Aerial Parts of Sonchus Asper (L) in Female Albino Rats

ANTIFERTILITY EFFECT OF

AERIAL PARTS OF SONCHUS ASPER (L) IN FEMALE ALBINO RATS

M. Pharm Dissertation Protocol

Submitted to the

RajivGandhiUniversity of Health Sciences,

Bangalore, Karnataka

By

Mr. B.PRASADA

B. Pharm.

Under the guidance of

MR.A. VEERANA GOUD

M.Pharm.

ASST. PROFESSOR.

POST GRADUATE DEPARTMENT OF PHARMACOLOGY

S.C.S.COLLEGE OF PHARMACY

HARAPANAHALLI – 583 131

2008-09

RajivGandhiUniversity of Health Sciences, Bangalore, Karnataka

Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

01 / Name and Address of the Candidate / MR.B.PRASADA.
S/O B.ANJINAPPA; JOSHI STREET
1ST WARD;OPP.PUNDI KOTTRAPPA SIR HOUSE; HARAPANAHALLI (p)
Pin-583131 DAVANAGERE (Dist).
02 / Name of the Institution / T. M. A. E. Society’s
S. C. S. College of Pharmacy,
Harapanahalli – 583 131.
Dsvanagere (Dist)
(Karnataka)
03 / Course of the Study
Branch / M. Pharm. (Pharmacology)
04 / Date of Admission to course / 30/05/2008
05 / Title of the Topic / ANTIFERTILITY EFFECT OF AERIAL PARTS OF SONCHUS ASPER (L)IN 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 – II

6.3. Objective of the Study

/ Enclosure – III
07 /

Materials and Methods

7.1. Source of data /

Enclosure – IV

7.2. Methods of collection of data

/ Enclosure – V
7.3. 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.
7.5. work plan details. / Yes, Registration No: 157 / 1999/ CPCSEA
(Copies enclosed)
08 /

List of References

/ Enclosure – VII
09 /

Signature of the candidate

/ (B.PRASADA)
10 /

Remarks of the Guide

/ The study is highly justifiable and is feasible to work in the institution. This work may throw light on the therapeutic utility of the aerial parts of avenue plant i.e. Sonchus asper (L) Hill.
11 /

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.,
Asst. Professor
Department of Pharmacology,
S.C.S.College of Pharmacy,
Harapanahalli-583 131.
Davanagere (Dist)
(Karnataka)

Dr. S. Ramachandra Setty

M.Pharm, Ph.D.
Professor,
Head of the Department of Pharmacology,
S.C.S.College of Pharmacy,
Harapanahalli-583 131.
Davanagere(Dist)
(Karnataka)
12 /

Remarks of the Principal

12.1Signature / (Dr. S. Ramachandra Setty)
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 effects. 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 V. Ravichandran, B.Suresh, M.N.Sathishkumar, K.Elango, R.Srinivasan12

Number of investigations has been carried out on traditionally claimed anti- fertility plants to validate their efficacy. The plant name sonchus asper (L)hill. Upon literature review, it was found that the plant possesses flavonoids and other poly phenolic compounds3, which are known as nonsteriodal phyto-estrogens and produced anti-fertility in animals2-3. 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 literature survey reveals that the plant SonchusAsper (L) Hill belongs to family Asteraceae or compositae with the synonyms lyon sheart, spiny sow thistle, prickly sow thistle have been reported to contain flavonoids1 (querecetin 3-o-glucoside, lutelolin 7-0-glucoside etc). The phyto-chemical profile of this plant reveals the presence of α-amyrin(4.0), β-amyrin(8.0) Germanicol(6.0), Taraxasterol(17.0), Lupeol 2 (28%) Stigmasterol, Apigenin luteolin3. There are reported that tender leaves of Sonchus Asper (L) Hill contains poly phenols4 (108.3). the plant can be used as an anti-oxidant & for the treatment of diabetes and cancer5. the local folklore practitioner suggested that the plant possess Hepatoprotective, Anti-Fertility, Anti-Inlammatory, Wound Healing properties, Anti-Ulcer activities. The plant has protective effect on cardiac abnormalities in experimental hypercholesterolemia and also has good cardiac protective effects6. At Loralai, the plant is pounded and applied to wounds or boils. In Spain; it is commonly used as an emollient7. The stem have been peeled and eaten raw like celery8. In literature, there are no clear cut report on the organ protective activity and anti-fertility activity of this plant and even phytochemical properties of this plant are incomplete, hence the present study is designed to explore the valuable pharmacological activities of the aerial part of the Plant Sonchus Asper (L) Hill.

