“EVALUATION OF GONADOPROTECTIVE EFFECTS OF CERTAIN MEDICINAL PLANTS ON DRUG-INDUCED REPRODUCTIVE TOXICITY IN RATS”
By
MOHD KHALED B.Pharm
M.Pharm. Dissertation Protocol
Submitted to the
Rajiv Gandhi University of Health Sciences,
Bangalore-560041, Karnataka.
In partial fulfillment
of the requirement for the Degree of
MASTER OF PHARMACY
IN
PHARMACOLOGY
Under the Guidance of
Dr. Benson Mathai K, M.Pharm, PhD.
Professor and Principal
Dept. of Pharmacology and Toxicology
St. John’s Pharmacy College
St. John’s Educational Institutions
Bangalore –560104.
2011 - 13.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1. / Name of the candidate &Address. / MOHD KHALED
I M Pharm
POST GRADUATE STUDENT,
ST. JOHN’S PHARMACY COLLEGE,
#6, 9TH CROSS, 2nd MAIN,
VIJAYANAGAR 2nd STAGE, BANGALORE-560104.
PHONE NO: 080 23300958/23300668.
2. / Name of the Institution. / St. John’s Pharmacy College
No 6, 9th cross, 2nd main, Vijayanagar,
2nd stage (Hampinagar),
Bangalore- 560104
Tel: 91-80-23300958/23300668
Email:
3. / Course of the study
& subject. / MASTER OF PHARMACY (M.PHARM)
Sub:- Pharmacology
4. / Date of admission. / 18th JULY 2011
5. / Title of the Topic:
“Evaluation of gonadoprotective effects of amla, tulsi, ashwagandha and shatavari against drug-induced reproductive toxicity in rats.”
6.
6.1
6.2
6.3 / Brief resume of intended work
Need of the work
Cytotoxic drugs are known to be spermiotoxic and almost all patients undergoing chemotherapy and radiotherapy in the treatment of cancer can cause temporary or permanent gonadal toxicity. The probability of permanent azoospermia is related to the specific agents used and their doses. [1] This is generally characterized by spermatogenic damage, germ cell apoptosis, Leydig cell dysfunction and testicular steroidogenic disorder. The side effects induced by cancer chemotherapeutic drugs on the reproductive organs are multifactorial in nature and with myriad etiological factors contributing towards the pathogenesis.[2]
The most damaging anticancer drugs are alkylating agents (particularly chlorambucil, procarbazine, Cyclophosphamide(CP), melphalan, and busulfan), cisplatin and radiation to the region of the testicles. [1] Cisplatin is known to cause to adverse effects on testes, kidneys, peripheral nerves, and the inner ear. [3,4] Doxorubicin (DXR) has recognized effectiveness against solid and non-solid malignant tumors. However it has been shown to disturb spermatogenesis in a dose-dependent manner. [5,6] CP treatment in patients is associated with oligospermia and azoospermia, as well as biochemical and histological alterations in the testes and epididymis of rats and humans. [7,8]
Amifostine is an efficient cytoprotector against toxicity caused by some chemotherapeutic drugs. However some studies have suggested that amifostine does not confer complete protection towards drug induced toxicity.[9] Cell culture and computer modeling studies does not offer a convincing answer towards prevention of the side effects with polychemical formulations. Therefore these studies are suitable to be performed in animal model of study.
Medicinal plants are known to offer gonadoprotective effects as there are fewer side effects with herbal drugs.[10-12] The present study plans to investigate for the same with amla, tulsi, ashwagandha and shatavari.
