hona spp.,
lladonna an
d Ayurvedic
of the count
is an import
, fruit etc. ar
the subconti
lants are so
ngladesh (Ud
ctive compo
he World H
mber are synt
d from plant
vincristrine
nd morphine
c medicine,
try and other
tant wealth i
re used to pr
inent are pr
far been en
ddin, 2014)
2
ounds
Health
thetic
ts are
e and
e and
also
rs are
in our
rotect
resent
listed

In vitro pha
Scientifi
Family:
Name: B
Distribu
Plant pa
Medicin
Scientifi
Family:
Name:
(english n
Distribu
Plant pa
Medicin
lessens b
Scientifi
Family:
Name: N
Distribu
Plant pa
Medicin
inflamma
loss treat
Scientifi
Family:
Name: G
Distribu
Plant pa
Medicin
and diges
armacological in
c Name: Ae
Rutaceae
Bel (local nam
tion: Cultiv
art used: Fru
al uses: dys
c Name: An
Annonaceae
Ata, Sharif
name)
tion: Cultiv
art used: Fru
al uses: jau
burning sensa
c Name: Az
Meliaceae
Neem
tion: Plante
art used: wh
al uses: an
ations, gastr
tment
c Name: Alo
Liliaceae
Ghritakumari
tion: Cultiv
art used: lea
al uses: pur
stive
nvestigations of
egle marmelo
me), Wood A
ated all over
uit
entery, diarr
nnona squam
e
fa (local n
ated all over
uit & leaf
undice; laxa
ation and sed
adirachta in
d all over Ba
hole plant
nthelmintic,
ric, fever, sk
oe barbaden
i (local name
ated, mainly
af
rgative, anth
f aqueous fracti
os
Apple (engli
r Bangladesh
rhoea
mosa
name); Cust
r Bangladesh
ative and an
dative to the
ndica
angladesh
used in
kin diseases
nsis
e), Aloe (eng
y in Natore
helmintic, c
ion of Coccinia
ish name)
h
tard Apple
h
nthelmintic;
e heart
ulcers and
and in hair
glish name)
carminative
cordifolia leavees 3

In vitro pha
Scientifi
Family:
Name: M
Distribu
Plant pa
Medicin
skin dise
Scientifi
Family:
Name: J
black-ber
Distribu
Plant pa
Medicin
asthma a
Scientifi
Family:
Name: A
(english n
Distribu
and Chitt
Plant pa
Medicin
antipyret
armacological in
c Name: La
Lythraceae
Mehedi (loca
tion: Cultiv
art used: bar
al uses: jau
ases
c Name: Syz
Myrtaceae
am, Kalojam
rry (english
tion: Plante
art used: wh
al uses: g
nd dysentery
c Name: He
Asclepiadac
Anantamul
name)
tion: Fores
tagong Hill T
art used: roo
al uses: bl
tic, antidiarrh
nvestigations of
wsonia inerm
al name), He
ated through
rk, leaf and s
undice, enla
zygium cumi
m (local nam
name)
d all over Ba
hole plant
good for so
y, cures dyse
emidesmus in
ceae
(local name
sts of Dhak
Tracts
ot
ood purifier
hoeal and pu
f aqueous fracti
mis
nna (english
hout the coun
seed
arged spleen
ini
me), Black Pl
angladesh
ore throat,
entery
ndicus
e), Indian S
ka-Tangail;
r, demulcen
urgative
ion of Coccinia
h name)
ntry
n, obstinate
lum, Indian
bronchitis,
Sarsaparilla
Chittagong
nt, diuretic,

In vitro pha
Scientifi
Family:
Name:
(english n
Distribu
Plant pa
Medicin
enlargem
anthelmin
bowels; i
Scientifi
Family:
Name: D
(english n
Distribu
Plant pa
Medicin
stomachi
Scientifi
Family:
Name: P
Distribu
Banglade
Plant pa
Medicin
dyspepsi
armacological in
c Name: Tri
Fabaceae
Methi, Met
name)
tion: Cultiv
art used: wh
al uses: use
ment of the
ntic, carmin
increase app
c Name: Pu
Punicaeae
Dalim, Anar,
name)
tion: Plante
art used: fru
al uses: cu
ic; astringent
c Name: Ca
Caricaceae
Pepe (local n
tion: W
esh
art used: Fru
al uses: dig
a, intestinal
nvestigations of
igonella foen
thishak (loc
ated in the w
hole plant
eful in drops
e spleen a
native, emol
etite, and cu
unica granatu
, Bedana (lo
d throughou
it, seed and
ures dyspep
t; strengthen
arica papaya
ame), Papay
Widely cu
uit, leaf and
gestive and
irritation an
f aqueous fracti
num-graecum
cal name);
western distr
sy, chronic
and liver;
llient, astrin
ure bronchiti
um
ocal name); P
ut the country
bark
sia; cardiac
ns gums; use
a
ya (english n
ultivated
seed
anthelminti
d ringworm
ion of Coccinia
m
Fenugreek
ricts
cough, and
antipyretic,
gent to the
s and piles
Pomegrante
y
tonic and
ed in piles
name)
throughout
ic; used in
In vitro pha
Scientifi
Family:
Name: A
Distribu
Plant pa
Medicin
Scientifi
Family:
Name:
name)
Distribu
Plant pa
Medicin
useful in
Scientifi
Family:
Name: T
Bay Leaf
Distribu
occationa
Plant pa
Medicin
diarrhoea
of the tee

armacological in
c Name: An
Bromeliacea
Anaras (local
tion: Cultiv
art used: lea
al uses: anth
c Name: Ad
Acanthaceae
Basak (loca
tion: Cultiv
art used: roo
al uses: exp
gonorrhoea
c Name: Cin
Lauraceae
Tejpata (loc
f (english na
tion: Cultiv
ally in other
art used: lea
al uses: stim
a, dyspepsia
eth, sore thro
nvestigations of
nanas sativus
ae
l name), Pine
ated all over
af
helmintic
dhatoda zeyla
e
al name), M
ated through
ot, bark and l
pectorant, an
and rheuma
nnamomum
cal name);
ame).
vated comm
places
af
mulant, carm
, anoerexia,
oat, piles, he
f aqueous fracti
s
eapple (engl
r Bangladesh
anica
Malabar Nu
hout Banglad
leaf
ntispasmodic
atism
tamala
Indian Cass
mercially in
minative; use
skin disease
art troubles
ion of Coccinia
lish name).
h
ut (english
desh.
c properties,
sia Lignea,
Sylhet and
ed in colic,
es, diseases

1.3 Vernacular Names of Coccinia cordifolia
Table 1.2: Showing the vernacular names of Coccinia cordifolia (Ajmal and Pandey, 2006;
Kumar et al., 2013)
Bangla Telakucha, Vinbu, Kakjhinga, Kawoaluli, Kanduri, Telakochu shag (Chakma),
Muss si (Murang)
English Ivy-Gourd, Scarlet Gourd
Hindi Kundaru ki bel, Tindora, Kovaikkai
Urdu Kunduru
Sanskrit Tundika
Assam Kawabhaturi
Malayalam Tendli, Ghiloda, Kundri, Kowai, Kovai, Kovakkai
Danish Skariagenagurk
Chinese Hong Qua
Japanese Yasai, karasuuri
Spanish Pepino, cimaron
Others Kovakka, Tindla, Kundri, Bhimb, Gentleman's Toes, Kowai, Tindori, Dondakaya,
Ghiloda, Little Gourd, Thainli, Tendli, Thendli, Manoli, Thondai
1.4 Taxonomy of C. cordifolia
Domain: Eukaryota
Kingdom: Plantae
Subkingdom: Tracheobionta
Superdivision: Spermatophyta
Division: Magnoliophyta
Class: Magnoliopsida
Order: Cucurbitales
Family: Cucurbitaceae
Genus: Coccinia
Species: Coccinia cordifolia
Botanical name: Coccinia cordifolia (Ayurveda Informatics, 2011; Pekamwar et al., 2013;
USDA, 2014)
1.5 Cucurbitaceae Family
Cucurbitaceae, the gourd family of flowering plants, belongs to the order Cucurbitales and
contains 118 genera and 845 species of food and ornamental plants. It includes the gourds,
melons, squashes, and pumpkins. Most species are prostrate or climb by tendrils. They are
annual or perennial herbs native to temperate and tropical areas. No member of the family
tolerates frost or cold soil. Most species are extremely sensitive to temperatures near freezing, a
factor that limits their geographic distribution and area of cultivation. The family includes such
economically important food plants as pumpkin, cucumber, gherkin, watermelon, muskmelon,
summer squash, winter squash, chayote, cassabanana, squash, and gourd. They are distributed in
tropical and subtropical regions (Encyclopaedia Britannica, 2014).
Members of the family are fast-growing, with long-stalked leaves that alternate along the stem.
Most species have unisexual flowers, which are borne in the leaf axils and have five white or
yellow petals. At the side of the leafstalk in annual species there is a simple, often branched,
spirally coiled tendril. It is generally regarded by most botanists to be a modified shoot. There
are five sepals in each flower; male flowers have up to five anthers, often fused or joined in a
complex way, and female flowers usually have three carpels. The fruit in most species is a
fleshy, many-seeded berry with a tough rind, often attaining considerable size. The seeds are
flattened and sometimes have beautiful wings (Encyclopaedia Britannica, 2014).
1.5.1 Species of Cucurbitaceae available in Bangladesh
Cucurbitaceae plants grow well in Bangladesh. They are found in plain areas as well as in hilly
areas like Sylhet and Chittagong. According to the recent reports of Bangladesh National
Harberium, the following Cucurbitaceae plants are available in Bangladesh as shown in table
below.

In vitro pha
Tab
Species:
Local na
Plant pa
Medicin
tapeworm
Species:
Local na
Plant pa
Medicin
against
diuretic
cures pai
cough, as
Species:
Local na
Plant pa
Medicin
renoprote
induced
ulcer and
armacological in
le 1.3: Some
Cucurbita m
ame: Mistiku
arts: Seeds
al or oth
ms
Lagenaria s
ame: Lau, K
arts: Fruits
al or other
doxorubici
activity, an
in, ulcers an
sthma and ot
Benincasa h
ame: Chalku
arts: Fruits, S
al or ot
ective activ
renal dama
d antifungal a
nvestigations of
e plants of C
maxima
umra
her uses:
siceraria
Kadu, Pani La
uses: Cardi
in induced
nti hyperlip
nd fever and
ther bronchi
hispida
umra
Seeds
ther uses
vity on isch
age, manag
activity
f aqueous fracti
Cucurbitacea
Treatment
au.
oprotective
d cardiotox
pidemic act
used for pe
al disorders
: Antidiar
hemia/reperf
gement of p
ion of Coccinia
ae family ava
t for
effect
xicity,
tivity,
ectoral
rrheal,
fusion
peptic
cordifolia leave
ailable in Ba
es
angladesh (UUddin, 2014)
9
).

Species: Cucumis sativus
Local name: Khira, Shasha
Plant parts: Fruits, Seeds
Medicinal or other uses: Removing constipation
and aid indigestion, has demulcent property,
cooling, tonic, diuretic and anthelmintic

Species: Cucumis melo
Local name: Kharmuj
Medicinal or other uses: Cooling, tonic,
laxative, aphrodisiac and diuretic; cures
biliousness, insanity, liver and kidney troubles,
bronchitis, burning of the throat, chronic fever,
painful discharges and suppression of urine

Species: Luffa aegyptiaca
Local name: Dhundul
Plant parts: Fruits
Medicinal or other uses: Expectorant, tonic,
laxative and diuretic; spleen diseases, leprosy,
piles, fever and bronchitis

Species: Momordica charantia
Local name: Korola
Medicinal or other uses: Anthelmintic,
antiemetic, carminative, purgative and for the
treatment of anemia, jaundice, malaria, cholera;
and also has antidiabetic, antioxidant property
Species: Trichosanthes cucumerina
Local name: Chichinga
Plant parts: Roots, Fruits, Seeds
Medicinal or other uses: Cure for bronchitis,
headache and boils, considered as cathartic, used
as an anthelmintic and stomach disorders.

