“SCREENING OF MARINE ALGAE (Sargassum polycystum, Acanthophora
spicifera, Gracilaria edulis, Chondrococcus hornemannii)
FOR THE ANTIOVULATORY AND POLYCYSTIC OVARY SYNDROME”
Submitted to
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE
KARNATAKA
In partial fulfillmentof the requirement for the Degree of
Master of Pharmacy in Pharmacology
Under the Guidance of
Dr. RAJU KONERI.
DEAN&HOD DEPT. OF PHARMACOLOGY
BY
OM PRAKASH KUMAR KARN
IstM.PHARM.
Department of Pharmacology
Karnataka College of Pharmacy, Bangalore-64
2013-15
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
1
KARNATAKA, BANGALORE.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
Name of the candidate and address
OM PRAKASH KUMAR KARN
S/o MR. AKHILESH LAL KARN
CHHAPKAIYA,
WARD NO.-1
BIRGUNJ
PARSA(DIST.)
NEPAL
2.
Name of the Institution
Karnataka College of Pharmacy,
THIRUMENAHALLI,
YELAHANKA HOBLI,
JAKKUR POST
BENGALURU-560064
KARNATAKA, INDIA
3.
Course of study and subject
M.Pharm-Pharmacology
4.
Date of the admission
13/09/2013
5.
Titleof the topic:
“SCREENING OF MARINE ALGAE ( Sargassum polycystum, Acanthophora spicifera,
Gracilaria edulis, Chondrococcus hornemannii)FOR THE ANTIOVULATORY AND
POLYCYSTIC OVARY SYNDROME”
2
S6. BRIEF RESUME OF THE INTENDED WORK
6.1 INTRODUCTION AND NEED FOR THE STUDY:
Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders among females of
reproductive ages. PCOS is a complex, heterogeneous disorder of uncertain etiology, but there is strong
evidence that it can, to a large degree, be classified as a genetic disease
[1-3]
. The name of the condition
comes from the appearance of the ovaries in most, but not all, women with the disorder - enlarged and
containing numerous small cysts located along the outer edge of each ovary (polycystic appearance).
PCOS produces symptoms in approximately 5% to 10% of women of reproductive age (12–45 years old
age). It is thought to be one of the leading causes of female subfertility[4-6] and the most frequent
endocrine problem in women of reproductive age [7].
The principal features are
(1) anovulation, resulting in irregular menstruation, amenorrhea, ovulation-related infertility;
(2) excessive amounts or effects of androgenic (masculinizing) hormones, resulting in acne
andhirsutism; and
(3) insulin resistance, often associated with obesity, Type 2 diabetes, and high cholesterol
levels[8-10].
Finding that the ovaries appear polycystic on ultrasound is common, but not an absolute requirement in
all definitions of the disorder. The symptoms and severity of the syndrome vary greatly among affected
women. Women with PCOS are at a higher risk for obesity, diabetes, high blood pressure, and heart
disease.
PCOS is a heterogeneous disorder of uncertain etiology[1, 2, 8-10].However there is strong evidence that it
is a genetic disease. PCOS may be associated with chronic inflammation,[8-10] with several investigators
correlating inflammatory mediators with anovulation and other PCOS symptoms[11,12]. Similarly, there
seems to be a relation between PCOS and increased level of oxidative stress[13].
The overall aim of the research is to take extract of marine algae species in pure form ad establish their
role in ameliorating the manifestation of PCOS.
Recent work done employing DPPH photometric assay has shown strong anti-oxidant property of
Chondrococcus hornemannii[14]. Like that hypolipidaemic effect of Acanthophora spicifera (red alga)
on albino rats have been seen[15], also work done in Sargassum polycystumwhich shows significant
reduce in oxidative stress induced by Acetaminophen in rats [16].
3
With these preliminary findings the investigators intend to investigate its effect on polycystic ovarian
syndrome, with specific emphasis on few of its etiological targets mentioned in the objectives.
6.2 Plant Material:
Fresh materials of marine algae species will be collected from intertidal regions of Leepuram,
Kanyakumarai, South East Coast of Tamilnadu, India, and authenticated by the Central Marine fisheries
Institute Mandapam Tamilnadu India. The freshly collected samples will be thoroughly cleaned using
sterilised sea water to remove the sand and salt contents. The sample will be also gently brushed with
soft brush to remove attached epiphytes, other marine organisms and debris. Dried seaweeds will be
powdered and soaked in different solvents (chloroform, methanol and water) (1:20, w/v) overnight,
filtered and concentrated to crude extract[14].
6.3- REVIEW OFLITERATURE:
Seaweeds are a group of non-flowering marine plants commonly referred to as marine macroalgae.
Taxonomically; they are classified into Rhodophyta (Red algae) phaeophyta (brown algae) and
chlorophyta (green algae) depending on their pigment and or chemical composition. Seaweeds contain
various inorganic and organic substances which can benefit mankind. various compound with biological
activities have been isolated and they possess pharmacological activities such as antibiotic,
antioxidant,antiproliferative,antitumoral,anticomplementary,antiflamatory,antibacterial,antifungal,antivi
ral,antihelmintic,antiprotozoan,antipeptic,hypolipidemic,antiadhesive and antifouling actives.
1)
Fresh materials of Chondrococcus hornemannii will be collected from intertidal regions of
Leepuram, Kanyakumarai, South East Coast of Tamilnadu, India, and authenticated by the Department
of Botany, Bangalore University, Bangalore, India. Chondrococcus hornemannii is among the most
widespread red seaweeds. Chondrococcus hornemannii forms flat and much branched purplish-red
fronds attaining a height of 10-15cm. It is mostly found in rocky habitats exposed to frequent wave
splash at low tide[17].
The following pharmacological studies have been done:

