ANTIFERTILITY EFFECT OF AERIAL PART OF
CROTALARIA VERRUCOSA
IN FEMALE ALBINO RAT
M. Pharm. Dissertation Protocol
Submitted to the
RajivGandhiUniversity of Health Sciences,
Bangaluru, Karnataka
by
MR.SUBODH KUMAR SINGH
B. Pharm.
Under the guidance of
MR. A. VEERANA GOUD
M.Pharm.
PROFESSOR.
POST GRADUATE DEPARTMENT OF PHARMACOLOGY
S.C.S.COLLEGE OF PHARMACY
HARAPANAHALLI – 583 131
2010-11
RajivGandhiUniversity of Health Sciences, Karnataka, Bangaluru
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
01 / Name and Address of the Candidate / SUBODH KUMAR SINGH S/O LATESHRI VASUDEV SINGH
H.NO.37,MANSAROVER COLLONY
PHASE-2(QUARSI)RAMGHAT ROAD
ALIGARH
02 / Name of the Institution / T. M. A. E. Society’s
S. C. S. College of Pharmacy,
Harapanahalli – 583 131
(Karnataka)
03 / Course of the Study
Branch / M. Pharm. (Pharmacology)
04 / Date of Admission to course / 15/07/2010
05 / Title of the Topic / ANTIFERTILITY EFFECT OF AERIAL PART OF CROTALARIA VERRUCOSAIN FEMALE ALBINO RATS
06 /
Brief resume of the intended work
6.1. Need for the Study /Enclosure – I
6.2. Review of the Literature / Enclosure – II6.3. Objective of the Study
/ Enclosure – III07 /
Materials and Methods
7.1. Source of data /Enclosure – IV
7.2. Methods of collection of data
/ Enclosure – V7.3. Does the study require any
investigations on animals?
If yes give details / Enclosure – VI
7.4. Has ethical clearance been
obtained form your institution
in case of 7.3. / Yes, Registration No: 157 / 1999/ CPCSEA
(Copies enclosed)
08 /
List of References
/ Enclosure – VII09 /
Signature of the candidate
/ Subodh kumar singh10 /
Remarks of the Guide
/ The present research work is original and not published in any of the journals. This work can be carried out in our Pharmacology laboratory11 /
Name and Designation of
(In Block Letters)11.1. Guide
11.2.Signature
11.3.Co-Guide (if any)
11.4.Signature
11.5. Head of the Department
11.6. Signature / Mr. A.VEERANA GOUD
M. Pharm.,
Professor
Department of Pharmacology,
S.C.S.College of Pharmacy,
Harapanahalli-583 131.
Karnataka
Mr.A. VEERANA GOUD
M.Pharm,
Professor And
Head of the P.G.Department of Pharmacology,
S.C.S.College of Pharmacy,
Harapanahalli-583 131.
Karnataka
12 /
Remarks of the Principal
12.1Signature / The present study is permitted to perform in the Pharmacology laboratory of our institution and the study protocol has been approved by IAEC.Principal
ENCLOSURE-I
06. Brief resume of Intended Work:
6.1 Need for the study.
The quest for the oral contraceptive agent that can control fertility is as old as recorded history. Although a wide variety of synthetic contraceptive agents are available, these cannot be used continuously due to their severe side effects1. Hence people are looking back to age old tradition of using herbal medicines, which have minimum side effects. So many plant preparations are reported to be used for fertility regulating properties in the ancient Indian literature. Research on Indian plants with antifertility activity has been exhaustively reviewed recently by Mali RG, Hundiwale JC, Gavit RS and Patil DA. et al2.
Number of investigations has been carried out on traditionally claimed anti- fertility plants to validate their efficacy. The plant nameCrotalaria verrucosa. Upon literature review, it was found that the plant possesses flavonoids3(β-sitosterol) and other alkaloids4,which are known as non-steriodal phyto-estrogens and produced anti-fertility in animals5. But there are no supportive scientific evidences to justify the claim. Keeping these facts in view the present study is planned. Therefore the study is justifiable.
ENCLOSURE-II
6.2. Review of Literature
The plant Crotalaria verrucosa (family: fabaceae) also knows as blue rattle pod is a branched annual herbs,50-100cm height,branches are acute and angled ovate to triangular leaves are 5-15cm long, leaf stalk 2-4mm,flower occur in 10-12 flowered racemes at the end of branches or laterally.
Flower tube is about 2cm bluish purple and white standards petal 1.5cm,ovate,veined with dark purple lines.
Pods are silky hairy, 2-4cm long, Flowering and fruiting occurs from July to February6.
Chemical constituents:
Its flowers contains flavonol glycoside-kaempferol-7,4-di-o-l-rhamnopyranoside and its leaves has a new necic acid lactone -2-methyl-3-(2-oxo[5H]-5-hydroxymethyl-5-methyl-furan-3-yl)propanoic acid7.
Its stem consists of taraxerol,β-sitosterol8, linoleic, palmitic, stearic, oleic, linolenic, arachidic, myristic and ricinoleic acids isolated from fixed oil of stems.
Its seeds consists of an alkaloids-crotalaburnine, secopyrrolizidine like crotaverrine and o-acetyl-crotaverrine, flavonoids-isovitexin, vitexin-4-xyloside, vitexin-o-glycoside and apigenin-o-glycoside-along with β-sitosterol9.
Medicinal uses:
The leaf decoction is given orally to cure jaundice10, present alkaloids used for skin infection and hepato toxic, it also used for cancer, stomach pain, hepaticpain and heart complaints (Ayurveda).
Review of literature till date regarding Crotalaria verrucosa was carried out by referring chemical abstracts,biological abstract,medicinal and aromatic plant abstracts and other scientific journals. The different part of the crotalaria verrucosa like flower, seeds, stem etc., has been possess antimicrobial, hepatotoxic11. However the present study is aimed to verify the claim of plant as antifertility activity of Crotalaria verrucosa.