1.6 Coccinia cordifolia
Coccinia cordifolia is one kind of species of Cucurbitaceae commonly known as telakucha, ivy
gourd, scarlet gourd, tindori, tindola, or kovai kai. It is native to north-central East Africa and is
also found wild in the Indo-Malayan region. Coccinia includes 29 additional species and they are
found only in tropical Africa (Singh, 1990).
In vitro pha
Coccinia
Australia
in these p
(Muniapp
of Southe
armacological in
a cordifolia w
a, Pacific Isl
parts of the w
pan et al., 2
east Asia (Sh
nvestigations of
Fig
was introduc
lands, the Ca
world becau
009). It is co
haheen et al.
f aqueous fracti
gure 1.1: Co
ced by huma
aribbean, an
use it is capab
onsidered as
., 2009).
ion of Coccinia
occinia cord
ans mostly as
nd southern U
ble of thrivin
s a valuable
cordifolia leave
difolia plant
s a food crop
United State
ng well in w
wild vegetab
es
p to several
es. It has be
warm, humid
able by the in

countries in
come natura
d, tropical re
ndigenous p
12
n Asia
alized
gions
eople
In vitro pha
Figure
L.S. fem
1.7 Syno
Coccinia
2011; Ku
1.8 Dist
C. cordif
Pakistan,
India in w
found in
2013).
armacological in
e 1.2: Coccin
male flower;
onyms of C
a indica; Ce
umar et al., 2
ribution of
folia is dist
, India and S
wild. It is m
areas like so
nvestigations of
nia cordifoli
E: Male flo
Coccinia co
ephalandra
2013; Pekam
f Coccinia c
tributed in A
Srilanka. It i
more common
outhern Asia
f aqueous fracti
ia, A: Twig w
ower with co
rdifolia
indica; Bryo
mwar et al., 2
cordifolia
Africa, tropi
is found in c
nly seen in a
an islands an
ion of Coccinia
with flower;
rolla split op
onia cordifo
2013).
ical Asia, a
climate that i
areas like Be
nd West Indi

cordifolia leave
; B: Male flo
pen; F: T.S.
olia; Coccin
and is comm
is warm and
engal, Bihar
ies and Haw
es
ower; C: Fem
Ovary (Sam
nia grandis
monly found
d humid. It i
and Orissa.
wain islands (
male flower;
mbamurty, 20
(Shivhare e
d in Bangla
is found in w
In generally
(Pekamwar e
13

D:
005)
et al.,
adesh,
whole
y it is
et al.,
In vitro pha
This plan
Hawaiian
Western
range of
Eastern P
througho
Figure
The urba
Azadirac
(Jaam/Ka
charantia
highest n
al., 2013
1.9 Hab
Coccinia
zones. It
adapted t
armacological in
nt has been
n and Mari
Australia, th
C. cordifoli
Papua, New
out the count
1.3: Region
freq
an and rural
chta indica
alojam tree)
a (Korola tr
number of ci
).
bitant of Co
a cordifolia
grows as a v
to tropical a
nvestigations of
n spread in
iana Islands
he northern t
a also found
w Guinea an
try in fallow
nal distributi
uently ment
l distribution
(Neem tree
, Terminalia
ree) and Swi
itations in bo
occinia cord
is a warm c
vine and can
and subtropi
f aqueous fracti
America an
s of the Pa
territory and
d in Philippin
nd Northern
lands and on
on of C. cor
ioned plants
n of C. cord
e), Trigonell
a chebula (H
ietenia mah
oth urban an
difolia
climate; shor
n be trellised
cal areas. C
ion of Coccinia
nd Pacific, b
acific. Smal
d the northern
nes, China,
n Territories
n fences (Pe

rdifolia (Syn
s in Banglade
difolia is com
la foenum-g
Haritaki tree)
agoni. It ha
nd rural regi
rt-lived pere
d when culti
C. cordifolia
cordifolia leave
but it has b
l population
n coastal par
Indonesia, M
(Australia)
ekamwar et a
nonym: Cocc
esh (Ocvirk
mpared with
graecum (M
), Ficus rac
as been foun
ions compar
ennial plant
ivated in a h
is growing
es
become inva
ns are scatt
rts of Queen
Malaysia, Th
. In Bangla
al., 2013; M
cinia indica)
et al., 2013)
h other com
Methi tree), S
cemosa (Dum
nd that C. c
red to other
that thrives
home vegetab
wild throug
asive only in
tered throug
nsland. The n
hailand, Viet
adesh it is f
BG, 2008).

) and other m
)
mmon plants
Syzygium cu
mur), Momo
cardifolia ha
plants (Ocv
s in rainy, h
ble garden. I
ghout Bangla
14
n the
ghout
native
tnam,
found
most
like,
umini
rdica
as the
irk et
humid
It has
adesh
In vitro pha
and is al
sheltered
trees. Th
up electri
1.10 Mo
C. cordif
1.10.1 S
It has ste
at the no
become s
1.10.2 L
Leaves h
smell. Th
armacological in
so cultivated
d position an
hese plants, w
icity poles, o
orphology o
folia is a dioe
Stem of C. c
ems and coil
odes. Initiall
swollen and
Leaf of C. c
have bright g
hey are trian
nvestigations of
d in various
nd a sandy s
when cultiva
over nearby
of Plant
ecious, peren
Figure 1.4
cordifolia
led tendrils.
ly, younger
semi-succul
ordifolia
green upper s
gular or pen
f aqueous fracti
parts of Ba
soil. It can fo
ated in garde
trees and int
nnial and he
4: C. cordifo
When stems
stems are
lent in nature
surface and p
ntagonal in sh
ion of Coccinia
angladesh. It
form dense m
ens, will qui
to suburban
erbaceous cli
folia leaves, f
s of C. cordi
slender, gre
e (Kumar et
pale-green u
hape (Kuma
cordifolia leave
t is an outdo
mats that rea
ickly spread
bush land (P
imber (Kum
fruits and flo
ifolia touch
een, and sm
al., 2013).
underneath, w
ar et al., 2013
es
oor plant bu
adily cover
d along fence
Pekamwar et
mar et al., 201
owers.
soil, they st
mooth but as
with charact
3).
ut prefers a s
shrubs and
es and roads
t al., 2013).
13).

trike roots re
s they grow
teristic odou
15
sunny
small
sides,
eadily
they
ur and
In vitro pha
1.10.3 F
The flow
borne sin
long) tha
tips. The
white, 3
(present a
armacological in
Flower of C
wers are acti
ngly in the le
at are joined
calyx has fi
4.5cm long
as staminode
nvestigations of
Fi
C. cordifolia
inomorphic
eaf forks on
together at t
five subulate
g, deeply d
es in female
Fi
f aqueous fracti
igure 1.5: L
a
and nearly a
stalks 1-5cm
the base and
, recurved lo
divided into
flowers). Th
igure 1.6: F
ion of Coccinia
Leaves of C.
always unise
m long. The
d usually hav
obes, each 2
five ovate
he ovary is i
lower of C.
cordifolia leave
cordifolia
exual. These
ey have five
ve five sprea
25mm long
lobes. Each
inferior (Kum
cordifolia
es

e white, tub
small narrow
ading petal lo
. The corolla
h flower ha
mar et al., 20

bular, flower
w sepals (6-
obes with po
a is campanu
as three sta
013).
16
rs are
-8mm
ointed
ulate,
amens
In vitro pha
1.10.4 F
The ivy
raw fruit
fruit (2.5
several p
Figure 1
1.10.5 S
Seeds are
grey (Shi
armacological in
Fruit of C. c
gourd fruit b
is green in c
5-6cm long a
pale, flattened
1.7: Fruits of
Seed of C. c
e obovoid an
ivhare et al.,
nvestigations of
cordifolia
belongs to th
color resemb
and up to 3
d seeds (Kum
f C. cordifol
cordifolia
nd rounded
, 2011).
F
f aqueous fracti
he berry typ
bles a small
.5cm wide)
mar et al., 20
lia-(a) unripe
at the apex,
Figure 1.8: S
ion of Coccinia
pe: oval and
dark green c
turn bright
013).
e fruit; (b) ri
, slightly pap
Seeds of C. c
cordifolia leave
hairless wit
cucumber wi
scarlet red
ipe fruit; (c)
pillose, muc

cordifolia
es
th thick and
ith paler strip
as they mat
cross-sectio
ch compress
d sticky skin
pes. These f
ture and con

on of unripe
ed and yello
17
n. The
fleshy
ntains
fruit.
owish
In vitro pha
1.10.6 R
The fresh
attached
shows ci
and thoro
2011).
1.11 Rep
This spec
from Au
self-fertil
create fru
fragment
common
dormancy
quickly,
1.12 Veg
Ivy gour
Coccinia
plant is M
The vari
armacological in
Root of C. c
h root is thic
to it. Roots
ircular outlin
ough perme
production
cies reprodu
gust to Sept
le. The plan
uit. Seeds m
ts can be sp
ly occurs by
y and usual
capable of g
getative pr
rd will grow
a cordifolia n
May-June an
iety is prop
nvestigations of
cordifolia
ck, tuberous,
s are flexible
ne and is ch
eation of par
F
n and dispe
uces by seed
tember. The
nts are dioec
may be dispe
read by floo
y humans w
ly germinate
growing 4 inc
ropagation
w in slightly
needs full su
nd Septemb
pagated thro
f aqueous fracti
, long taperi
e, soft and b
haracteristic
renchyma w
Figure 1.9: R
ersal of Coc
and also ve
species is d
cious, which
ersed by bir
ods or in dis
hen we deli
e within 24
ches per day
of Coccini
y sandy loa
un and consi
ber-October.
ugh vegetat
ion of Coccinia
ing, more or
break with a
of storage ty
with vascular
Roots of C. c
ccinia cord
egetatively v
dioecious an
h means the
rds and othe
scarded gard
iberately cul
4 weeks at 2
y (Bird, 1990
ia cordifolia
am soils wit
stent moistu
The plants
tion. Plantin
cordifolia leave
r less tortuou
a fibrous fra
ype. Parench
r elements i
cordifolia
difolia
via stem frag
nd pollinated
ey need two
er animals t
den waste. L
ltivate these
20C. Ivy g
0).
a
th good dra
ure. So, the s
should be i
ng the three
es
us with a few
acture. A tra
hyma is full
is observed
gments. C. c
d by insects.
o vines of d
that eat the
Long distanc
plants. Seed
gourd takes h
ainage. For
suitable plan
irrigated dur
e-noded cutt
w fibrous ro
ansaction of
l of starch g
(Shivhare e
cordifolia flo
. The plant i
different sex
fruit, while
ce dispersal
ds do not ex
hold and sp
the best har
nting time fo
ring hot wea
tings is the
18
otlets
f root
grains
et al.,
owers
is not
xes to
stem
most
xhibit
reads
rvest,
or this
ather.
e best
method. The selected cuttings should be planted in pits of 60cm diameter dug at a spacing of 2m
by 2m. Two or three cuttings may be planted in each pit. The most important thing is that care
should be taken to keep the root zone sufficiently moist. It is observed that manure along with
70g nitrogen and 25g each of phosphorus and potash per pit would provide the best result. The
plants should be allowed to climb over pandal or trellises (Gautam et al., 2014).
1.13 Nutritional value of Coccinia cordifolia
The nutritional value per 100gm of edible portion of Coccinia cordifolia is shown in table below.
Table 1.4: Nutritional value of per 100gm of edible portion of C. cordifolia (Gautam et al.,
2014)
Components Amount
Energy 21 (KCal)
Protein 1.4 (g)
Carbohydrate 3.4 (g)
Fat 0.2 (g)
Calcium 25 (mg)
Iron 0.9 (mg)
1.14 Local uses of C. cordifolia in Bangladesh
Table 1.5: Use of C. cordifolia in different parts of Bangladesh (Rahmatullah, 2010)
Local
Name
Tribe Village District
Part(s)
Used
Ailment
Nichu-
bang
Marma The fruit is given for respiratory
problems and lung disorders.
Telakucha Khasia Juice and paste of the leaves to be
taken with honey for diabetes.
Telakucha Dashuria
village
Pabna Whole
plant
Diabetes, carminative, hypertension,
fever.
Telakucha Boro bon
gram
Rajshahi Leaf,
stem
For diarrhea, juice of young leaves is
(1/32kg) is taken with a little sugar
Local
Name
Part(s)
Used
Ailment
village every morning till cure.
For blood dysentery, 1/32kg of
leaves is taken and juice squeezed
out. The juice is heated in an iron pot
and mixed with 1/64kg of sugar. To
be taken every morning on an empty
stomach.
For dizziness from sunstroke, juice
from leaves mixed with a little water
is applied to forehead and top of
head.
Telakucha Rajshahi Leaf The fried leaves are taken for
diabetes. Paste of leaves is applied to
hands or head if they feel hot.
Telakucha Muslim Rajshahi Root In case of persistent bleeding
following menstruation, the roots are
cut into small pieces and taken with
molasses sherbet.
Telakucha Kodra
village
Jessore Leaf For dysentery, leaf juice is taken
with a little salt for a week.
Telakucha Bajua
danga
village
Jessore Root For leucorrhea and menstruation
problems, pills are made from a
combination of root of Coccinia
cordifolia, root bark of Abroma
augusta, root of Achyranthes aspera,
whole plant of Ipomoea paniculata
along with honey and opium. Three
tablets are to be taken in the morning
on an empty stomach.
Local
Name
Part(s)
Used
Ailment
Telakucha Natore Leaf For cough relief and anti-pyretic
effect, 1 glass of leaf juice is taken
with sugar thrice daily.
Telakucha Narshingdi Juice from the leaves is used in
diabetes.
Telakachu Garo Sherpur For diabetes, mix enough water with
leaf and stem to make 125ml of
juice. Take two times daily (morning
before meal, and night after meal).
Kawla
kochu
pata
Noakhali Leaves The leaves are washed and crushed
to make a semi-solid paste. Pills
made from the paste are dried and
given to patients for controlling
diabetes mellitus.
Note: the housewives claim that this
preparation can cure diabetes
mellitus.
1.15 Toxicology of C. cordifolia
No toxic effect was seen on human body with C. cordifolia consumption. A study was planned to
assess the acute toxicity of Coccinia indica (Synonym: Coccinia cordifolia, Coccinia grandis)
leaves. Rats were orally administrated single dose of 100, 500 and 1000mg/kg of ethanolic
extract of Coccinia indica. Mortality, signs of toxicity, body weight, food consumption and gross
findings were observed for 14 days post treatment of Coccinia indica extract. In addition, no
significant differences were noticed in the body and organ weights between the control and
treated groups. These results state that ethanol extract of Coccinia indica is toxicologically safe
by oral administration (Baghe et al., 2011).


