INTRODUCTION:

Reproduction is an important phenomenon of all living organism and is the vital process that enables a species to represent itself in the species to represent itself in the species is to reproduce at a particular season (breeding season) and such animals are called seasonal breeders. This process in a way restricts the growth of population of such species. But in a few animals i.e. humans, breeding is not confined to any part of the year. This provides a favourable environment for an un-interrupted conception throughout the year resulting in increased population.¹

The population in India has been growing rapidly. It is doubled from 344 million in 1947 to 685 million in 1980 and 1027 million in 2001.

There is increased demand on food and other articles. There cannot be effective measures to check on crimes, diseases, destructive activities, illiteracy, deforestation, air and water pollution and land effluents waste products. These may contribute to population explosion. According to UN projection 2001, world population is expected to be 7973 million at 2025 and 9322 million at 2050^2,3.

Today there is need to limit the family size at a personal level and to control the population at national level. Today's almost all contraceptives are expensive having side effects and complications. Contraceptive agents (oral) like Mala-D, Mala-N, Ovral-L are combination of estrogen and progesterogen⁴ have lot of side effects like intermenstrual spoting in the first three days more, scanty menstrual bleeding, CA of endocervix, variation of libido, suppression of lactation, pituitary adenoma, nausea, vomiting, reduces irritability, increased weight, 7-10 times more frequency of thromboembolic disorders, monilial vaginatis etc. carbohydrate tolerance, headache, migraine, depression.⁵ Due to these hazardable effects women are worried about these contraceptives. They need effective, safe, economic, reliable, acceptable contraceptives with little skill to use.

The use of medicinal plants and their products for regulation of fertility in India and other countries is still continuing.⁶PoineerAyurvedic scholars have practiced with different safe contraceptive since many centuries. Which are explained in Bhavaprakash, Yogaratnakar, Bhaishajya Ratnavali etc. single and combination of drugs like Pippali (Piper longum)⁷, Vidanga (Embeliaribes) and ShodhitaTankana(processed borax) (in equal part) with milk given during Rutu Samaya which are expected to act as contraceptive medicines.^8,9,10

Hence "Evaluation of oral contraceptive effect of Pippali, Vidangaand Tankana on albino rats" was undertaken. i) To evaluate oral contraceptive effect of Pippali, Vidanga and Tankana i) To elucidate the probable mechanism of action of Pippali, Vidanga and Tankana as a contraceptive agent. ii) To contribute an effective, safe and economic contraceptive medicine to do Clinical research and National Health Programme likes family planning.

MATERIALS AND METHODS:

Materials:

1) Animals: Wister stain albino rats of both sexes 2) Drugs: a) Pippali, Vidanga, Tankana,and Milk (milk is mentioned as anupana) 3) Equipment/glass wares 4) Chemicals

1) Animals:

Inclusive criteria

l. Healthy albino rats of child bearing age and should have normal estrous cycle in case of anti implantation study and healthy female albino rats 25-30 days old eighing 40-45g for screening of estrogenic activity

2. Both male and female rats of known fertility will be taken in case of anti- implantation study

Exclusive criteria:

Unhealthy albino rats Weight range below 150gm and above 210g. Sterile male and female rats.

18 female albino rats, 36 male albino rats and 45 litters from central animal house, BLDE’S AVS Ayurveda Mahavidhyalaya, Vijayapur formed the material for the experimental study. All the experimental animals were maintained at standard laboratory conditions fed with balanced diet as per CFTRI formula and water ad libitum at room temperature of 28±20% with a lighting scheduled of 2hrs light and 12hrs darkness. Animals under different group of experiments were caged separately.

Drugs were obtained from Teaching pharmacy and authenticated by department of Dravya Guna, of BLDE’S AVSAyurvedaMahavidhyalaya, Vijayapur.

Dose fixation: The doses of the drugs were calculated by extrapolating the therapeutic dose to rat dose on the basis of body surface area ratio (conversion factor 0.18 for rats) by referring to the table of “Paget and Barns" (Paget and Barnes, 1964). Human dose is 3gm (54 mg/200gm body weight of albino rats).