ENCLOSURE III

6.3. Objectives of the study:

The main objective of the study is to evaluate anti-fertility activity of aerial parts of Sonchus Asper (L) in a scientific manner using different experimental models.

The present study is therefore undertaken with following objectives.

1.To prepare various extracts of aerial parts of Sonchus Asper (L) by using different solvents.

2.To carry out preliminary phytochemical screening of crude extracts.

3.To determine the acute toxicity studies of the crude extracts in mice.

4.To evaluate extracts for anti-fertility activity in female rats using different experimental models like;

i)Estrogenic activity in immature female rats.

ii) Anti-implantation and early abortifacient activity in female rats.

ENCLOSURE – IV

1. Material & methods:
2. Source of data:

The source of data is from experiments which involves

1)Extraction of shade dried aerial parts of Sonchus Asper(L)Hillwith different solvents. (Petroleum ether, 70% alcohol and distilled water)

2)Evaluation of the extracts for antifertility 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:

a) Chemical studies:

For the present study the fresh plant will be collected from the fields of Harapanahalli and dried under shade. Dried aerial parts 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:

1.Acute toxicity studies.

Fixed dose method (OECD guideline Annexure no.420) of CPCSEA will be followed to carry out acute toxicity studies for the three extracts. The animals used for this will be female albino mice weighing between 20-25gm.

2. 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

I. Evaluation of Anti-fertility Activity.

Estrogenic activity on immature female rats16

Immature female rats of wistar strain 21-23 days old weighing 40-60gms were used. They were divided in to eight groups of six animals each. The various groups were treated as follows:

Group I - Control (Vehicle Tween 80, 1% 5 ml/kg p.o.)

Group II - Standard (Ethinyl estradiol 1g/rat/day in olive oil(s.c.)

Group III & IV -70% ethanolic extract of aerial parts of Sonchus Asper (L) of two selected doses (p. o.)

Group V & VI - Petroleum ether extract of aerial parts of Sonchus Asper (L) of two selected doses (p.o.)

Group VII & VIII - Aqueous extract of aerial parts of Sonchus Asper (L) of two selected doses (p.o.)

All the above treatments were given for 7 days. Vagina and the vaginal smears were examined in all the animals in the treated groups for 7 days of treatment. 24 hrs of last treatment all the animals were sacrificed by decapitation and uteri were dissected out, cleared off the adhesive tissue, blotted on filter paper and weighted quickly on a sensitive balance. The tissues were fixed in Bouin’s fixative for 24 hrs, dehydrated in alcohol and embedded in paraffin. The paraffin blocks were sectioned at 6 and stained with haemotoxylene-eosin solution (H & E Stain) for histological observations17 The diameter of the uterus, thickness of endometrium, and the height of endometrial epithelium were measured in 10 randomly selected sections using a calibrated ocular micrometer.

Statistical analysis:

Results will be expressed as mean  SEM, (n=6). Statistical analysis will be performed with one way analysis of variance (ANOVA) followed by Turkey-Kramer Multiple Comparison test by using Graph Pad Instat software. P value less than 0.05 was considered to be statistically significant. *P<0.05, **<0.01 and ***0.001, will be compared with control and toxicant group as applicable.

II. Anti-implantation and early abortifacient activity in rats.

The method of Khanna and Chowdhary19 will be adopted with the modification for the anti-implantation and early abortifacient activities of Petroleum ether, 70% ethanol and aqueous extracts of aerial parts of Sonchus Asper (L).

Female albino rats (Wistar strain) weighing 150-200gms will be used to assess anti-implantation and early abortifacient activity. All the animals will be maintained under controlled standard animal house condition with access to food and water ad libitum. Vaginal smears from each rat was monitored daily. Only the rats with normal oestrous cycle will be selected for the experiment.

Female rats of proestrus phase will be kept with male rats of proven fertility for mating in a ratio of 2:1. The females will be examined the following morning for evidence of copulation. The animals exhibiting thick clumps of spermatozoa in vaginal smears will be separated from male partner. That day when spermatozoa were detected in the vaginal smear was considered as day one of gestation.

The separated pregnant rats will be divided into seven groups of six rats each.

The various groups will be treated as follows:

Group I - Control (Vehicle Tween 80, 1% 5 ml/kg p.o.)

Group II & III -70% ethanolic extract of aerial parts of Sonchus Asper (L) of two selected doses (p. o.)

Group IV &V - Petroleum ether extract of aerial parts of Sonchus

Asper (L) of two selected doses (p. o.)

Group VI &VII - Aqueous extract of aerial parts of Sonchus Asper (L) of two selected doses (p.o.)