Chapter Two
LITERATURE REVIEW

2.1 Phytochemical constituents
Phytochemical screening and antimicrobial activity of Coccinia cordifolia L. plant
The medicinal plant, Coccinia cordifolia L. was analyzed in a research for its chemical
composition. Chemical analysis showed that the plant is rich in nutrients, especially antioxidant
compounds such as total phenol, vitamin C and -carotene. Phytochemical screening showed that
the methanolic extract contains the bioactive constituents such as tannins, saponins, phenols,
flavonoids and terpenoids (Khatun et al., 2012).
Studies on phytochemical constituents, quantification of total phenol, alkaloid content and
In-vitro anti-oxidant activity of Coccinia cordifolia
In a study extracted dried plant of Coccinia cordifolia in hexane, ethyl acetate, ethanol (70% v/v)
and methanol was collected and these extracts were checked for their phytochemical
constituents. The whole plant of Coccinia cordifolia revealed the presence of steroids,
triterpenoids, glycosides, saponins, tannins, alkaloids, saponins, phenols and carbohydrates but
extract of Coccinia cordifolia give the negative results for quinines and oils. The methanolic
extract has more phenolic content than other extracts and the ethanol (70% v/v) extract has more
alkaloid content than other extracts (Ganga et al., 2011).
2.2 Pharmacological properties
2.2.1 Anti-diabetic and hypoglycemic activity
2.2.1.1 In vivo study on human model
Blood Sugar Lowering Effect of Coccinia grandis (L.) J. Voigt: Path for a New Drug for
Diabetes Mellitus
Double-blind phase I clinical trial was conducted at the general hospital and a private hospital in
Matara in August 2009. Sixty-one healthy volunteers were given a meal for dinner containing
20g of leaves of Coccinia grandis which was mixed with a measured amount of scraped coconut
and table salt for breakfast, and other 61 were given the placebo meal for dinner which also
contained scraped coconut and salt. They maintained a 10-hour fasting period. Glucose tolerance
test was performed blindly for the two groups. Results showed that overall blood sugar levels of
the experimental group were also significantly lower than those of the control group (F(1,117)
5.56, P < 0.05). Increase in the blood sugar levels from fasting to one hour (F(1,117) 6.77, P <
0.05) and two hours (F(1,117) 5.28, P < 0.05) postprandially was statistically significant for
participants who were in the control group than those of in the experimental group. The mean
difference of postprandial blood sugar levels (mg/dL) after one hour (20.2, 95% confidence
interval, 4.81 to 35.5) and two hours (11.46, 95% confidence interval; 1.03 to 21.9) was
statistically significant between the two groups. This proved that Coccinia grandis has a blood
sugar lowering effect (Munasinghe et al., 2011).
Effect of Supplementation of Coccinia cordifolia Extract on Newly Detected Diabetic
Patients
This study aimed to evaluate the effectiveness of Coccinia cordifolia on blood glucose levels of
incident type 2 diabetic patients requiring only dietary or lifestyle modifications. A double blind,
placebo control, randomized study trial was carried out. Sixty incident type 2 diabetics (aged 35
60 years) were recruited. The subjects were randomly assigned into the placebo or experimental
group and were provided with 1g of an alcoholic extract of the herb for 90 days. Anthropometric,
biochemical, dietary and physical activity assessment were carried out at baseline and were
repeated at day 45 and day 90 of the study. All the subjects were provided with standard dietary
and physical activity advice for the control of their blood sugars. Results showed a significant
decrease in the fasting, post prandial blood glucose and glycosylated hemoglobin of the
experimental group when compared to the placebo group. The fasting and post prandial blood
glucose levels of the experimental group at day 90 significantly decreased by 16% and 18%
respectively. This study suggests that Coccinia cordifolia extract has a potential hypoglycemic
action in patients with mild diabetes (Kuriyan et al., 2008).
Coccinia indica in the treatment of patients with diabetes mellitus
A double blind control trial was conducted with preparation from the leaves of the plant,
Coccinia indica, on uncontrolled, maturity onset diabetics. The trial lasted for six weeks for an
individual patient. Out of the 16 patients who received the experimental preparations 10 showed
marked improvements in their glucose tolerance while none out of the 16 patients in the dummy
group showed such a marked improvement. This difference is highly significant (kappa 2 with
Yates' correction = 11.7, P < 0.001) (Khan et al., 1979).

2.2.1.2 In vivo study on animal model
Comparative efficacy of Telakucha (Coccinia indica) leaves and Amaryl(R) Tablet
(Glimepiride) in induced diabetes mellitus in rat
Thirty healthy rats of both sexes weighing between 150 to 200gm were selected from among the
offspring and randomly divided into six equal groups. Rats of Group A and Group B were kept
as non-hypoglycemic control and hypoglycemic control, respectively. After acclimatization
hyperglycemia was induced in five groups of rats (B, C, D, E and F) by administering
streptozotocin (STZ) intraperitoneally at a dose of 55mg/kg body weight. After fifteen days of
STZ injection, four groups of rats (C, D, E and F) were administered with Telakucha (Coccinia
indica) and Amaryl
(R)
(Glimepiride) as per schedule dose and all the control and treated rats were
closely observed during 14 days of treatment. The oral administration over 14 days of Telakucha
(Coccinia indica) leaves extract significantly lowered blood glucose level but was not as potent
as patent drug Amaryl
(R)
(Glimepiride). The herbal preparation also increased body weight but
not to the extent caused by the patent drug Amaryl
(R)
. The Telakucha leaves at 750mg/kg body
weight significantly reduced (30.73%) the blood glucose level from 31.240.36mmol/L to
21.640.17mmol/L and significantly increased (5.45%) the body weight from 181.9621.10g to
191.8712.42g (Amanullah et al., 2008).
Effect of Ethanol Extract of Coccinia grandis Lin leaf on Glucose and Cholesterol Lowering
Activity
Glucose and cholesterol lowering effect of the ethanol extract of C. grandis leaf was evaluated
using the alloxan-induced diabetic rat and compared the activity with diabetic control and
antidiabetic drug (Glibenclamide). Ethanol extract (25mg/kg) of C. grandis and Glibenclamide
were administered to normal and experimental diabetic rats for the duration of 10 days. In the
alloxan-induced diabetic rat model, C. grandis (25mg/kg) significantly (p<0.05) lowered fasting
blood glucose levels. Body weight was increased significantly (p<0.05) in the C. grandis treated
diabetic group. These results suggest that the ethanol extract of C. grandis leaf possesses
significant glucose lowering activity in animal model (Al-Amin et al., 2013).

Protective effect of Coccinia indica on changes in the fatty acid composition in
streptozotocin induced diabetic rats
The present study was undertaken to investigate the effect of Coccinia indica on blood glucose,
plasma insulin, cholesterol, triglycerides, free fatty acids and phospholipids and fatty acid
composition of total lipids in liver, kidney and brain of normal and streptozotocin (STZ) diabetic
rats. Oral administration of the ethanolic extract of Coccinia indica leaves (200mg/kg body
weight, CLEt) for 45 days to diabetic rats decreased the concentrations of blood glucose whereas
plasma insulin was elevated. These results suggest that CLEt exhibits hypoglycaemic effect in
STZ induced diabetic rats. The effect of CLEt at 200mg/kg body weight was better than that of
glibenclamide. The results indicate that the administration of CLEt to diabetic animals
normalizes blood glucose and causes marked improvement of altered carbohydrate metabolic
enzymes during diabetes (Pari and Venkateswaran, 2003).
2.2.2 Hypolipidemic and antidyslipidemic activity
Protective effect of Coccinia indica on changes in the fatty acid composition in
streptozotocin induced diabetic rats
The present study was undertaken to investigate the effect of Coccinia indica on cholesterol,
triglycerides, free fatty acids and phospholipids and fatty acid composition of total lipids in liver,
kidney and brain of normal and streptozotocin (STZ) diabetic rats. Oral administration of the
ethanolic extract of Coccinia indica leaves (200 mg/kg body weight, CLEt) for 45 days to
diabetic rats decreased the concentrations of lipids and fatty acids, viz., palmitic, stearic, and
oleic acid whereas linolenic and arachidonic acid were elevated. These results suggest that CLEt
exhibits hypolipidaemic effects in STZ induced diabetic rats. It also prevents the fatty acid
changes produced during diabetes. The effect of CLEt at 200 mg/kg body weight was better than
that of glibenclamide (Pari and Venkateswaran, 2003).
Antidyslipidemic activity of polyprenol from Coccinia grandis in high-fat diet-fed hamster
model
Ethanolic extract was fractionated into chloroform, n-butanol and water-soluble fractions and
were evaluated. Activity was proved to be concentrated in chloroform-soluble fraction.
Chloroform-soluble fraction containing active component was subjected to repeated column
chromatography, furnished a polyprenol characterized as C(60)-polyprenol(1) isolated for the
first time from this plant. It significantly decreased serum TG by 42%, total cholesterol (TC)
25% and glycerol (Gly) 12%, accompanied HDL-C/TC ratio 26% in high-fat diet (HFD)-fed
dyslipidemic hamsters at the dose of 50mg/kg body weight. Results are comparable to standard
drug, fenofibrate at the dose of 108mg/kg. So the compound polyprenol(1) isolated from leaves
of C. grandis possess marked antidyslipidemic activity (Singh et al., 2007).
Ameliorative potential of Coccinia grandis extract on serum and liver marker enzymes and
lipid profile in streptozotocin induced diabetic rats
The present study was undertaken to evaluate the potential of Coccinia grandis extract on serum
and liver marker enzymes (ALP, AST, ALT and LDH) and lipid profile (total cholesterol,
phospholipids, triglycerides and free fatty acids in serum and liver) in streptozotocin induced
diabetic animals. The experimental animals were treated with methanolic extract of Coccinia
grandis and the levels of marker enzymes and lipid profile were estimated. The ALP, AST, ALT
and LDH levels were increased in diabetic rats and restored to near normal levels after
administration of plant extract. The lipid profile increased in diabetic group and after the
treatment with the plant extract the levels were reverted to near normal. Thus the methanolic
extract of Coccinia grandis has a potent ability to restore the marker enzymes and the lipid
profile was reverted to near normal levels (Krishnakumari et al., 2011).
Effect of Ethanol Extract of Coccinia grandis Lin leaf on Glucose and Cholesterol Lowering
Activity
Glucose and cholesterol lowering effect of the ethanol extract of C. grandis leaf was evaluated
using the alloxan-induced diabetic rat and compared the activity with diabetic control and
antidiabetic drug (Glibenclamide). Ethanol extract (25mg/kg) of C. grandis and Glibenclamide
were administered to normal and experimental diabetic rats for the duration of 10 days. C.
grandis extract (25mg/kg) produced significant (p<0.05) total cholesterol lowering and HDL
increasing (p<0.05) effects. Surprisingly, body weight was increased significantly (p<0.05) in the
C. grandis treated diabetic group. These results suggest that the ethanol extract of C. grandis leaf
possesses significant cholesterol lowering activity in animal model (Al-Amin et al., 2013).

2.2.3 Anti-inflammatory activity
Anti-inflammatory, analgesic and antipyretic activity of aqueous extract of fresh leaves of
Coccinia indica
This study was aimed to evaluate both post- and pre-treatment anti-inflammatory activities of the
aqueous extract of fresh leaves of Coccinia indica in rats using the carrageenan-induced paw
oedema method at various dose levels. Ceiling effect of the extract was observed at 50mg/kg in
pre-treatment carrageenan test. In post-treatment studies, a dose-dependent anti-inflammatory
effect was observed in the dose range of 25-300mg/kg. The effect was equivalent to diclofenac
(20mg/kg) at 50mg/kg but it was significantly pronounced at higher doses. Effectiveness of
extract in the early phase of inflammation suggests the inhibition of histamine and serotonin
release. In conclusion, this study has established the anti-inflammatory activity of C. indica and,
thus, justifies the ethnic uses of the plant (Niazi et al., 2009).
Evaluation of Anti-inflammatory activity of Coccinia indica leaves extracts
The effects of Coccinia indica leaves extracts on different phases of acute inflammation were
examined. Investigations were performed using different phlogistic agents-induced paw edema
viz.Carrageenan-induced paw oedema and Dextran- induced paw oedema in rats. Various
extracts (ethanol and aqueous) of Coccinia indica leaves extracts at a dose of 250mg/kg and
500mg/kg orally were tested. Diclofenac sodium at the dose of 10mg/kg was used as standard.
Both the extracts showed significant activity (*p<0.0 & **p<0.01) compared with the control in
both of these models (Sutar et al., 2010).
A study on Anti-inflammatory activity of the leaf and stemextracts of Coccinia grandis L.
Voigt
The Aqueous extracts of Coccinia grandis L. Voigt leaves and stem were investigated in
chemically-induced inflammation rodents model. The extracts inhibited formaldehyde-induced
paw edema in rats. These inhibitions were statistically significant (p<0.05, 0.01, 0.001) as
compared to control. Aqueous extract of leaves showed highest activity (Deshpande et al., 2011).

2.2.4 Analgesic and antipyretic activity
Anti-inflammatory, analgesic and antipyretic activity of aqueous extract of fresh leaves of
Coccinia indica
Analgesic and antipyretic properties were evaluated using tail flick model and yeast-induced
hyperpyrexia, respectively. The extract produced marked analgesic activity comparable to
morphine at 300mg/kg, which suggests the involvement of central mechanisms. A significant
reduction in hyperpyrexia in rats was also produced by all doses of extract with maximum effect
at 300mg/kg comparable to paracetamol. In conclusion, this study has established the analgesic
and antipyretic activity of C. indica (Niazi et al., 2009).
2.2.5 Anti-bacterial and anti-fungal activity
Antimicrobial activity of the fruit extracts of Coccinia indica
The bioactive compounds of fruits of Coccinia indica were investigated for antibacterial activity
against some pathogenic bacteria. The aqueous extracts did not show much significant activity,
while the organic extracts (petroleum ether and methanol) showed the highest activity against the
test bacteria. The activity was more pronounced on gram-positive organisms with
Staphylococcus aureus being more susceptible and Salmonella paratyphi A being more resistant
(Shaheen et al., 2009).
Antibacterial Activity of the Leaves of Coccinia indica (W. and A) Wof India
The aim of the present research was focused on investigating the antibacterial properties of
Coccinia indica (W.A.) via in vitro approach. The aqueous and organic solvent (Petroleum ether,
chloroform and ethanol) extracts from the leaves of Coccinia indica (Cucurbitaceae) were tested
against Enterobacter aerogenes, Pseudomonas aeruginosa, Staphylococcus epidermidis, Bacillus
subtilis and Salmonella typhimurium by agar well diffusion method and broth dilution method.
Results showed promising antibacterial activity against the bacteria tested. Among these, ethanol
and aqueous extracts were found to have a more potent inhibitory effect comparing with the
other extracts. This proves that there is potentiality in the plant extracts for the treatment of
various skin and gastrointestinal infections in humans (Hussain et al., 2010).