Methods:

Experimental Design:

Experimental protocol:

1) Screening for vaginal opening 2) Screening for vaginal cornification 3) Uterine weight assay 4) Ovarian weight assay 5) Screening for anti-implantation activity 6) Histological study on isolated rat ovary.

Anti implantation activity was conducted by following Choudary and Khanna method¹¹. It involves 6 steps.

a) Taking vaginal smear.

b) Examination of smear to know the phase of oestrous cycle.

c) Allowing animals for mating 1: 2 (female: male) ratio.

d) Observation for sperm clumps to confirm mating.

e) Drug administration

f) On 10^th day of drug administration rats were subjectedto laparotomy to observe for implantation.

Sample size: n= 6 in each group

Criteria for assessment

1) Anti-implantation activity:

Anti-implantation activity assessment is based on the observation of effect of the drug treatment on the continuation of pregnancy. If incidence pregnancy of pregnancy and number and size of implantations of rats delivering at full term is markedly decreased in comparison to control group, then anti-implantations, (anti fertility) activity is considered to be present in the test drug.

2)Vaginal opening and cornification:

If vaginal opening and comifications of vaginal epitheial cells taken place after drug administration in immature female rats, then its is considered that the drug possess estrogenic activity. The vaginal conalisation (opening) in test drug given group is compared with those of control and relative standard given groups.

3) Uterine and ovary weight assay:

Effect of test drug on the weight of uterus and ovary in immature female rats is noted and the data obtained was compared to uterine and ovary weight data from control and relative standard groups. Drugs possessing estrogenic activity markedly increases the weight of uterus and ovary.

4) To assess permanent contraception:

The animals of study drug groups in which ant implantation activity was observed, again such animals were let for mating for second time. This time the study drug was not administered. Even then if "anti implantation" activity possessed, then it is it is said the study drug posses permanent contraception activity. Histology study: Cyto-architecture of slides were seen and for number and nature of

1) Mature, immature follicles

2) Graffian follicles

3) Corpus luteum

4) Corpus albicans

Based on the observations mechanism of action and toxic effect is postulated.

Observations and Results:

For experimental study animals were selected from central Animal House of AV. Samithi's Ayureda Mahavidyalaya considereing inclusive and exclusive criteria during the period from August 2003 to July 2004. During the study following facts were noted

Table1: Showing effect of Drug on vaginal opening in immature rats

No of animals showing vaginal opening/ No, of animals treated
		Day of examination
Group	Drug	1	2	3
Control Group	Milk	0/5	0/5	0/5
Study group	Pippali+Vidanga + Tankana	0/5	0/5	0/5

Observations In the above table reveals that the test drug has got no impact on vaginal opening.

Table2: Showing effect of drug on vaginal cornification in immature rats

	No of animals showing vaginal cornification/ No. of animals treated Day of examination
Group	Drug	1	2	3
Control Group	Milk	0/5	0/5	0/5
Study group	Pippali+ Vidanga + Tankana	0/5	0/5	0/5

Observation in the above tble reveals that the test drug has got no impact on vaginal cormification.

Table 3: Showing the drugs effect on uterine weight in immature Rats

Group	Drug	Uterine weight Mean t SEM (Mg)
Control Group	Milk	97.5 ±0.22
Study group	Pippali+ Vidanga + Tankana	105.5±0.22

The observations in the above table reveals that the test drug has got no statistical significant difference in the weight of uterus.

Table4: Showing the drugs effect on ovary weight in immature Rats

Group

Drug

Ovary weight

Mean t SEM (Mg)

Control Group

Milk

11.5±0.22

Study group

Pippali+ Vidanga + Tankana

14.5±0.22

The observations in the above table reveals that the test drug has got no statistical significant difference in the weight of ovary

The above table reveals the Anti - implantation activity in 6/6 female albino rats in study group but it was noted that the mated vaginal smear had only a few sperms in it.

The above table reveals a significant difference in number of implantations and number of births in study groups in comparison with control group

DISCUSSION:

In present study Pippali, Vidanga and Tankana combination was tested for anti-implantation and pregnancy interruption properties with oral doses. The experimental study was carried out in four phases to achieve the aim and objectives of the study

a) Screening for-Anti-implantation activity: to assess contraceptive activity

b) Screening for vaginal opening, vaginal cormnification, ovary and uterine weight assay to assess probable mechanism of action

c) Histology of isolated rat ovary: to assess probable mechanism of action and toxic effect.

d) Screening of anti-implantation activity for second time on study drug group: to assess temporary or permanent contraceptive effect.