ENCLOSURE III
6.3. Objectives of the study:
01.To prepare various extracts of crotalaria verrucosa (petroleum ether extract, 70% alcoholic extract, aqueous extract.) and subject them to preliminary phytochemical studies to detect the presence of type of phytochemical constituents in the aerial part of the plant.
02. To determine the acute toxicity studies for the crude extracts.
03. To evaluate the extracts for antifertility activity in female rats.
ENCLOSURE – IV
- Material & methods:
- Source of data:
The source of data are from experiments which involves.
1)Extraction of shade dried aerial part of the cotalaria verrucosawith different solvents.(petroleum ether , 70% alcoholic, aqueous ).
2)Evaluation of the extracts for antifertillity activity using different experimental animal models.
The data from the literature is also used to generate and interpret the experimentally generated data.
ENCLOSURE – V
7.2Method of collection of data: (Including sampling procedure if any)
a)Chemical studies:
For the present study the fresh plant will be collected from the fields of Harapanahalli and dried under shade. Dried aerial part of the plant will be coarsely powdered and extracted by soxhlation to get sufficient crude extracts. The extracts obtained will be subjected to preliminary phytochemical tests.
b)Pharmacological studies:
- Acute toxicity studies.
Fixed dose method (OECD guideline no. 420) of CPCSEA will be followed to carry out acute toxicity studies for the three extracts12. The animals used for this will be female albino mice weighing between 20-25 gm.
- Evaluation of the extracts for antifertility activity by adopting the screening methods reported in the literature. The experimental models (Screening methods) used in the study are
- Study of estrogenic activity13, 14.
1)Status of Animals: In this model healthy immature female rats of 21-23 days old weighing between 30-50 gm will be used.
2)For the above model animals will be divided into experimental and control groups of six animals each.
3)The uterine weight and histological data of tissue will be employed in this model to get the data.
4)No re-use of animals in this model.
- Anti implantation and Abortifaicent activity13,15.
1)Status of Animals : In this model healthy wistar strain of female rats weighing 150-200 gm will be used.
2)For the above model animals will be divided in to experimental and
control groups of six animals each.
3)Method of Khanna and Chowdhary will be adopted with suitable modification to study, % Anti-implantation, % Early abortifacient and % Antifertility activities.
4)No re-use of animals in this model.
Number of animals on which proposed research is planned may be
Rats-90
Mice-30
The data thus obtained from the above antifertility studies will be subjected to ANOVA.
c) Work plan details
Total duration for the completion of proposed research work may be ten months
1. Collection of plant material. - One month.
2. Duration of experimentation on animals including - Five months.
preparation of crude extracts.
3. Literature collection. - Two months.
4. Dissertation writing and communication of research - Two months.
papers
ENCLOSURE – VI
7.3Does the study require any investigation or interventions to be conducted on patients or other humans or animals? If so, please describe briefly.
The proposed study requires investigation on albino mice for acute toxicity study and female albino rats for antifertility activity.
7.4Has ethical clearance been obtained from your institution in case of 7.3?
The study is Approved from institutional animal ethics committee (IAEC Certificate enclosed).
ENCLOSURE – VII
8.0 List of references:
1.Perry B Molinoff, Reymond WR. The pharmacological basis of therapeutics.10Th edition, USA. Mc Graw-hill companies; 2001; p1597-1634.
2.Mali RG, Hundiwale J C, Gavit RS, Patil DA, PatilKS. Herbal abortifacients used in north maharashtra. Natural Product Radiance 2006; 5(4): p315-18.
3.Ram Rastogi P,Mehrotra B.N.Compendium Of Indian Med.Plants; 1991; 2: p637.
4.Arseculeratne. SN, Gunatilaka.AAL,and Panabokke. RG,
J.Eethanopharmacology 1999; (4) :p159-77.
5.William charles evan. Pharmacognosy. Sydney : Harcourt Publishers limited; 2002; p247.
6.Madhava Chetty K, Sivaji K, Julasi Rao K. Flowering Plants of Chittor District Andhra Pradesh (India); 2008; p87.
7.Rastogi P,Mehrotra B.N. Compendium Of Indian Med.Plants; 1995; 4:p233-34.
8.Rastogi P,Mehrotra B.N. Compendium Of Indian Med.Plants; 1998; 5: p259.
9.Rastogi P,Mehrotra B.N. Compendium Of Indian Med.Plants; 1990; 1:p131.
10.Senthilkumar. M, Gurumoorthi P, and Janardhanan K. Natural Product Radiance; 2006; 5(5): p385.
11. Kirtikar KR, Basu BD. Indian Medicinal Plants; 1999; 1: p694-95.
12.Mrs .prema veeraraghavan. Expert consultant. CPCSEA, OECD guideline no.420.
13. RavichandranV, Suresh B, Sathishkumar M.N, Elango K, Srinivasan R.
Antifertility Activity of Hydroalcholic Extract of Ailanthys Excelsa(Roxb): An
ethnomedicines Used by Tribals of Nilgiris Region on Tamilnadu. Journal of
Ethanopharmacology 2007; 112: p189-91.
14.Shivayogi P. Hiremath, Shrishailappa Badami, Shanmukhaswamy K.Hunasagatta,
Sarawathi B.Patil. Anti Fertility and Hormonal Properties of Flavones of Striga
Orobanchioides: European Journal of Pharmacology. 2000: 391; p193-97.
15.Jagadish V. Kamath, Rana AC. Anti Fertility Activity of Calotropis Procera Root
in Female Rats. Fitoterapia; 2002; 73: p111-15.