The extracts will be administered orally from first day to seventh day of gestation. The control animals receives only vehicle. On the tenth day laparotomy was carried out under light ether anesthesia in semisterile condition. The uteri were examined to determine the number of implantation sites. The number of corpora lutea in ovaries will be also recorded. The abdomen will be sutured and animals left in cages. The drugs will be administered orally again for 3 days (day 14 to 16). On the eighteenth day laprotomy will be carried out once again under light ether anesthesia for the abortifacient study.

The percentages of anti-implantation and early abortifacient activities will be calculated by using the following formulas.

No. of implantation

% age of anti-implantation activity = 100 – ------x 100

No. of Corpora lutea

No of resorbtions No of resorbtions

% age of abortifacient activity = ------x 100

No of Corpora lutea

Parameters of anti-fertility activity will be:

In Estrogenic activity on immature female rats;

1.Gravimetric changes: uterine weight.

2. Vaginal changes: vaginal opening, proestroeus or estrous conditions number of cornified cells in vaginal smear.

3. Micrometric changes in the uterus:uterotrophic responses including diameter of the rat uterus, thickness of the endometrium and height of the endometrial epithelium.

Anti-implantation and early abortifacient activity

Number of implantation sites and increase in number of resorbtions.

Statistical analysis will be performed with one way analysis of variance (ANOVA) followed by Tukey-Kramer Multiple Comparisons test by using Graph Pad Instat software.

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 albino rats of both sex for antifertility activity.

7.4Has ethical clearance been obtained from your institution in case of 7.3?

The study is cleared from institutional animal ethics committee (IAEC Certificate enclosed).

7.5 Work plan details

Total duration for the completion of proposed research work may be ten months

1.Collection of plant materials including authentication -one month.

2.Preparation of crude extracts & duration of experimentation -Five months

On animals including

3.Literature collection -Two months

4.Dissertation writing and communication of research -Two months

Papers.

\

ENCLOSURE – VII

8.0 List of references:

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1. Ram Rastogi P, Mehrotra. Compendium of Indian Med. Plants; 1993; 3: 600.

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1. The Localfood-Nutraceuticals Consortium .Understanding Local Mediterranean Diets: A Multisciplinary Pharmacological and Ethnobotanical Approach.

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1. Meenakshi Dhanawat, Gajendra Kamal Singh, Arindam Paul.Pharmacology and Potential Therapeutic Uses of Quercetin-A Plant Flavonoid. Indian Journal of Natural Products. 2005; 2(21): 3-11.

1. Varathajan Sudhar, Sekar Ashok Kumar et al. Protective Effect of Lupeol and its Ester on Cardiac Abnormalities in Experimental Hypercholesterolemia. Vascular Pharmacology. 2007; 6(46): 412-418.\

1. Kirtikar KR, Basu BD. Indian Medicinal Plants. 1999; 2: 1441-1445.

1. Moerman D. Native American Ethnobotany Timber Press. Oregon. 1998; 453-459.

1. Maria Mamani –Matusda, Jerome Rambert, Denis Maivy et al. Quercetin Induces Apoptosis of Trypannosoma Brucei Gambiense and Decrease the Proinflammatory Response of Human Macrophages, Antimicrobial Agents And Chemotherapy.

1. Shivakumar SI, Suresh HM, Kumar GS. Heptoprotective Activity of Fruits of Coccina Grandis against CCl4 Induced Hepatotoxicity. Adv.Pharmacol Toxicol. 2006; 7(1): 7-9.

1. Suja SR,Latha PG. Assessment of Hepatoprotective and Free Radical Scavenging Effect of Rhinacanthus Nausta(Linn)Kurtz in Rats Journal of Natural Remadies. 2004; 4(1): 66-72.

1. V. Ravichandran, B.Suresh, M.N.Sathishkumar, Elango K, R.Srinivasan. Antifertility Activity of Hydroalcholic Extract of Ailanthys Excelsa(Roxb): An ethnomedicines Used by Tribals of Nilgiris Region on Tamilnadu. Journal of Ethanopharmacology. 2007; 112: 189-191.

1. 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; 193-197.

1. Jagadish V. Kamath, Rana AC. Anti Fertility Activity of Calotropis Procera Root in Female Rats. Fitoterapia; 2002; 73: 111-115.

1. Mrs .prema veeraraghavan. Expert consultant. CPCSEA, OECD guideline no.420.

1. Padmasali B, Vaidya VP, Vagdevi HM, Satyanarayana ND. Antifertility efficacy of the plant Balanites roxburghii (Balanitaceae) in female rats. Ind J Pharm Sci 2006; 347-50.

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1. Khanna V, Chowdhary RR. Antifertility screening of plants, part-I: investigation of Butea monosperma Lam (Kuntze). Indian J Med Res 1968; 56: 1575-80.