Chondrococcus hornemannii are studied for its anti-oxidant activity [14].
4

Antitumor activity of Chondrococcus hornemanniare studied on Dalton’s lymphomaascites in
mice [18].

Bioactive compounds obtained from the seaweed Chondrococcus hornemanni has shown
antibacterial activity of on ichthyopathogenic bacteria affecting marine ornamental fish [19].

Antihepatotoxic potential of marine red algae Chondrococcus hornemanni are seen[20].
2) Gracilaria edulis, a major Indian agarophyte and an edible marine alga is commonly found in Indian
coast Tamil Nadu.G. edulis is represented by different morphological forms in the Mandapam region.
The morphological variation of selected characters has been analyzed and quantified. Characters
includes basal branch constriction, branch endig, branch attenuation, stoutness of the thallus, angle of
divergence of branches, branching index, size of medullary, subcortical and cortical cells, and gradation
of cell size from cortex to medulla. All the characters showed continuous variation; however, branching
index and branching attenuation showed significant negative correlations between them[21].
Gracilaria edulis extract induces apoptosis and inhibits tumor in Ehrlich Ascites tumor cells in vivo.
Alcoholic extract of the algae was prepared as described earlier and the presence of biologically active
components including alkaloids, flavonoids, sterols, terpenoids, proteins, saponins, phenols, coumarins,
tannins and glycosides was documented[22-27].
3) Sargassum polycystum (sargassaceae) is a marine species found in the large communities on rocks in
lower intertidal zones in relatively calm water. It is collected from Rameswaram Coast, Tamil Nadu. The
erect branches have numerous spines on the stem. Leaves are lanceolate to oblong with serratious, and
vesicles are spherical. The plants are 1-2m high.
The following pharmacological studies are done:

Antioxidant property of S. polycystum has been studied[28].

Investigation has shown hypolipidemic influence of Sargassum subrepandum on hyperlipidemic
rats[29].

Antihepatotoxic potential of Sargassum polycystum(Phaeophyceae)on antioxidant defense status
has been seen in Dgalactosamine-induced hepatitis in rats[30].