Review of Literature:
Many indigenous plants were used by Ayurvedic physicians in India from the time immemorial for the treatment of several ailments. Composite extract of Withania somnifera and Ocimum sanctum was found to be protective against swimming-induced oxidative stress in testis and male secondary sex organs in rats. [13] A.A Al-Qarawi et al reported the effect of extracts of Cynomorium coccineum and Withania somnifera on Gonadotrophins and Ovarian Follicles of immature Wistar rats.[15] Camellia sinensis was known to exert protective effects against doxorubicin-induced spermatogenic disorders. [10] Study of the histological and biochemical changes in the testes after treatment with Azadirechta indica leaves and its pattern of recovery, revealed its possible reversible antiandrogenic property.[15]
Drugs used for the cancer chemotherapy are well known to produce acute toxic side effects in multiple organ systems. CP treatment in cancer patients is associated with oligozoospermia and azoospermia, as well as biochemical and histologic alterations the testes and epididymis of rats and humans.[16] Masao Horimoto et al has reported that Ethylene glycol monoethyl ether (EGEE), Sulfasalazine (SASP), and 2,5-hexandione (2,5-HD) are known to produce changes in sperm production at high exposure levels.[17]
DXR is a very potent chemotherapeutic drug which has been used against a variety of cancers. Despite its efficiency, DXR has been shown to cause death of healthy cells, especially those undergoing rapid proliferation. It has been shown that DXR causes germ cell death and seminal alterations. [18, 19]
Reproductive toxicity studies are increasingly including assessments of sperm parameters including motility, morphology, and counts. These assessments can provide valuable information for the determination of potential reproductive toxicity.[20,17] Howell et al reported that adult male patients treated with CP for more than 4months have diminished sperm counts and absence of spermatogenic cycles in their testicular tissue. [21]
Aim of the study:
To evaluate the gonadoprotective effects of amla, tulsi, ashwagandha and shatavari against drug-induced reproductive toxicity in rats.
Objectives:
The objectives of the present study are as follows :
1. To determine the gonadoprotective effects of amla, tulsi, ashwagandha and shatavari against the Doxorubicin-induced reproductive toxicity by measuring Epididymal sperm count, sperm motility and epididymal weight.
2. To determine gonadoprotective effects of amla, tulsi, ashwagandha and shatavari against the Cisplatin-induced reproductive toxicity by measuring Epididymal sperm count, sperm motility and epididymal weight.
3. To compare the chemoprotective effects of amla, tulsi, ashwagandha and shatavari.
4. To establish the mechanism for possible chemoprotective effects of amla, tulsi, ashwagandha, and shatavari.
7.
8. / Materials & Methods
7.1 Source of data
Whole work is planned to generate data from laboratory studies i.e.; experiments are performed as described in reference, experimental studies in journals and in textbooks available with college, Indian Institute of Science (IISc) library, Rajiv Gandhi University of Health Sciences (RGUHS) digital library (Helinet), websites like
www.sciencedirect.com,
www.ncbi.nlm.nih.gov/pubmed,
www.google.com,
www.ijp-online.com,
were used to obtain related information regarding this research protocol.
7.2 Chemicals – All chemicals of standard quality and grade will be procured.
7.3 Methods of collection of data
The experiments will be conducted using laboratory animals and the data will be collected by analyzing appropriate parameters. All experiments are planned in accordance with Committee for the purpose of control and supervision of experiments on animals (CPCSEA), Chennai, India. The present study will be carried out after approval from Institutional Animal Ethical committee (IAEC).
(a) Experimental animals
Ø Species Wistar rats
Ø Age 2-3months
Ø Weight 150-200gm
Ø Gender Male
Animals will be housed in polypropylene cages on clean paddy husk bedding till the completion of study. Animals will be maintained under controlled temperature at 25ºC ± 2ºC with 12 hr light/dark cycle with food and water provided ad libitum. Animals which do not comply with above criteria and also which are found to be diseased will be excluded from the study.