Antibacterial, cytotoxic and antioxidant activity of chloroform, n-hexane and ethyl acetate
extract of plant Coccinia cordifolia
Disc diffusion technique was used for in vitro antibacterial screening against gram positive and
gram negative human pathogenic bacteria. Here kanamycin disc (30g/disc) was used as
standard. The chloroform extract of Coccinia cordifolia showed good antibacterial activity with
the average zone of inhibition 9-12mm. The n-hexane and ethyl acetate extract showed average
zone of inhibition 7-10mm and 7-11mm respectively (Bulbul et al., 2011).
Antibacterial activity of Coccinia grandis leaf extract on selective bacterial strains
To assess the antibacterial activities of Coccinia grandis leaf extract on selective bacterial strains
under in-vitro conditions. The antibacterial activity was tested against five bacterial strains by
agar well diffusion method. The crude extract showed a broad spectrum of antibacterial activity
by inhibiting both the gram positive and gram negative groups. The antibacterial activity of C.
grandis leaf extract using solvents such as acetone, ethanol, methanol, aqueous and hexane was
evaluated against five bacterial sp. Ethanol leaf extract of C. grandis showed high antibacterial
activity against S. aureus, B. cereus, E coli, K pneumoniae and S. pyogens. Minimal inhibitory
concentration of the leaf extract against each test organism was also studied by observing their
growth on Mueller Hinton Agar containing the extract at various incremental levels, equivalent
to 31.25g/ml to 1000g/ml of the extract. The highest activity was observed in ethanol extracts
against S. aureus, E. coli, and K. pneumoniae with an inhibitory concentration below 31.5g/ml.
The significance of the study was conducted to investigate the in vitro antibacterial activity of
folklore medicinal plant and to evaluate scientific base of their applications (Sivaraj et al., 2011).
Antimicrobial activity of protease inhibitor isolated from leaves of Coccinia grandis (L.)
Voigt.
A 14.3kDa protease inhibitor PI exhibited marked growth inhibitory effects on colon cell lines in
a dose-dependent manner. PI was thermostable and showed antimicrobial activity without
hemolytic activity. PI strongly inhibited pathogenic microbial strains, including Staphylococcus
aureus, Klebsiella pneumoniae, Proteus vulgaris, Eschershia coli, Bacillus subtilis and
pathogenic fungus Candida albicans, Mucor indicus, Penicillium notatum, Aspergillus flavus
and Cryptococcus neoformans. Examination by bright field microscopy showed inhibition of
mycelial growth and sporulation. Morphologically, PI treated fungus showed a significant
shrinkage of hyphal tips. Reduced PI completely lost its activity indicating that disulfide bridge
is essential for its protease inhibitory and antifungal activity. Results reported in this study
suggested that PI may be an excellent candidate for development of novel oral or other anti-
infective agents (Satheesh and Murugan, 2011).
Phytochemical screening and antimicrobial activity of Coccinia cordifolia L. plant
The antimicrobial activities of the methanol, water, ethanol and ethyl acetate extracts of Coccinia
cordifolia L. plant were evaluated against some Gram positive bacteria (Sarcina lutea, Bacillus
subtilis and Staphylococcus aureus), Gram negative bacteria (Salmonella typhi, Shigella
dysenteriae and Escherichia coli) and fungi (Candida albicans, Aspergillus niger and
Penicillium notatum). In the methanolic extract of the plant, promising antimicrobial potential
was observed against the tested microorganism. Methanolic extract showed highest activity
against Shigella dysenteriae, Escherichia coli, Staphylococcus aureus, and Candida albicans
compared to the other extracts. Water extract showed less antimicrobial activity as compared to
other extractants (Khatun et al., 2012).
2.2.6 Anthelmintic Activity
Evaluation of Anthelmintic Activity of Coccinia indica (fruits)
The present study was designed to explore the anthelmintic activity of different extracts of plant
Coccinia indica (fruits) using petroleum ether, ethyl acetate methanol and water as solvents.
Various concentrations (25 and 50mg/ml) of all the extracts were tested, which involved
determination of time of paralysis and time of death of the worms. It was compared with
Albendazole as standard reference and normal saline as control. The study indicated the potential
usefulness of Coccinia indica against earthworm infections (Shivhare et al., 2011).
2.2.7 Cytotoxic, antitumer and pesticidal activity
The main aim of this study was to find out the antibacterial, cytotoxic and antioxidant activity of
chloroform, n-hexane and ethyl acetate extracts of Coccinia cordifolia (Family: Cucurbitaceae).
The Brine shrimp lethality bioassay method was used to determine the cytotoxic activity and
vincristine sulphate was used as positive control. The LC
50
values of standard vincristine
sulphate, chloroform, n-hexane and ethyl acetate extract were 7.55g/ml, 23.96g/ml,
14.12g/ml & 15.49g/ml respectively which indicate the plants extracts compounds are
promisingly cytotoxic and they might have antitumer and pesticidal activity. Among the extracts
the chloroform extract showed the highest cytotoxic activity with LC
50
23.96g/ml (Bulbul et al.,
2011).
2.2.8 Antioxidant activity
Effect of Coccinia indica leaf extract on plasma antioxidants in streptozotocin- induced
experimental diabetes in rats
The present study was carried out to investigate the antioxidant effect of an ethanolic extract of
Coccinia indica leaves. Oral administration of Coccinia indica leaf extract (CLEt) (200mg/kg
body weight) for 45days resulted in a significant reduction in plasma thiobarbituric acid reactive
substances, hydroperoxides, vitamin E and ceruloplasmin. The extract also caused a significant
increase in plasma vitamin C and reduced glutathione, which clearly shows the antioxidant
property of CLEt (Venkateswaran and Pari, 2003).
Antioxidant activity test of the crude extracts were assessed by means of DPPH free radical
scavenging method where ascorbic acid was used as standard with IC
50
43.22 g/ml. The ethyl
acetate fraction of Coccinia cordifolia showed strongest antioxidant activity with IC
50
value of
50.98g/ml (Bulbul et al., 2011).
Studies on phyto chemical constituents, quantification of total phenol, alkaloid content and
In-vitro anti-oxidant activity of Coccinia cordifolia
In the present study dried plant of Coccinia cordifolia was extracted in hexane, ethyl acetate,
ethanol (70%v/v) and methanol. The selected plant extracts were produced concentration
dependent percentage inhibition of superoxide radical and produced maximum activity at a
concentration of 160g and there after the percentage inhibition were raised gradually to its
maximum level with higher concentrations. Among the four types of C. cordifolia extracts, the
methanolic extract showed better activity than remaining extracts at 160g concentrations
(Ganga et al., 2011).
2.2.9 Chemoprotective activity
Chemoprotective potential of Coccinia indica against cyclophosphamide-induced toxicity
The present study was aimed to investigate the chemoprotective potential of Coccinia indica
against CP-induced oxidative stress, genotoxicity, and hepatotoxicity. Rodents were orally pre-
treated with Coccinia indica extract (200, 400, and 600mg/kg) for five consecutive days. On 5th
day, these animals were injected with CP (5mg/kg i.p) and sacrificed after 24hrs for the
evaluation of oxidative stress, hepatotoxicity, micronucleus formation, and chromosomal
aberrations. It was found that the CP significantly increased malondialdehyde (MDA) and
decreased catalase and glutathione (GSH) levels in brain, and it was significantly reversed by
Coccinia indica extract (400 and 600mg/kg). Further, pre-treatment with Coccinia indica extract
(200, 400, 600mg/kg) significantly and dose-dependently reduced micronuclei formation and
incidence of aberrant cells. It was also found that the CP-induced increase in the serum
biomarker enzymes like alkaline phosphatase (ALP), alkaline aminotransferase (ALT), and
aspartate aminotransferase (AST) were significantly reduced by Coccinia indica extract. Thus,
the results indicate the protective effect of Coccinia indica extract against CP-induced oxidative
stress, genotoxicity, as well as hepatotoxicity (Nitharwal et al., 2013).
2.2.10 Antiulcer and cytoprotective activity
Antiulcerogenic and antioxidant effects of Coccinia grandis (Linn.) Voigt leaves on aspirin-
induced gastric ulcer in rats
The effect of Coccinia grandis (Linn.)Voigt leaves powder, its methanol and aqueous extracts
were investigated on aspirin-induced gastric ulcer model in rats. The leaf powder showed a
significant dose related decrease in ulcer index, with significant increase in mucus secretion and
decrease in level of Lipid peroxidation (LPO) and Superoxide dismutase (SOD) activity.
Methanol extract at an equivalent dose to that of the powder also showed a significant decrease
in ulcer index with significant changes in mucus secretion, LPO and SOD. However, aqueous
extract was found to be non-significant in reducing ulcer index. The group, receiving standard
drug Famotidine, showed no effect on the mucus secretion induced in this experimental model.
These observations confirm the antiulcerogenic potential of this plant, probably due to increased
mucus secretion and antioxidant property (Mazumder et al., 2007).
Anti-ulcer activity of ethanolic, aqueous and total aqueous extracts of Coccinia grandis
Linn. Voigt in pyloric ligature induced ulcers in albino rats
The present study was designed to investigate the antiulcer potential of ethanolic, aqueous and
total aqueous extracts of Coccinia grandis Linn. Ulcer was induced by pylorus ligature in Wistar
albino rats. Drugs were administered in two different dose levels (200mg/Kgbwt,
400mg/Kgbwt). Though all three extracts of Coccinia grandis, dose dependently reduced, the
total acidity, ulcer index, and increased pH of gastric juice, ethanol extracts exhibited markedly
significant results. However, ethanol extract has shown (78.57%) a highly significant ulcer
curative potential and decreased ulcer formation also. A preliminary phytochemical analysis
revealed the presence of different phytoconstituents such as alkaloids, carbohydrate, glycosides,
phyto sterol, saponins, volatile oil, tannins etc. which may impart their anti-ulcer activity by
acting as anti-secretory and cytoprotective agents. The present result suggests that both anti
secretory and cytoprotective mechanisms of different extracts of Coccinia grandis exerted
protective effect (Santharam et al., 2013).
2.2.11 Anti-hepatotoxic activity
Antihepatotoxic activity of Coccinia indica
Aqueous, light petroleum, chloroform, alcohol, benzene and acetone extracts of the leaves of
Coccinia indica (Family: Cucurbitaceae) were screened for antihepatotoxic activity.The extracts
were given after the liver was damaged with CCl
4
. Liver function was assessed based on liver to
body weight ratio, pentobarbitone sleep time, serum levels of transaminase (SGPT, SGOT),
alkaline phosphatase (ALP) and bilirubin. Alcohol and light petroleum extracts were found to
have good anti-hepatotoxic activity (Gopalakrishnan et al., 2001).
Protective effect of Coccinia indica leaf extract against alcohol combined with carbon
tetrachloride and paracetamol induced liver damagein rats
The aim of our study was to investigate the effect of leaf extract of Coccinia indica against
Alcohol combines with CCl
4
and Paracetamol induced hepatotoxicity. The effects of oral
treatment with Coccinia indica (CI) leaf extracts (100mg/kg and 200mg/kg for 7 days) were
studied on hepatic damage induced by alcohol (40% alcohol 2.0ml/100g, p.o. for 21 days) and
CCl
4
(0.1ml/kg, s.c. on 20th day) and also with paracetamol (750mg/kg ip.) in rats. Biochemical
parameters in serum like glutamate oxaloacetate transaminase (SGOT), total billirubin (TB),
alkaline phosphatase (ALP) and total proteins (TP) were estimated to assess the liver function.
Alcohol CCl
4
and paracetamol treatment produced an increase in SGOT, ALP, Total billirubin
and decrease in total proteins indicating the liver damage. These effects were progressively
reduced (SGOT, ALP and Total billirubin) and increased (Total proteins) by treatment doses of
(100mg/kg and 200mg/kg) CI leaf extracts. These biochemical observations were supplemented
by histopathological examination of liver sections. CI leaf extract protected the liver from
alcohol-CCl
4
and paracetamol induced hepatic damage (Maheswari et al., 2011).
Evaluation of hepatoprotective activity of ethanol extract of Coccinia grandis (L.)
Voigt.leaves on experimental rats by acute and chronic models
The aim of this work was to study the hepatoprotective effect of crude ethanolic extract from the
leaves of C. grandis against liver damage induced by Paracetamol and CCl
4
in rats.
Administration of paracetamol (750mg/kg/day) and CCl
4
(3ml/kg/day) showed a marked
increase in SGOT, SGPT, ALP, bilirubin (total, direct), total proteins, globulin, cholesterol and
decrease in albumin in comparison with the normal control group. The effect of ethanol extract
of C. grandis at 150mg/kg and 300mg/kg doses reduced the serum activities caused by
paracetamol and CCl
4
, which were observed to be statistically significant when compared with
that of the control group. Silymarin provided a better inhibition or exhibition of the biochemical
parameters induced by paracetamol and carbon tetrachloride in rats. The activity may be due to
the presence of either alkaloids or triterpenoids or reducing sugars or their combinations, as
obtained from the preliminary phytochemical screening of the leaves of the plant. The extract
showed no signs of acute toxicity up to a dose level of 3.2gkg
1
in rats by oral route. Thus, it
could be concluded that ethanolic extract of C. grandis leaves possessed significant
hepatoprotective activity (Kundu et al., 2012).
Hepatoprotective Activity of Aqueous Fruit Extract of Coccinia indica against Paracetamol
Induced Hepatotoxicity in rats
To evaluate the hepatoprotective activity of the aqueous fruit extract of Coccinia indica against
paracetamol induced hepatotoxicity in albino rats. Hepatotoxicity was induced in albino rats by
p.o of paracetamol (2gm/kg for 3 days). The aqueous fruit extract of Coccinia indica was
administered to the experimental animals at two selected doses for 14 days. The hepatoprotective
activity of the extract was evaluated by the liver function marker enzymes in the serum
(asparitate transaminases AST, alanine transaminase ALT, alkaline phosphatase ALK.P, total
bilirubin TB, and histopathological studies of liver. Both the treatment groups showed
hepatoprotective effect against paracetamol induced hepatotoxicity by significantly restoring the
levels of serum enzymes to normal which was comparable to that of silymarin group. The oral
administration of Coccinia indica significantly ameliorates paracetamol hepatotoxicity in rats
(Sanapala and Kumar, 2013).
2.2.12 Fertility inducing activity
Fertility inducing effect of aerial parts of Coccinia cordifolia L. in female rats
The present study was undertaken to evaluate the fertility inducing effect of aerial parts of
Coccinia cordifolia L. in female rats. The effect on serum estrogen level, serum progesterone
level and reproductive tract was also evaluated in normal healthy female rats. The extract was
administered orally at two different doses of 500mg/kg and 1000mg/kg. In hyperprolactinemia
induced infertility model, the numbers of uterine implants were almost 10 times more in the
extract treated groups as compared to control. The high dose of extract also produced a
significant increase in serum estrogen levels (p<0.01) and number of corpus luteum (p<0.05) in
healthy female rats. The extract at both doses was effective in reducing the weight of endometrial
implants by 70-80%, but failed to induce fertility in rats with endometriosis. The extract was
ineffective in inducing fertility in androgen-induced infertility model. Thus the aqueous extract
of Coccinia cordifolia L. induces fertility in hyperprolactinemia induced infertility model in
female rats (Jha et al., 2010).
2.2.13 Larvicidal, repellent and egg hatching inhibition property
Effect of leaf essential oil of Coccinia indica on egg hatchability and different larval instars
of malarial mosquito Anopheles stephensi
The larvicidal potential of C. indica leaf essential oil was evaluated against 1st, 2nd, 3rd and 4th
instars larvae of An. stephensi using WHO protocol. The 24hr LC
50
and LC
90
values of the
essential oil were determined following probit analysis. The egg hatching inhibition activity was
also tested at 10, 20, 40, and 60mg/L. The IC
50
value of essential oil was determined against eggs
of An. stephensi. The results showed that essential oil extracted from C. indica possessed
excellent larvicidal and egg hatching inhibition activity against An. stephensi. The bioassays
showed LC
50
-LC
90
of 54.3-140.3, 65.5-155.6, 86.8-180.7 and 95.3-192.6 for 1st, 2nd, 3rd, and
4th larval instars, respectively. The 50% egg hatching inhibition concentration (IC
50
) was noted
at 16.5mg/L. The present finding suggests that the C. indica leaf essential oil provided an
excellent potential for controlling An. stephensi mosquito at earlier stage of their life cycle
(Rajkumar et al., 2011).
Ovicidal and repellent properties of Coccinia indica Wight and Arn. (Family:
Cucurbitaceae) against three important vector mosquitoes
The ovicidal activity was determined against three mosquito species to various concentrations
ranging from 50-300 ppm under the laboratory conditions. The hatch rates were assessed 48hrs
post treatment. The repellent efficacy was determined against three mosquito species at three
concentrations viz., 1.0, 2.5 and 5.0 mg/cm
2
under the laboratory conditions. Among five solvent
extracts tested, the methanol extract have most promising ovicidal activity. The methanol extract
exerted zero hatchability (100% mortality) at 150 ppm for Cx. quinquefasciatus, at 200 ppm for
Ae. aegypti and An. stephensi. The methanol extract of C. indica found to be more repellency
than the other extracts. A higher concentration of 5.0mg/cm
2
provided 100% protection up to
270min against Cx. quinquefasciatus and 210min against Ae. aegypti and An. stephensi,
respectively. The results clearly show that repellent activity was dose dependent. From the
results it can be concluded the crude extract of C. indica was an excellent potential for
controlling Cx. quinquefasciatus, Ae. aegypti and An. stephensi mosquitoes (Govindarajan,
2011).
2.2.14 Antilithiatic activity
In-vivo study of antilithiatic activity on the fruit extracts of Coccinia indica (Wight & Arn)
ethylene glycol induced lithiatic in rats
The aim of this study was to investigate the effect of ethanolic extract of fruits Coccinia indica
Wight & Arn. (Cucurbitaceae) on urolithiasis in rats. Thirty-six male rats were randomly divided
into six groups (n = 6). Ethylene glycol (EG) 0.75% in drinking water were fed to all groups
(Groups II-VI) except normal control (Group I) rats for 15-28th days to induce urolithiasis.
Group III-VI rats were treated with ethanolic extract Coccinia indica of at doses 100, 200, 300
mg/kg, respectively, for 28 days. Positive control (Group II) rats were treated with EG alone.
Group I rats were administered drinking water and distilled water (6l/g) by gavage. After 15-
28th days, blood samples were collected and analyzed for serum concentrations of calcium,
inorganic phosphorus, blood urea, and creatinine. The kidneys were removed and sectioned for
histopathological examination. In vivo study, renal stone inducing treatment to male rats resulted
in hyperoxaluria. Ethanolic extract of Coccinia indica (at 100 and 200mg/kg) exhibited a dose
dependent significant anti-lithiatic activity on treatment. The extract dose of 100mg/kg also
caused reduction of calcium, oxalates, phosphorus and creatinine in blood serum level the results
were found statistically significant. The antilithiatic effect of ethanol extract at was found
effective than the reference standard. Histopathology of the kidneys in Groups V and VI revealed
less tissue damage and were almost similar to Group I rats (Kumar et al., 2014).