Anti-obesity property of the Sargassum polycystum using an in vivo animal model has been
studied [31].
5
4)Acanthophora spicifera is a species of marine red algae in the family Rhodomelaceae. Acanthophora
spicifera is one of the most common nonindigenous algal species collected from Rameswaram Coast,
Tamil Nadu, India.
A. spicifera has a large, irregularly shaped holdfast for attachment to hard bottoms. From the holdfast,
erect fronds begin to branch out. The main branches have short, determinate branchlets that are
irregularly shaped and spinose. Branchlets are hook-like, brittle and fragment easily under heavy wave
action. Color is highly variable, and can be shades of red, purple, or brown (Littler and Littler, 1989). A.
spicifera grows upright to approximately 25 cm.
The following pharmacological studies are done:

Antibacterial Potential of Hexane Extract from Red Algae, Acanthophora spicifera has been
studied [32].

Hypolipidaemic effect of Acanthophora spicifera (red alga) on albino rats have been seen [15].

Anti-oxidant property of Acanthophora spicifera has been studied [33].
6.4 OBJECTIVE OF THE STUDY:
The objectives of the investigation are as follows:
1.
To prepare methanolic extracts of the marine algae species.
2.
To study the effect of marine algae species on Estradiol Valerate (EV) and Letrozole induced
PCOS.
3.
To study the toxicity profile of the compound(s) as per OPPTS guideline(no. 870.1100-Acute
Oral Toxicity: Up- and Down Procedure).
4.
To study the antiovulatory activity of marine algae species.
7.MATERIALS AND METHODS
7.1SOURCE OF DATA:
Whole work is planned to generate data from laboratory studies (experiments performed as described in
references), experimental studies in journals and in text books available with college and other libraries.
Literature is obtained from various web sites in the internet.
6
7.2METHODOLOGY:
7.2.1 Extraction and Isolation:
The marine algae species will be isolated, chopped into small pieces and dried under shade, powdered.
This powder will used for the preparation of Methanolic extract. A 95% w/v methanolic extract will
prepared by soxhlet extraction method. The dried powdered of marine algae (200g) will be extracted
with 95%v/v methanol for 21hrs. Using soxhlet extractor. The combined extracts will concentrated.
7.2.2 EXPERIMENTAL DESIGN FOR PCOS :
7.2.2.1 Acute Oral Toxicity Study:
The acute oral toxicity study will be performed according to the OPPTS (Office of prevention, pesticides
and toxic substance) guidelines following the up and down procedure [34].
7.2.2.2 Estradiol valerate (EV) induced PCOS models [35,36]:
Induction of PCO:
Female wistar rats with regular estrous cycle will be selected and divided into six groups with six
animals in each group. Vaginal smears of the rats will be taken for 18 days and those rats which
exhibited three regular cycle will be selected. PCO will be induced in rats by a single dose of
intramuscular injection of EV at a dose of 2mg.
Group classification: 60 healthy rats will be divided into 10 groups,
Group 1 (N=6): vehicle control
Group 2 (N=6): EV control
EV 2mg/kg (i.m.).
Group 3 (N=6):
EV 2mg/kg( i.m.) + Extract of Sargassum polycystum low dose orally daily.
Group 4 (N=6):
EV 2mg/kg (i.m). + Extract of Sargassum polycystum high dose orally daily.
Group 5 (N=6):
EV 2mg/kg ( i.m.) + Extract of Acanthophora spicifera low dose orally daily.
7
Group 6 (N=6):
EV 2mg/kg (i.m.) + Extract of Acanthophora spicifera high dose orally daily.
Group 7 (N=6):
EV 2mg/kg( i.m.)+ Extract of Gracilaria edulis low dose orally daily.
Group 8 (N=6):