The first method was done according to technique of Choudhary and Khanna. The method involves placing of mature breeding female rats with males and then the occurrence of conception was established by recording the number of implantations, by performing laprotomy. The normal incidence in the study group was then compared with control group.

In the study this, in nallipara, the control group had 8.833±031 number of implants, and study group had no implants, control group had mean number of births 7.167±0.31 and study group have no births. With these 2 parameters i.e. number of implants and number of births demonstrated significant effect in anti-implantation activity may be due to the presence of steroid in the combination (Pippali, Vidanga and Tankana).

The control group had mean weight of litters 6.583 ±

0.15 and study group no litters. In control group mean number of litters died within two days is 1.667± 0.21 and in study there were no litters. These facts shown statically significant of difference.

The anti-implantation activity of the drug combination might be postulated as follows

· Progesterone is essential in augmentation of carbonic anhydrase, an essential factor in the adhesive stage of blastocyst implantation.

· Progesterone is essential in all phases of pregnancy¹¹

Hence,inhibition of progesterone synthesis or blockage of receptor of binding will result in the failure of blastocyst implantation and interruption of early pregnancy.¹²Hence Anti-implantation activity of Pippali, Vidanga and Tankana might be due to anti-progestional activity either as i) Inhibition in bio-synthesis or ii) As a blocker in receptor binding.

In elucidating the estrogenic and anti-estrogenic property of drug. 25-30 days old immature female albino rats weighing 40-45g were subjected for vaginal opening and vaginal cornification study for 3 days. But both in control group and study group no rat responded positively with 0/5, while in standard group rats have shown 5/5 of vaginal opening and cornification.

Secondly in uterine weight assay control group had 97.5±0.22 and study group had 105.5±0.22, ovary weight assay control group had 11.5±0.22 and study group had 14.5±0.22. The drug combination has shown no statistically significant increase in both ovary and uterine weight. However, in 5/5 there was slight gain in weight was noted in comparison with control group. In standard group statistically significant increase of weight was noted. Based on this estrogenic activity of test drug cannot be postulated.

Pippali, Vidanga and Tankana have shown the presence of saponins, steroids, flavanoids and tannins, benzene, chloroform and aqueous extract of this fruits shows antifertility activity, anti- oestrogenic and anti ovulatory effect.^13,14,15,16

CONCLUSION:

The herbo- mineral combination Pippali, Vidanga and Tankana in a dose 54mg/200gm body weight of albino rats shown significant anti implantation activity. The mean implantation 8.833±0.31 in control group and where as it was 0,00 in study group. The mean number of births 7.167±0.31 in control group and 0.0±0.0 in study group in albino rats were noted.Hence significance of difference was noted between control group and study group in number of implants and births may be due to the presence of steroid in the combination (Pippali, Vidanga and Tankana). There was no significant difference in the weight of uterus and ovary in study group compared to standard. The study drug has demonstrated only temporary contraceptive activity.In histological study the cytoarchitecture of the ovary of the test drug group indicates the drug had suppressed the maturation of graffian follicle.

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2. Howkins and Bourme (V.G Padubidri Shrish N Daftry).: Chapter18, Shaw’s Textbook of Gynaecology, Birth control and medical termination of pregnancy. Churchill Livingston, Edinburg. 9th Edn.1978.

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14. Biswal S. Phytochemical analysis and a study on the antiestrogenic antifertility effect of leaves of Piper betel in female albino rat. Anc Sci Life. 2014;34(1):16–22. doi:10.4103/0257-7941.150770

15. Priyanka Soni, Anees Ahmad Siddiqui, Jaya Dwivedi, Vishal Soni., Antiovulatory and Estrogenic Activity of Stem of Musa paradisiaca. in Female Albino Rats. J App Pharm Sci. 2013; 3 (08): 102-106.

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Received on 11.12.2021 Modified on 17.06.2022

Research J. Pharm. and Tech 2023; 16(7):3099-3103.

DOI: 10.52711/0974-360X.2023.00509