Rationale and objective of the work
Coccinia cordifolia is an important medicinal plant in Bangladesh. In the rural areas the different
parts of the plant are extensively used to treat varieties of ailments, such as diabetes, diarrhea,
dysentery, hypertension, menstrual problems and fever (Rahmatullah, 2010). The plant parts
proved to be effective for the treatment of various diseases and so it is profoundly used for its
medicinal value. This makes C. cordifolia a potential study material for both phytochemical and
pharmacological investigations. So experimental studies were carried out to evaluate different
pharmacological activities of the methanol extract of C. cordifolia leaves.
The aim of research project was to carry out
Phytochemical fractionation of the methanolic extract of Coccinia cordifolia (leaves) using
n-hexane, carbon tetrachloride, chloroform and ethyl acetate, consecutively.
Evaluation of antioxidant property of aqueous fraction of methanolic extract of Coccinia
cordifolia leaves by DPPH free radical scavenging
Evaluation of total phenol content of Coccinia cordifolia leaf extract
Evaluation of total flavonoid content Coccinia cordifolia leaf extract
Evaluation of cytotoxic activity of Coccinia cordifolia leaf extract by brine shrimp lethality
bioassay
Evaluation of antimicrobial activity of Coccinia cordifolia leaf extract against different
Gram positive bacteria, Gram negative bacteria and fungi.


Chapter Three
METHODS AND MATERIALS

In vitro pha
3.1 Coll
Plant sam
identifica
sun dried
low temp
high cap
Pharmacy
3.2 Extr
About 65
liters) an
plug and
stirring.
paper and
The conc
extract ob
armacological in
lection and
mple (leaves
ation of plan
d for several
perature for
pacity grind
y, East West
raction of t
50gm of the
nd soaked in
d aluminum f
The whole m
d the filtrate
centrated ex
btained from
nvestigations of
d preparatio
) of Coccini
nt sample w
l days. The
better grind
ing machine
t University
the plant m
e powdered
n 3.5 liter of
foil and kep
mixture was
thus obtaine
Figure 3.1
tract was th
m the powder
f aqueous fracti
on of plant
ia cordifolia
was done by
plant mater
ding. The dr
e in the Ph
.
material
material wa
f methanol. T
pt for a perio
s then filtere
ed was conc
: Drying of
en air dried
red whole pl
ion of Coccinia
t material
a was collect
an expert ta
rials were th
ried leaves w
hytochemica
as taken in s
The contain
od of 15 day
ed through c
entrated at 3
extract using
to solid res
lant was 25g
cordifolia leave
ted from Shr
axonomist. T
hen oven dri
was then gro
al Research
separate cle
ner with its c
ys accompan
cotton follow
39C with a r
g rotary evap
sidue. The w
gm respectiv
es
reepur, Gazi
The leaves o
ied for 24hr
ound in coar
Laboratory
ean, round b
content was
nying occasi
wed by Wha
rotary evapo

porator
weight of the
vely.
ipur. Then p
of the plant
s at conside
rse powder u
y, Departme
bottomed fla
sealed by c
onal shaking
atman No.1
oration.
e crude meth
39
proper
were
erably
using
nt of
ask (5
cotton
g and
filter
hanol
3.3 Preparation of mother solution
5gm of methanol extract was triturated with 90ml of methanol containing 10ml of distilled water.
The crude extract was dissolved completely. This is the mother solution.
3.4 Partition of mother solution
The mother solution was then partitioned off successively by four solvents of different polarity.
3.4.1 Partition with n-hexane
The mother solution was taken in a separating funnel. 100ml of the n-hexane was added to it and
the funnel was shaken and then kept undisturbed. The organic portion was collected. The process
was repeated thrice (100ml 3). The n-hexane fraction was then air dried for solid residue.
3.4.2 Partition with carbon tetrachloride
To the mother solution left after partitioning with n-hexane, 12.5ml of distilled water was added
and mixed. The mother solution was then taken in a separating funnel and extracted with carbon
tetrachloride (CCl
4
). The process was repeated thrice (100ml 3). The CCl
4
fraction was then air
dried for solid residue.
3.4.3 Partition with chloroform
To the mother solution that was left after partitioning with n-hexane and carbon tetrachloride
16ml of distilled water was added and mixed uniformly. The mother solution was then taken in a
separating funnel and extracted with CHCl
3
(100ml 3). The CHCl
3
soluble fractions were
collected together and air dried.
3.4.4 Partition with ethyl acetate
To the mother solution that left after washing with n-Hexane, CCl
4
and CHCl
3
, was then taken in
a separating funnel and extracted with Ethyl acetate (100ml 3). The Ethyl acetate soluble
fractions were collected together and air dried.

In vitro pha
Figure 3

n-
Ethy
C
armacological in
3.2: Schemat
-Hexane solu
(Upper L
CCl
4
solubl
(Upper L
yl acetate so
(Lower L
CHCl
3
solub
(Upper L
nvestigations of
tic represent
uble fraction
Layer)
le fraction
Layer)
oluble fractio
Layer)
ble fraction
Layer)
M
Extra
f aqueous fracti
tation of the
card
Aqueous me
Crude ex
n
on
Methanol (90m
action with n
Extractio
Extracti
Extract
ion of Coccinia

partitioning
difolia leaves
ethanol solut
xtract (5gm)
ml) + Water
n-Hexane (10
on with CCl
ion with CH
ion with Eth
cordifolia leave
g of methano
s
tion
)
Aq
(L
A
(
A
A
(
(10ml)
00ml 3)
l
4
(100ml X 3
HCl
3
(100ml
hyl acetate (1
es
olic crude ex
queous fracti
Lower Layer
queous fract
(Lower Laye
Aqueous frac
(Upper Laye
queous fract
(Lower Laye
3)
3)
100ml 3)
+ Water
+ Wat
xtract of Cocc
ion
r)
tion
er)
ction
er)
tion
er)
r 12.5ml
ter 16ml
41
cinia
In vitro pha
3.4.5 Co
After pa
remainin
for differ
3.5 Anti
3.5.1 DP
3.5.1.1 P
The 1,1-
radical sc
hydrazin
or metha
antioxida
Resulting
was scav
molecule
acceptan
diamagne

armacological in
ollection of
artitioning th
ng at the end
rent pharmac
ioxidant Ac
PPH (1, 1-d
Principle
-diphenyl-2-p
cavenging c
e when it re
anol solution
ant activity b
g from a col
venged by
e. In the radi
ce of an elec
etic molecul
F
nvestigations of
f Aqueous F
he mother s
d was collect
cological pro
ctivity
diphenyl-2-
picrylhydraz
capacity of a
eacts with hy
n. With this m
by measurem
lor change fr
an antioxid
ical form thi
ctron or hyd
le (Miller et
Figure 3.3: M
f aqueous fracti
Fraction
solution with
ted and air d
operties (ant
-picrylhydr
zyl radical (
antioxidants.
ydrogen don
method it w
ment of the d
rom purple t
dant, throug
s molecule h
drogen radica
al., 2000).
Mechanism o
ion of Coccinia
h the four
dried. This a
ioxidant, cyt
razyl) radi
(DPPH) has
DPPH free
nors. DPPH c
was possible
decrease in th
to yellow th
h donation
had an absor
al from an a
of free radica
cordifolia leave
different so
aqueous frac
totoxic and a
ical test
s been wide
e radical is r
can make st
to determine
he absorbanc
he absorbanc
of hydroge
rbance at 51
antioxidant c
al scavengin
es
olvents the
ction was fur
antibacterial
ly used to e
reduced to th
table free rad
e the antirad
ce of DPPH
ce decreased
en to form
7nm which
compound to
ng activity
aqueous fra
rther investi
l).
evaluate the
he correspon
dicals in aqu
dical power
at 517nm.
d when the D
a stable D
disappeared
o become a s

42
action
gated
e free
nding
ueous
of an
DPPH
DPPH
d after
stable
3.5.1.2 Apparatus and reagents
1,1-diphenyl-2-picrylhydrazyl (DPPH) UV-Spectrophotometer
Methanol Foil paper
Aqueous fraction Test tube
Light-proof box Pipette and pipette pumper
Micropipette Beaker
Distilled water Amber reagent bottle

3.5.1.3 Procedure
DPPH solution preparation
DPPH was used to evaluate the free radical scavenging activity (antioxidant potential) of various
compounds and medicinal plants (Choi et al., 2000). 4mg of DPPH was weighed and dissolved
in 10ml methanol to get a DPPH solution having a concentration 400g/ml. The solution was
prepared in the amber reagent bottle and kept in the light-proof box or in a dark place for 30
minutes.
Extract solutions preparation
4mg or 0.004gm of aqueous extract was taken and dissolved in 40ml methanol. This extract
solution was then used to prepare 5 different extract concentrations using different amounts of
methanol.
Determination of DPPH radical scavenging activity
To each of the five extract solutions of different concentrations (20g/ml, 40g/ml, 60g/ml,
80g/ml and 100g/ml) taken in separate test tubes 100l of DPPH was added. A typical blank
solution was made containing 5ml methanol and 100l of DPPH. The test tubes were kept in the
dark for 30 minutes to allow the reaction to reach completion. Then the absorbance of the
solution was measured at 517nm using a spectrophotometer against blank.