EV 2mg/kg( i.m.)+ Extract of Gracilaria edulis high dose orally daily.
Group 9(N=6):
EV 2mg/kg( i.m.) + Extract of Chondrococcus hornemannii low dose orally daily.
Group 10(N=6):
EV 2mg/kg( i.m.) + Extract of Chondrococcus hornemannii high dose orally daily.
On the 61st day, 24 hours after last treatment, the animals from each group will be anesthetized and
sacrificed. The blood will be collected from retro-orbital plexus into clean and dry centrifuge tubes. The
sera will aspirated with Pasteur pipettes into clean,dry,sample bottles and will be used for the estimation
of bio-chemical parameters like serum glucose, triglycerides, total cholesterol using kits (Prism
Diagnostics PVT.LTD.). Immediately after blood collection, animals will be sacrificed. Ovaries will be
dissected out, freed from extra deposition, and will be weighed on a sensitive balance. Ovaries will be
processed for the estimation of catalase, superoxide dismutase and lipid peroxidation.
7.2.2.3 Letrozole Induced PCOS Model [37]:
Induction of PCOS:
Female wistar rats with regular estrous cycle will be selected and will be divided into 10 groups with six
animals in each group. Vaginal smears of rats will be taken for 18 days ad those rats which exhibited 3
regular cycle will be selected. PCO will be induced in the rats with Letrozole orally at a dose of
6mg/100gm body weight.
Group classification: 60 healthy rats will be divided into 10 groups,
Group 1 (N=6): vehicle control
Animals will receive 1 percent CMC orally.
Group 2 (N=6): LET control
LET 0.5mg/kg body weight in 1% CMC orally for 21 days.
8
Group 3 (N=6):
LET 0.5mg/kg body weight in 1% CMC +Extract of Sargassum polycystum low dose orally daily.
Group 4 (N=6):
LET 0.5mg/kg body weight in 1% CMC +Extract of Sargassum polycystum high dose orally daily.
Group 5 (N=6):
LET 0.5mg/kg body weight in 1% CMC + Extract of Acanthophora spicifera low dose orally daily.
Group 6 (N=6):
LET 0.5mg/kg body weight in 1% CMC + Extract of Acanthophora spicifera high dose orally daily.
Group 7 (N=6):
LET 0.5mg/kg body weight in 1% CMC + Extract of Gracilaria edulis low dose orally daily.
Group 8 (N=6):
LET 0.5mg/kg body weight in 1% CMC + Extract of Gracilaria edulis high dose orally daily.
Group 9 (N=6):
LET 0.5mg/kg body weight in 1% CMC + Extract of Chondrococcus hornemannii low dose orally
daily.
Group 10 (N=6):
LET 0.5mg/kg body weight in 1% CMC + Extract of Chondrococcus hornemannii high dose orally
daily.
On the 22nd day, 24 hours after last treatment, the animals from each group will be anesthetized and
sacrificed. The blood will be collected from retro-orbital plexus into clean and dry centrifuge tubes. The
sera will aspirated with Pasteur pipettes into clean, dry, sample bottles and will be used for the
estimation of bio-chemical parameters like serum glucose, triglycerides, total cholesterol using kits
(Prism Diagnostics PVT.LTD.). Immediately after blood collection, animals will be sacrificed. Ovaries
will be dissected out, freed from extra deposition, and will be weighed on a sensitive balance. Ovaries
will be processed for the estimation of catalase, superoxide dismutase and lipid peroxidation.
9
7.2.3ANTIOVULATORY ACTIVITY [38, 39, 40, 41, 42]:
Vaginal smears from each rat will be examined daily for 18 days, and those rats exhibited three regular
cycles (4-5 days)
[43]
will be selected for the study. Then, rats will be divided into nine groups of six
animals each.
The treatment will started when the animals is in estrous phase.
Group 1: -Received vehicle only (1%Tween 80, p.o. daily) and served as control.
Group 2:- Received methanolic extract of Sargassum polycystumat 250mg/kg body weight.(p.o. daily)