(b) Administration of drugs
Doxorubicin (15mg/kg b.wt) i.v.[22]
Selenium (100mg/kg b.wt) oral[23]
Cisplatin (single dose of 7.5mg/kg b.wt) i.v.[24]
Amla, Tulsi, Ashwagandha, Shatavari (1000mg/kg b.wt) oral[25]
(c) Animal groups
Experiment 1: Gonadoprotective effects of Indian Medicinal plants against the Doxorubicin-induced reproductive toxicity:[22]
Group 1: Normal rats / 06
Group 2: Doxorubicin only (15 mg/kg b. wt) / 06
Group 3: Selenium (100 mg/kg b. wt.) + Doxorubicin only (15 mg/kg b. wt) / 06
Group 4: Ashwagandha (1000 mg/kg b.wt) + Doxorubicin only (15 mg/kg b. wt) / 06
Group 5: Amla (1000 mg/kg b.wt) + Doxorubicin only (15 mg/kg b. wt) / 06
Group 6: Tulsi (1000 mg/kg b.wt) + Doxorubicin only (15 mg/kg b. wt) / 06
Group 7: Shatavari (1000 mg/kg b.wt) + Doxorubicin only (15 mg/kg b. wt) / 06
Total animals required / 42
Experiment 2: Gonadoprotective effects of Indian Medicinal plants against cisplatin-induced reproductive toxicity:[23]
Group 1: Normal rats / 06
Group 2: Cisplatin a single dose of 7.5mg/kg / 06
Group 3: Selenium (100 mg/kg b.wt) + Cisplatin a single dose of 7.5mg/kg / 06
Group 4: Ashwagandha (1000 mg/kg b.wt) + Cisplatin a single dose of 7.5mg/kg / 06
Group 5: Amla (1000 mg/kg b.wt) + Cisplatin a single dose of 7.5mg/kg / 06
Group 6: Tulsi (1000 mg/kg b.wt) + Cisplatin a single dose of 7.5mg/kg / 06
Group 7: Shatavari (1000 mg/kg b.wt) + Cisplatin a single dose of 7.5mg/kg / 06
Total animals required / 42
Total animals for two models (42x2) / 84
(d) Methodology
The animals will be allocated to seven groups (n=6, respectively) as specified above. A single dose of the cytotoxic drug will be administered (doses as specified above).[23,24] Twelve hours after the administration of the cytotoxic drugs the animals will be orally fed with the medicinal plant (amla, tulsi, ashwagandha and shatavari) or selenium (positive control) daily for the next 42 days. At six weeks after initiation of the experiment, the animals will be sacrificed under halothane administration.
The bilateral testes blood and epididymis of each animal will be collected and following parameters are assessed:[21,18]
· Epididymal sperm count
· Sperm motility
· Epididymal weight
§ Testes and epididymis will be weighed and the relative organ weight will be calculated.
§ Epididymal sperm count, motility and abnormality will be assessed immediately thereafter.
§ Epididymal sperm will be collected by cutting the one epididymis into small pieces in 5mL of physiological saline at 32ºC. A sperm viability test will be done using the method described by World Health Organization (WHO). Epididymal sperm count and motility will be evaluated.
§ Epididymal weights also vary because of individual variation of weights and ages among tested rats. Therefore, sperm numbers will be calculated relative to epididymal weights (sperm per gram).
Statistical analysis:-
The data obtained from experimentation will be subjected to one way Analysis Of Variance (ANOVA) followed by suitable post hoc test.
Total No. of animals required:
o No of the rats for Doxorubicin induced Reproductive Toxicity = 42
(7gps × 6 animals)
o No of rats for Cisplatin induced Reproductive Toxicity =42
(7gps × 6 animals)
o Total No of rats ( 42 + 42) = 84
7.4 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so. Please describe briefly?
Yes, the study requires investigation on male wistar rats.
7.5 Has ethical clearance been obtained from your institution in case of 7.3?
Institutional Animals Ethical Committee (IAEC) meeting will be held in our Instituition in February and the approval letter will be sent to university after obtaining the approval.
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8. Kaur F,SanghaGK,Bilaspuri GS. Cyclophosphamide-inducedstructuraland biochemical changes in testis and epididymidis of rats. Indian J Exp Biol 1997;35:771-5.
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16. Kirkland RT, Bongiovanni AM, Cornfield D, Mc Cormic JB, Parks JS, Tenore A. Gonodotropin responses to leutinizing releasing factor in boys treated with cyclophosphamide for nephritic syndrome. J Pediatr 1976;89:941-4.
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