In vitro pha

3.5.2 To
3.5.2.1 P
The cont
Ciocalteu
alkaline c
1
ex
4
20
armacological in
otal phenol
Principle
tent of total p
u Reagent (
condition ph
Ta
1ml
tract
+
4ml
g/ml
nvestigations of
Figure
ic content
phenolic com
(FCR). The
henols ionize
able 3.1: Co
L
Sodium
2ml
extrac
+
3ml
40g/m
f aqueous fracti
e 3.4: Schem
mpounds in
FCR actua
e completely
omposition o
Ingredien
Water
Lithium Sulf
m Tungstate
ct
ml
100l D
concentr
Abs
ion of Coccinia

matic diagram
plant metha
lly measure
y.
of 100mg Fo
nt
fate
Dihydrate
3ml
extract
+
2ml
60g/ml
DPPH was ad
ration and ke
sorbance was
max
= 517n
cordifolia leave
m of DPPH t
anolic extrac
es a sample
olin-Ciocalte
Am
57
15
10
dded in each
ept in dark fo
s taken at
nm
es
test
cts was deter
s reducing
eu Reagent
mount
7.5ml
5.0mg
0.0mg
4ml
extract
+
1ml
80g/ml
h
or
rmined by F
capacity. In
5
ext
100
44
Folin
n the

ml
tract
g/ml
In vitro pha
When FC
Usual co
chemical
heteropo
reduction
change is
phenolic
3.5.2.2 A
3.5.2.3 P
Standar
Ascorbic
a concen
and 2.0m
mixture w
armacological in
P
CR is used in
lor of FCR i
l nature
lyphosphotu
n reactions
s measured i
content of th
Apparatus
Folin-C
Ascorbi
Na
2
CO
3
Methan
Distille
Procedure
rd curve pr
c acid was us
ntration rang
ml of Na
2
CO
was incubate
nvestigations of
Hydro
Phosphoric A
Molybdic
n this ionize
is yellow and
of the F
unstates - m
lead to blue
in a spectrop
he compoun
and reagen
Ciocalteu rea
ic acid
3
solution (7
nol
d water
reparation
sed here as s
ing from 12
O
3
(7.5% w/
ed for 20 mi
f aqueous fracti
Ingredien
ochloric Aci
Acid 85% so
c Acid Sodiu
ed phenolic s
d after the ox
FCR is n
molybdates.
e species, p
photometer a
nd (Singleton
nts
agent (10 fold
.5%)
standard. Di
0g/ml to 8
/v) solution
nutes at room
ion of Coccinia
nt
d>=25%
olution in wa
um Dihydrate
solution the
xidation pro
not known,
Sequences
possibly (PM
at 765nm. Th
n et al., 1999
d diluted)
fferent ascor
0g/ml. 2.5m
was added
m temperatu
cordifolia leave
Am
10
ater 5
e 2
reagent will
ocess the solu
, but it
s of revers
MoW
11
O
40
)
4-
he absorban
9; Vinson et
UV-sp
Beaker
Test tu
Microp
Cuvett
rbic acid sol
ml of FCR (
to 0.5ml of
ure. After 20
es
mount
0.0mg
.0mg
.5mg
l readily oxi
ution becom
is believ
sible one-
-
. The inten
nce value wil
al., 2005).
pectrophotom
r (100 & 200
ube
pipette (50-2
te
lutions were
(diluted 10 t
f ascorbic ac
0 minutes the
idize the phe
me blue. The
ved to co
or two-ele
sity of the
ll reflect the
meter
0ml)
200l)
e prepared ha
times with w
cid solution
e absorbance
45
enols.
exact
ontain
ectron
color
e total

aving
water)
. The
e was
measured at 765nm. After plotting the absorbance in ordinate against the concentration in
abscissa a linear relationship was obtained which was used as a standard curve for the
determination of the total phenolic content of the test samples.
Sample preparation
2mg of the Coccinia cardifolia aqueous fraction was taken and dissolved in 1ml of methanol to
get a sample concentration of 2mg/ml.
Determination of total phenol content
1.0ml of plant extract (200g/ml) of different concentrations (120g/ml, 110g/ml, 100
g/ml, 90g/ml and 80g/ml) was taken in test tubes.
5ml of Folinciocalteu (Diluted 10 fold) reagent solution was added into the test tube.
4ml of Sodium carbonate solution was added into the test tube.
The test tubes containing the samples were incubated for 1hr at the room temperature to
complete the reaction.
The same process was repeated for ascorbic acid sample solutions.
Then the absorbance of the solutions were measured at 765nm using a spectrophotometer
against blank.
Absorbance of a typical blank solution containing methanol was taken.
3.5.3 Total flavonoid content
3.5.3.1 Principle
Aluminium chloride (AlCl
3
) colorimetric method is incorporated to determine the total flavonoid
contents of the crude plant extract. The basic principle of the assay method is that aluminium
chloride forms acid stable complexes with the C-4 keto group and either the C-3 or C-5 hydroxyl
group of flavones and flavonols of the crude extract. In addition aluminium chloride also forms
acid labile complexes with the ortho-dihydroxyl groups in the A or B-ring of flavonoids. The
formed flavonoid-aluminium complex between flavonoid of the crude extract and aluminium
chloride has an absorptivity maximum at 510nm. Therefore, the amount of flavonoid in the crude
extract can be quantified by measuring the absorbance of reaction mixture at 510nm using a UV-
visible spectrophotometer against a blank containing all reagents except the extracts. Quercetin
at various concentrations was used as standard (Chang et al., 2002).
Flavonoid (Extract) + AlCl
3
(reagent)= Formation of flavonoid-aluminium complex (
max
510nm)
3.5.3.2 Apparatus and reagents
Aluminium chloride Spatula
Methanol Analytical balance
Ascorbic acid Pipette and pumper
Sodium hydroxide Aqueous fraction
Sodium nitrite Test tubes and beaker
3.5.3.2 Procedure
Aluminium chloride (10%) solution preparation
10mg of aluminium chloride (AlCl
3
) was taken into a 100ml of a volumetric flask and the
volume was adjusted by distilled water.
NaOH (4%) solution preparation
4mg of sodium hydroxide (NaOH) was taken into a 100ml volumetric flask and the volume was
adjusted by distilled water.
NaNO
2
(5%) solution preparation
5mg of sodium nitrite NaNO
2
was taken into a 100ml of a volumetric flask and the volume was
adjusted by distilled water.
Standard solution preparation
The stock solution was prepared by taking 0.025gm of ascorbic acid and dissolved into 5ml of
ethanol. The concentration of this solution was 5g/l of ascorbic acid. The experimental
concentrations from this stock solution were prepared by the following manner.

Table 3.2: Different concentrations of ascorbic acid solution preparation
Concentration
(g/ml)
Solution taken from
stock solution (l)
Volume adjusted by
methanol (ml)
Final volume
(ml)
250 250 4.75 5
200 200 4.80 5
150 150 4.85 5
100 100 4.90 5
50 50 4.95 5
Extract solution preparation
5mg of plant extract was taken and dissolved into 5ml of methanol. The concentration of the
solution was 1mg/ml of plant extract.
Determination of total flavonoid content
1.5ml extract was taken in a test tube and then 6ml of distilled water was added. Then 5% of
NaNO
2
was added and incubated for 6 minutes. 10% AlCl
3
was added and incubated for 6
minutes. 4% NaOH and 0.6ml distilled water was added. Then it was incubated for 15 minutes.
For blank solution 1.5ml methanol was taken and the same procedure was repeated. Then the
absorbance of the solution was measured at 510nm using a spectrophotometer against blank.
In vitro pha
3.6 Brin
3.6.1 Pr
Brine shr
compoun
pharmaco
products
simply to
(in-vivo)
used as
bioactive
for their
of pharm
Artemia
Nueza,

armacological in
F
ne shrimp l
rinciple
rimp lethalit
nds and natu
ological act
etc. Bioact
oxicology at
lethality, a
a convenie
e natural pro
bioactivity b
macological a
of aquatic c
2013; Rishik
1.
nvestigations of
Figure 3.5:
lethality bio
ty bioassay i
ural product
ivities e.g.
tive compou
t a lower dos
simple zool
ent monitori
oducts. Natu
by this meth
activity of n
crustaceans.
kesh et al., 2
.5ml extract
0.45ml NaN
0.45ml AlC
0.6ml of
f aqueous fracti
Schematic d
oassay
is a recent d
extracts, wh
anticancer,
unds are alm
se or toxicol
logical organ
ing for scre
ural product
hod. This bio
natural produ
Artemia is t
2013).
(1mg/ml) in
6ml of
NO
2
(5% w/v
Cl
3
(10% w/v
6ml NaOH
f distilled wa
max
510
ion of Coccinia
diagram of fl
development
hich indicate
antiviral, an
most always
logy is simp
nism, (Brine
eening and
extracts, fra
oassay is ind
ucts. Brine s
the only gen
n methanol /
f distilled wa
v) taken and
v) taken and
H (4% w/v)
ater and incu
0nm absorba
cordifolia leave
lavonoid con
t in the assay
es cytotoxicit
nd pharmaco
s toxic in h
ply pharmac
e shrimp nap
fractionatio
actions or pu
dicative of c
shrimp is the
nus in the fam
1.5ml of me
ater
d incubated fo
d incubated f
taken
ubated for 15
ance
es
ntent test
y procedure
ty as well as
ological acti
high doses.
ology at a h
pulii- Artem
on in the di
ure compoun
cytotoxicity a
e English na
mily Artemi
ethanol
for 6min
for 6min
5min
for the bioa
s a wide ran
ivities of na
Pharmacolo
higher dose.
ia salina) ca
iscovery of
nds can be t
and a wide r
ame of the g
iidae (Olowa
49

active
nge of
atural
gy is
Thus
an be
f new
tested
range
genus
a and
3.6.2 Apparatus and reagents
Artemia salina leach (brine shrimp eggs) Pipettes & Micropipette
Sea salt (NaCl) Glass vials
Small tank with perforated dividing dam to hatch
the shrimp
Magnifying glass
Test samples
Lamp to attract shrimps Dimethyl sulfoxide (DMSO)
3.6.3 Procedure
3.6.3.1 Preparation of sea water
To hatch the brine shrimp nauplii for the assay, sea water representing brine should be prepared
at first. To prepare sea water 38gm of pure NaCl was dissolved in distilled water and then the
volume made up to 1000ml by distilled water in a 1000ml beaker for Artemia salina hatching. 1-
2 drops of NaOH solution of 0.1N was added with a dropper to obtain the pH 8.4 as sea water.
3.6.3.2 Hatching of brine shrimp
A rectangular tank was divided in to two unequal compartments by a porous separator. The
larger compartment was darkened while the smaller one was kept illuminated. Then dry
preserved eggs of Artemia salina Leach was added in the artificial sea water. Oxygen was
supplied through an air pump and a table lamp was placed near the beaker. The eggs of Artemia
salina were hatched at room temperature (25-30C) for 18-24hrs. The larvae (nauplii) were
attracted by the light and moved to the smaller compartment through the holes. 10 living shrimps
were then collected by a pipette and then added to each of the test tubes containing 5ml of
seawater. Those freshly hatched free-swimming nauplii were used for the bioassay.
In vitro pha
3.6.3.3 P
Clean tes
tube for
tamoxife
Prepara
All the t
(DMSO)
containin
prepared
concentra
100l sa
concentra
50g/ml,
dilutions
Prepara
In the pre
is dissolv
made us
6.25g/m
armacological in
Preparation
st tubes were
each conce
n for ten con
ation of tes
test samples
in vials to
ng 5ml of si
solution in
ations were
ample was a
ations of the
, 25g/ml, 1
.
ation of pos
esent study t
ved in DMS
sing DMSO
ml, 3.125g/m
nvestigations of
Figu
n of test so
e taken. The
ntration) of
ncentrations
t samples o
s of 4mg w
get stock s
imulated sea
n the first te
prepared fr
added to tes
e obtained s
12.5g/ml, 6
sitive contr
tamoxifen is
O to get an
O to get 40
ml, 1.5625
f aqueous fracti
ure 3.6: Arte
olutions
ese test tubes
f test sample
of it and ano
of experim
were taken a
solutions. T
awater and
est tube was
rom the stoc
st tube and
solution in e
6.25g/ml, 3
rol group
s used as the
initial conce
00g/ml, 20
g/ml and 0.7
ion of Coccinia
emia salina 2
s were used
es and ten t
other one tes
ental plant
and dissolve
hen 100l o
10 shrimp n
s 400g/ml.
ck solution
fresh 100
each test tub
3.125g/ml,
positive con
entration of
0g/ml, 100
78125g/ml
cordifolia leave
24 hours old
for ten diffe
test tubes w
st tube for co
t
ed in 200l
of solution
nauplii. Thu
. Then a se
by serial di
l DMSO w
be were 400
1.5625g/m
ntrol. Measu
f 20g/ml fro
0g/ml, 50
l. Then ten l
es