for 18 days.
Group
3:-
Received
methanolic
extract
of
Sargassum
polycystumat
500
mg/kg
body
weight.(p.o.daily)for 18 days.
Group 4:- Received methanolic extract of Acanthophora spicifera at 250 mg/kg body
weight.(p.o.daily)for 18 days.
Group 5:- Received methanolic extract of Acanthpphora spicifera at 500 mg/kg body
weight.(p.o.daily)for 18 days.
Group 6:- Received methanolic extract of Gracilaria edulis at 250 mg/kg body weight.(p.o.daily)for 18
days.
Group 7:- Received methanolic extract of Gracilaria edulis at 500 mg/kg body weight.(p.o.daily)for 18
days.
Group 8:- Received methanolic extract of Chondrococcus hornemannii at 250 mg/kg body
weight.(p.o.daily)for 18 days.
Group 9:- Received methanolic extract of Condrococcus hornemannii at 500 mg/kg body
weight.(p.o.daily)for 18 days.
Vaginal smear from each animals will be observed every morning between 9-10 A.M. and stage of
oestrous cycle will be recorded after 250mg and 500mg/kg body weight of methanolic extract of above
marine algae species’s administration and Body weight will be recorded every day.
On the 19th day, 24 hours after the last treatment, the animals from each group will be sacrificed. Blood
will be collected from retro-orbital plexus into clean and dry centrifuge tubes. The serum will be later
aspirated with Pasteur pipettes into clean, dry, sample bottles and will be then used for the hormonal
10
assay. Serum FSH, LH, prolactin, estradiol,progesterone levels will be measured[44] by Fully automated
Chemi Luminescence immunoassay- C.L.I.A.
Immediately after blood collection, Animals will besacrificed. Ovary and uterus will be dissected out,
freed from extra deposition, and weighed on a sensitive balance One Ovary and one horn of the Uterus
from each animal will be fixed in 10% formalin buffer for histopathological studies[45].
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41. Al-Said MS, Al-Khamis KI, Islam MW, Parmar NS, Tariq M, Ageel AM, Post-coital antifertility activity of
the seeds of Coriandrumsativum in rats. Journal of Ethnopharmacology 1987, 21(2): 165-173.
13
42. Hafez ES, Reproduction in breeding techniques for laboratory animals. Philadelphia: Lea and
febiger1970;2: 23-93.
43. Tortora G J, Sandra Reynolds Grabowski. Development and inheritance: Principles of Anatomy and
physiology. 10th ed. N Y, USA: Von Hoffmann Press.2003: 1011-1061.
44. Yakubu, Effect of Cnidoscolousaconitifolius (Miller) I.M.Johnston leaf extract on reproductive hormones
of female rats. Iranian J Reproductive Med 2003, 6(3):149-55.
45. Saraswati S D. Evaluation of antifertility activity of Momardica cymbalaria roots in female
rats(dissertation).visveswarapura institute of pharmaceutical sciences:Rajeev Gandhi
University,Bangalore:2004.
14
9.0
SIGNATURE OF THE CANDIDATE:
(OM PRAKASH KUMAR KARN)
10.
REMARKS OF THE GUIDE
The topic selected for dissertation is satisfactory.
Adequate equipment and chemicals are available to
carry out the project work.
11.
Dr. RAJU KONERI
11.1 NAME AND DESIGNATION
DEAN & HOD DEPT. OF PHARMACOLOGY,
OF GUIDE
KCP, BANGALORE-560 064.
11.2 SIGNATURE OF GUIDE:
(Dr. RAJU KONERI)
11.3 CO-GUIDE (IF ANY)
Nil
11.4 SIGNATUREOFCO-GUIDE
Nil
Dr. RAJU KONERI
11.5 HEAD OF THE DEPARTMENT
HOD DEPT. OF PHARMACOLOGY,
KCP, BANGALORE-560 064.
11.6SIGNATURE OF HOD
(Dr. RAJU KONERI)
12.
12.1 REMARKS OF THE DIRECTOR:
All required facilities will be provided to carry out the
dissertation work under the supervision of the guide.
12.2 SIGNATURE
Dr. K. RAMESH
DIRECTOR,
KCP, BANGALORE-560 064.
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