d
erent concen
were taken f
ontrol test.
of pure dim
was taken i
us, final con
eries of solu
ilution meth
was added t
0g/ml, 200
ml and 0.78
ured amount
om which se
g/ml, 25g
living brine
ntrations (on
for standard
methyl sulfo
in test tube
ncentration o
utions of va
hod. In each
to vial. Thu
g/ml, 100
125g/ml fo
t of the tamo
erial dilution
g/ml, 12.5
shrimp naup
51
ne test
drug
foxide
each
of the
arying
h case
us the
g/ml,
or 10
oxifen
ns are
g/ml,
plii in
5ml simulated seawater are added to the positive control solutions in the pre-marked test-tubes to
get the positive control groups.
Preparation of negative control group
100l of DMSO was added to the pre-marked test tube containing 5ml of simulated seawater and
10 shrimp nauplii to use as control groups. If the brine shrimps in these vials show a rapid
mortality rate, then the test is considered as invalid as the nauplii died due to some reason other
than the cytotoxicity of the compounds.
3.6.3.5 Counting of nauplii
After 24hrs, the vials were inspected using a magnifying glass and the number of survived
nauplii in each vial was counted. From this data, the percent (%) of lethality of the brine shrimp
nauplii was calculated for each concentration.
3.7 Antimicrobial activity by disc diffusion method
3.7.1 Principle
The disk diffusion susceptibility method is simple and well-standardized. Bacterial inoculums
are applied to the surface of a large agar plate. Antibiotic discs and disc of test materials are
placed on the inoculated agar surface. Plates are incubated for 1624hrs at 35C prior to
determination of results. The test materials having antimicrobial property inhibit microbial
growth in the media surrounding the discs and thereby yield a clear, distinct area defined as zone
of inhibition. The zones of growth inhibition are measured to the nearest millimeter around each
of the antibiotic disks. The diameter of the zone is related to the susceptibility of the isolate and
to the diffusion rate of the drug through the agar medium (Barry, 1976).
3.7.2 Apparatus and reagents
3.7.2.1 Materials
Filter paper discs Screw cap test tubes
Petri dishes Nose mask and Hand gloves
Inoculating loop Laminar air flow hood
Sterile cotton Autoclave
Sterile forceps Incubator
Spirit burner Ethanol
Micropipette Nutrient Agar Medium
3.7.2.2 Test sample of Coccinia cordifolia
Aqueous fraction of methanolic extract of Coccinia cordifolia leaves were taken as test sample.
3.7.2.3 Test organisms
The bacterial strains used for the experiment were collected as pure cultures from the East West
University microbiology laboratory. Gram positive bacteria, Gram-negative bacteria and fungi
organisms were taken for the test and they are listed in the following table.
Table 3.3: List of microorganisms
Type of microorganism Name of microorganism
Gram +ve bacteria Beta-hemolytic streptococcus
Bacillus subtilis
Gram ve bacteria Escherichia coli
Salmonella paratyphi
Pseudomonas aeruginosa
Fungi Saccharomyces cerevisiae
3.7.3 Procedure
3.7.3.1 Preparation of the medium
To prepare required volume of this medium, 5.6gm of agar medium was taken in a bottle with a
cap and distilled water was added to it to make 200ml volume. The contents were then
autoclaved to make a clear solution.
In vitro pha
3.7.3.2
In order
antimicro
maintain
and other
15-lbs/sq
sterilized
armacological in
Sterilizatio
to avoid any
obial screen
ed. UV light
r glassware
q. inch for 2
d.
nvestigations of
on procedu
y type of co
ning was do
t was switch
were steriliz
20 minutes.
f aqueous fracti
Figure 3.7:
ure
ontamination
ne in lamin
hed on one h
zed by autoc
Micropipet
ion of Coccinia
Autoclave m
n and cross
nar hood an
hour before w
claving at a
tte tips, cott
cordifolia leave
machine
contaminati
nd all types
working in th
temperature
ton, forceps
es
on by the te
of precauti
he laminar h
e of 121C a
, blank disc

est organism
ions were h
hood. Petri d
and a pressu
cs etc. were
54
ms the
highly
dishes
ure of
e also
In vitro pha
3.7.3.3
The test
melted an
mixed w
taken and
with the h
3.7.3.4 P
Three typ
armacological in
Preparatio
organisms w
nd sterilized
with normal
d dipped into
help of this c
Preparation
pes of discs w
nvestigations of
on of test pl
were transfe
d agar mediu
saline with
o the suspen
cotton bud.
n of discs
were used fo
f aqueous fracti
Figure 3
late
erred from th
um. The bac
the help of
nsion. Then t
or antimicrob
ion of Coccinia
.8: Laminar
he subcultur
cterial and f
f vortex mac
the bacterial
bial screenin
cordifolia leave
hood
re to petridi
fungal suspe
chine. Then
l/fungal samp
ng.
es

sh containin
ensions were
a sterilized
mple is applie
ng about 10m
e taken by a
d cotton bud
ed to the petr
55
ml of
a loop
d was
ridish
Standard discs: These were used as positive control to ensure the activity of standard
antibiotic against the test organisms as well as for comparison of the response produced by
the known antimicrobial agent with that of the test sample. In this investigation, azithromycin
disc was used as the reference.
Blank discs: These were used as negative controls which ensure that the residual solvent
(left over the discs even after air-drying) and the filter paper were not active themselves.
Sample discs: These discs were soaked with solutions of test samples of known
concentration, dried and used to determine the anti-activity of the samples.
3.7.3.5 Preparation of test sample
Measured amount of test sample was dissolved in specific volume of solvent to obtain the
desired concentrations in an aseptic condition. Sterilized metrical (BBL, Cocksville, USA) filter
paper discs were taken in a blank petri dish under the laminar hood. Then discs were soaked with
solutions of test samples and dried.
3.7.3.6 Application of test samples
Standard azithromycin discs were used as positive control to ensure the activity of standard
antibiotic against the test organisms as well as for comparison of the response produced by the
known antimicrobial agent with that of produced by the test sample. Methanol discs were used as
negative controls which ensure that the residual solvents (left over the discs even after air-
drying) and the filter paper were not active themselves.
3.7.3.7 Diffusion and incubation
The sample discs, the standard antibiotic discs and the control discs were placed gently on the
previously marked zones in the agar plates pre-inoculated with test bacteria. The plates were then
inverted and kept in an incubator at 37C for 24hrs.
In vitro pha
3.7.3.8 D
The antim
of the m
incubatio
diameter

armacological in
Determina
microbial po
microorganis
on, the antim
of the zones
nvestigations of
ation of ant
otency of the
sms surroun
microbial act
s of inhibitio
f aqueous fracti
Figure
timicrobial
e test agents
nding the d
tivities of th
on in millime
ion of Coccinia
e 3.9: Incuba
l activity by
are measure
discs which
he test mate
eter with a tr
cordifolia leave
ator
y measurin
ed by their a
gives clea
rials were d
ransparent sc
es
ng the zone
activity to pr
ar zone of
determined b
cale.
e of inhibiti
revent the gr
inhibition.
by measurin
57

ion
rowth
After
ng the


Chapter Four
RESULTS AND DISCUSSION

4.1 Antioxidant test results
Antioxidant tests are classified by various methods. Samples were subjected to various standard
methods to determine various scavenging capacity and amount that is equivalent to the standard
like ascorbic acids. Antioxidant property of the aqueous fraction of methanolic extract of C.
coccinia (leaves) was determined by following methods-
Determination of DPPH radical scavenging assay
Determination of total phenolic content
Determination of total flavonoids content
4.1.1 DPPH test results
One of the quick methods to evaluate antioxidant activity is the scavenging activity on DPPH, a
stable free radical. DPPH is one of the free radicals widely used for testing preliminary radical
scavenging activity of a compound or a plant extract. In DPPH assay method is based on the
reduction of alcoholic DPPH solution (dark blue in colour) in the presence of a hydrogen
donating antioxidant converted to the non-radical form of yellow colored diphenyl-
picrylhydrazine. Lower the absorbance higher the free radical scavenging activity
The aqueous fraction of Coccinia cordifolia leaves was evaluated for their free radical
scavenging activity. Here, ascorbic acid (AA) was used as reference standard. Then percentage
inhibition was calculated to determine the amount of free radical scavenged by the extract or
standard at different concentrations using the following formula,

% inhibition =

blank absorbance sample absorbance
blank absorbance
100
In vitro pha
4.1.1.1 P
Abso
of b
0

%

I
n
h
i
b
i
t
i
o
n

armacological in
Preparation
orbance
blank
C
(
.164
F
0
20
40
60
80
100
120
0
%
nvestigations of
n of standa
Table 4.1: %
Conc.
g/ml)
Ab
of
0
20
40
60
80
100
Figure 4.1: R
20
DPPH
f aqueous fracti
ard curve
% inhibition
bsorbance
f ascorbic
acid
0.164
0.049
0.037
0.030
0.015
0.009
Regression l
4
Concen
H Free Ra
ion of Coccinia
and IC
50
val
% Inhibi
samp
ab
ine and R
2
v
40
ntration (
adical Sc
(Standar
cordifolia leave
lues of ascor
ition= (blan
ple absorba
bsorbance)
0
70.12
77.44
81.71
90.85
94.51
value of asco
60
g/ml)
cavengin
rd)
es
rbic acid
k absorban
ance/blank
100
orbic acid
y = 0.77
R =
80
ng Assay
nce IC
50
(g/ml

25.19
7x + 30.604
= 0.6778
100
y
59
l)
9

In vitro pha
4.1.1.2 P
T
Absorb
bla
0.1
Fi

%
I
n
h
i
b
i
t
i
o
n
armacological in
Preparation
Table 4.2: %
bance of
ank
C
(
164
igure 4.2: R
0
20
40
60
80
100
120
0
%

I
n
h
i
b
i
t
i
o
n

nvestigations of
n of aqueo
% inhibition a
Conc.
g/ml)
Ab
e
0
20
40
60
80
100
egression lin
20
DPPH
f aqueous fracti
us fraction
and IC
50
valu
sorbance
of the
extract
0.164
0.079
0.070
0.056
0.042
0.033
ne and R
2
va
4
Con
Free Rad
ion of Coccinia
n curve
ues of aqueou
% Inhibitio
sampl
abs
alue of aqueo
40
ncentratio
dical Sca
(Extract
cordifolia leave
us fraction o
on= (blank
le absorban
sorbance)
0
51.83
57.31
65.85
74.40
79.88
ous fraction
60
on (g/ml
avenging
t)
es
of Coccinia c
absorbance
nce/blank
100
of Coccinia
y = 0.6795
R = 0
80
)
g Assay
cordifolia
e IC
50
(g/m

42.82
cordifolia
5x + 20.903
0.7782
100
60
ml)
2

In vitro pha
4.1.1.3 D
Table 4
Sta
Ext
In this in
IC
50
valu
The IC
50

be said t
ascorbic
armacological in
Discussion
4.3: Free rad
Samp
andard (Asco
tract (Aqueo
nvestigation,
ues 25.19g/m
Figure
value of aqu
that the ext
acid.
0
5
10
15
20
25
30
35
40
45
I
C
5
0

(
g
/
m
l
)

I
nvestigations of
dical scaveng
ple
orbic acid)
us fraction)
standard and
ml and 42.82
e 4.3: Comp
ueous fractio
tract has ve
Standard (A
C
50
(g/m
f aqueous fracti
ging capacity
cord
Linea
e
y = 0.7
y = 0.67
d aqueous fr
2g/ml resp
arison betwe
on is more th
ery low anti
scorbic acid)
25.19
ml) value
and
ion of Coccinia
y of ascorbic
difolia leaves
ar regression
equation
77x + 30.60
795x + 20.9
raction exhib
ectively.
een IC
50
valu
han that of s
ioxidative co
Extract
es of both
ascorbic
cordifolia leave
c acid and aq
s
n R
2
v
04 0.6
903 0.7
bited antioxi
ues of stand
tandard as s
ompounds a
(Aqueous fract
42.82
h aqueou
acid
es
queous fract
value IC
6778
7782
idative activi
ard and extr
hown in Fig
and has low
tion)
2
us fraction
tion of Cocci
C
50
(g/ml)
25.19
42.82
ities with the

ract
gure 4.3. So i
wer potency
n
61
inia
e
it can
than
In vitro pha
4.1.2 To
The aque
content p
4.1.2.1 P
A linear
shown in
calculate
F
armacological in
otal phenol
eous fraction
present. Here
Preparation
Concentr
relationship
n Figure 4.4.
d in Microso
Figure 4.4: G
2.2
2.4
2.6
2.8
3
3.2
80
A
b
s
o
r
b
a
n
c
e

a
t

7
6
5
n
m

nvestigations of
content te
ns of Coccini
e, ascorbic a
n of standa
Table 4.
ation (g/m
80
90
100
110
120
was observ
This linear
oft Office Ex
Graphical rep
85
f aqueous fracti
est results
ia cordifolia
cid (AA) wa
ard curve
.4: Total phe
ml) Absorb
2.40
2.47
2.76
3.05
3.08
ed when the
curve was c
xcel 2010.
presentation
90 95
Con
Totla P
(
ion of Coccinia
a leaves were
as used as re
enol content
bance R
06
y =
73
67
57
80
e absorbance
onsidered as
n of assay of
5 100
ncentratio
Phenol C
Standar
cordifolia leave
e subjected t
eference stan
t of ascorbic
Regression l
= 0.0193x + 0
es were plott
s a standard
phenolic con
y
105 1
n (g/ml)
Content
rd)
es
to determine
ndard.
acid
line R
2
0.8246 0
ted against c
curve. Regr
ntent of asco
y = 0.0193x + 0
R = 0.937
110 115

e total pheno
2
value
0.9372
concentration
ression analy

orbic acid
0.8246
72
120
62
olic
ns, as
ysis is
In vitro pha
4.1.2.2 T
Based on
compared
content p
Tab
4.1.2.3 D
Fi
armacological in
Total phen
n the absorba
d with the
present in the
ble 4.5: Tota
Concentrat
(g/ml)
80
90
100
110
120
Discussion
igure 4.5: C
1
1
2
2
2
2
2
3
A
b
s
o
r
b
a
n
c
e

a
t

7
6
5
n
m

nvestigations of
ol content
ance values
standard sol
e extract is c
al phenolic c
tion Abs
2
2
2
omparison b
.7
.9
.1
.3
.5
.7
.9
.1
70
Abs
f aqueous fracti
present in
of the extrac
lutions of a
calculated an
content of aq
sorbance
1.886
1.969
2.097
2.098
2.286
between abso
con
80 9
Conce
orbances
diffe
ion of Coccinia
aqueous fr
ct solution, r
ascorbic acid
nd given in th
queous fracti
X val
(mg of AA
of dried e
54.9
59.2
65.9
65.9
75.7
orbances of s
ncentrations
90 100
entration
of standar
rent conce
cordifolia leave
raction
reacted with
d equivalent
he table belo
ion of leaves
lue
AE/gm
extract)
(
99
29
93
98
72
standard and
0 110
(g/ml)
rd and ext
entrations
Absorba
Absorba
es
h Folin-Cioc
ts (AAE), th
ow.
s of Coccinia
Average X
(mg of AAE
dried extr
64.38
d extract ove
120
tract over
s
nce (Ascorbic acid
nce (Aqueous frac
alteu reagen
he total phe
a cordifolia
X value
E/gm of
ract)
8

er different
d)
ction)
63
nt and
enolic

The absorbance was found to be directly proportional to the concentration in both standard and
aqueous fraction samples. In both extract and standard the absorbance increased with the
increase in concentration indicating increase in phenolic content. Overall absorbance of the
extract is very low compared to that of the standard, as shown in Figure 4.5.
Based on the absorbance values of extract solution and using the regression line equation of the
standard curve, 64.38mg of AAE/gm of dried extract of phenol content was found in the aqueous
fraction of Coccinia cordifolia (leaves).
4.1.3 Total flavonoid content result
The aqueous fractions of Coccinia cordifolia leaves were subjected to determine total flavonoid
content present. Here, ascorbic acid (AA) was used as reference standard.
4.1.3.1 Preparation of standard curve
Table 4.6: Total flavonoid content of ascorbic acid
Concentration
(g/ml)
Absorbance Regression line R
2
value
50 0.05
y = 0.0017x-0.042 0.991
100 0.13
150 0.19
200 0.29
250 0.39
After absorbances were taken of different solution of ascorbic acid of concentrations ranging
from 50g/ml to 250g/ml, a linear relationship was observed when the absorbances were
plotted against concentrations, as shown in Figure 4.6. This linear curve was considered as a
standard curve. Regression analysis is calculated in Microsoft Office Excel 2010.
In vitro pha
F
4.1.3.2 T
Based on
standard
Tab
4.1.3.3 D
To deter
1mg/ml c
dried ext
A
b
s
o
r
b
a
n
c
e
a
t
5
1
0
n
m
armacological in
igure 4.6: G
Total flavo
n the absorb
curve, the to
ble 4.7: Tota
Samp
Aqueous fra
Coccinia co
Discussion
mine the to
concentratio
ract of flavo
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
50
A
b
s
o
r
b
a
n
c
e

a
t

5
1
0
n
m

nvestigations of
Graphical rep
noid conte
ance value o
otal flavonoi
al flavonoid c
ple C
action of
ordfolia

otal flavonoi
on of aqueou
onoid conten
To
f aqueous fracti
presentation
nt present
of extract so
id present in
content of aq
Concentrati
(mg/ml)
1
d content o
us fraction of
nt was found
100
Concen
otal flav
(Sta
ion of Coccinia
of assay of f
in aqueous
olution and u
n the extract i
queous fract
on Absorb
0.31
f the test sa
f Coccinia c
. So this extr

150
tration (
oniod co
andard)
cordifolia leave
flavonoid co
s fraction
using the re
is calculated
tion of leave
bance
con
o
19
amples the s
cordifolia (le
ract contains
y =
g/ml)
ontent

es
ontent of asc
gression lin
d and is give
es of Coccini
Total flavo
ntent (mg of
of dried ext
212
standard cur
eaves) 212m
s antioxidati
0.0017x - 0.04
R = 0.991
200
corbic acid
e equation o
n in Table 4
ia cordifolia
noid
f AAE/g
tract)
rve was use
mg of AAE/g
ve compoun
42
250
65

of the
4.7.
a
ed. In
gm of
nds.
4.2 Brine shrimp lethality bio-assay result
The aqueous fraction of the methanolic extract of Coccinia cordifolia leaves were subjected to
brine shrimp lethality bioassay following the procedure Meyer et al., (1982). After 24hrs, the test
tubes were inspected using a magnifying glass and the number of survivors counted.
The effectiveness of the concentration and % mortality relationship of plant product was
expressed as a median Lethal Concentration (LC
50
) value. This represents the concentration of
the standard or aqueous extract that produces death in half of the test subjects after a certain
period. The percentage mortality at each concentration was determined using the following
formula:
% mortality = (number of dead nauplii/total number) 100
The LC
50
of the test samples was obtained by a plot of percentage of the shrimps died (%
Mortality) against the logarithm of the sample concentration (Log C) and the best-fit line was
obtained from the curve data by means of regression analysis. The concentration-% mortality
data were analyzed by using Microsoft Office Excel 2010.

In vitro pha
4.2.1 Pr
Tamoxife
Tes
tube
no.
1
2
3
4
5
6
7
8
9
10
Fig
armacological in
reparation
fen was used
T
t
e
.
Concen
(C) (
40
20
10
5
2
12
6.
3.1
1.5
0.78
gure 4.7: Plo
0
20
40
60
80
100
120
-0.5
%

M
o
r
t
a
l
i
t
y

nvestigations of
of standar
as positive c
able 4.8: Re
ntration
g/ml)
00
00
00
50
25
2.5
25
125
625
8125
ot of % mort
0
0
0
0
0
0
0
0 0
Brine
f aqueous fracti
d curve
control.
esults of the
Log C N
2.602
2.301
2.000
1.699
1.398
1.097
0.796
0.495
0.194
-0.107
tality and pre
0.5 1
e shrimp
(St
ion of Coccinia
bioassay of
Number of
nauplii
alive
0
1
2
3
5
5
6
7
8
9
edicted regre
1 1.5
Log C
p lethalit
tandard)
cordifolia leave
Tamoxifen
Number o
nauplii
dead
10
9
8
7
5
5
4
3
2
1
ession line o
y =
2
ty bioass
)
es
(standard)
of %
Mortal
100
90
80
70
50
50
40
30
20
10
of Tamoxifen
= 33.021x + 12
R = 0.9891
2.5
say
lity
LC
50
(g/m
13.38

n (standard)
2.806
1
3
67
0

ml)
8
In vitro pha
4.2.2 Pr
Tes
tube
no.
1
2
3
4
5
6
7
8
9
10
Figur
armacological in
reparation
Tab
t
e
.
Concen
(C) (
40
20
10
5
2
12
6.
3.1
1.5
0.78
re 4.8: Plot
0
20
40
60
80
100
120
-0.5
%

M
o
r
t
a
l
i
t
y

nvestigations of
of aqueous
ble 4.9: Resu
ntration
g/ml)
00
00
00
50
25
2.5
25
125
625
8125
of % mortal
0
0
0
0
0
0
0
0 0
Brine
f aqueous fracti
s fraction c
ults of the bi
Log C N
2.602
2.301
2.000
1.699
1.398
1.097
0.796
0.495
0.194
-0.107
ity and pred
0.5 1
e shrimp
(E
ion of Coccinia
curve
ioassay of aq
Number of
nauplii
alive
1
1
2
3
3
4
6
8
8
9
icted regress
1.5
Log C
p lethalit
Extract)
cordifolia leave
queous fracti
Number o
nauplii
dead
9
9
8
7
7
6
4
2
2
1
sion line of a
y
2
ty bioass
es
ion (extract)
of %
Mortal
90
90
80
70
70
60
40
20
20
10
aqueous frac
y = 32.417x + 1
R = 0.9521
2.5
say
)
lity
LC
50
(g/m
12.39

ction (extrac
14.56
1
3
68
0

ml)
9
ct)
In vitro pha
4.2.3 Di
In Brine
different
proportio
concentra
an increa
concentra
as shown
Table 4
Stand
Extract
In this in
values 13
aqueous
against b
armacological in
scussion
Shrimp Let
concentrati
onal to the
ation in both
ase in conce
ation of 400
n in Table 4.6
.10: Cytotox
Sample
dard (Tamox
(Aqueous fr
nvestigation,
3.38g/ml a
fraction the
brine shrimp
Figure
11.5
12
12.5
13
13.5
L
C
5
0

(
g
/
m
l
)

nvestigations of
thality bioas
ons of the t
concentrat
h standard an
ntration of t
0g/ml, whe
6 and Table
xic activity o
xifen)
fraction)
, standard an
nd 12.39g/
R
2
value is
nauplii com
e 4.9: Compa
Standard (T
LC
50
val
f aqueous fracti
ssay, varying
test samples
tion ranging
nd aqueous
the test samp
ereas the lea
4.7.
of Tamoxifen
Linear re
equa
y = 33.021
y = 32.417
nd aqueous
/ml respectiv
closer to on
mparable to th
arison betwe
Tamoxifen)
13.38
lues of bot
extract (
ion of Coccinia
g degree of
s. The degre
g from the
fraction sam
ples. Maxim
ast mortalitie
n and aqueou
egression
ation
x + 12.806
7x + 14.56
fraction exh
vely as show
ne which ind
he standard (
een LC
50
val
Extract
th Tamoxif
(aqueous f
cordifolia leave
lethality wa
ee of lethali
e lowest co
mples. Morta
mum mortalit
es at lowest
us fraction o
R
2
valu
0.989
0.952
hibited cytot
wn in Table
dicates that t
(Tamoxifen)
lues of stand
(Aqueous fract
12.39
fen (stand
fraction)
es
as observed
ity was foun
oncentration
ality increase
ties took pla
concentratio
of Coccinia c
ue LC
50

1
1
toxic activiti
4.10. For b
the extract h
).
dard and extr
tion)
9
dard) and
with exposu
nd to be dir
to the hi
ed gradually
ace at the hi
on 0.78125
cordifolia le
(g/ml, 24h
13.38
12.39
ies with the
both standard
has potent ac
ract
69
ure to
rectly
ighest
y with
ighest
g/ml
eaves
hr)
LC
50

d and
ctivity
From the above figure it can be concluded that for aqueous fraction the lethal concentration
required to kill 50% of the sample population is lower than the standard. So the extract is more
potent than ascorbic acid at lower concentration.
4.3 Antimicrobial test results
The antimicrobial activities of aqueous fraction of methanolic extract of Coccinia cordifolia
leaves were examined in the study against various Gram positive bacteria, Gram negative
bacteria and fungi. The aqueous fraction was subjected to the various bacterial and fungal
cultures and from that zones of inhibition were measured. Here Azithromycin was used as
standard reference.
4.3.1 Zone of inhibition of standard and aqueous fraction
Table 4.11: Antimicrobial activity of standard sample (Azithromycin) and aqueous fraction
Type of microorganism
Zone of inhibition (mm)
Standard sample Aqueous fraction
Gram positive
bacteria
Beta-hemolytic streptococcus 18 11
Bacillus subtilis 20 12
Gram negative
bacteria
Escherichia coli 18 8
Salmonella paratyphi 25 11
Pseudomonas aeruginosa 18 9
Fungi Saccharomyces cerevisiae 18 6

In vitro pha
4.3.2 Di
Figure 4
Aqueous
antimicro
aqueous
4.10. Am
against B

Z
o
n
e
o
f
i
n
h
i
b
i
t
i
o
n
(
m
m
)
armacological in
scussion
4.10: Comp
fraction o
obial activit
fraction is e
mong all the
Beta-hemolyt
0
5
10
15
20
25
Z
o
n
e

o
f

i
n
h
i
b
i
t
i
o
n

(
m
m
)

nvestigations of
arison of ant
of methanol
ty when com
equal to Azit
microbiolog
tic streptoco
f aqueous fracti
timicrobial a
lic extract o
mpared to
thromycin a
gical cultures
ccus (11mm
Antimic
ion of Coccinia
activity betw
of Coccinia
Azithromyc
against any b
s, the fractio
m) comparabl
crobial a
cordifolia leave
ween Azithro
a cordifolia
cin. None o
bacteria or fu
on showed th
le to the stan
activity
es
omycin and a
a showed m
of the zone
fungi as show
he best antim
ndard (18mm
Stan
Aqu
aqueous frac
moderate to
of inhibitio
wn in the Fi
microbial ac
m).
ndard sample
ueous fraction
71

ction
low
on of
igure:
ctivity

Chapter Five
CONCLUSION

5.1 Conclusion
The results obtained in this study indicate that the aqueous fraction of the leaves of Coccinia
cordifolia have significant cytotoxic activity. Experimental evaluation showed that the leaves of
this plant also possess antimicrobial and antioxidant properties. Investigations performed on the
aqueous extract proved that the leaves contain phenolic and flavonoid compounds. Since C.
cordifolia leaves exhibited potent cytotoxic activity, so the leaves can be further evaluated for
anticancer, pesticidal and antitumor properties. Detailed investigations can be carried out to
isolate and identify the active compounds present in the leaf extract that are responsible for such
kind of pharmacological activity for development of novel and safe drugs. Further tests can be
performed to evaluate whether the leaves possess some other potent pharmacological activities.


Chapter Six
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IN V

DECLARATION BY THE CANDIDATE

CERTIFICATION BY THE SUPERVISOR

ENDORSEMENT BY THE CHAIRPERSON

1.11 Reproduction and dispersal of Coccinia cordifolia 18

2.2.11 Anti-hepatotoxic activity 33

3.5.2 Total phenolic content 44

3.7.2.2 Test sample of Coccinia cordifolia 53

4.1.2.2 Total phenolic content present in aqueous fraction 63

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