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Evaluation of anti-fertility activity ofTabernaemontana divaricata (Linn)R.Br. leaves in ratsSachin Jain a , Avijeet Jain a , Lokesh Deb a , K.R. Dutt a & DeepakKumar Jain ba Department of Pharmacognosy, Herbal Drug Discovery Division,BRNSS-Contract Research Center–SIRO, B.R. Nahata College ofPharmacy, Mhow-Neemuch Road, Mandsaur 458001, MadhyaPradesh, Indiab Truba Institute of Pharmacy, Karond Bypass, Bhopal, MadhyaPradesh, India

Version of record first published: 18 Mar 2010.

To cite this article: Sachin Jain, Avijeet Jain, Lokesh Deb, K.R. Dutt & Deepak Kumar Jain (2010):Evaluation of anti-fertility activity of Tabernaemontana divaricata (Linn) R.Br. leaves in rats,Natural Product Research: Formerly Natural Product Letters, 24:9, 855-860

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Natural Product ResearchVol. 24, No. 9, 20 May 2010, 855–860

Evaluation of anti-fertility activity of Tabernaemontana divaricata(Linn) R.Br. leaves in rats

Sachin Jaina*, Avijeet Jaina, Lokesh Deba, K.R. Dutta and Deepak Kumar Jainb

aDepartment of Pharmacognosy, Herbal Drug Discovery Division, BRNSS-ContractResearch Center – SIRO, B.R. Nahata College of Pharmacy, Mhow-Neemuch Road,Mandsaur 458001, Madhya Pradesh, India; bTruba Institute of Pharmacy, Karond Bypass,Bhopal, Madhya Pradesh, India

(Received 16 April 2009; final version received 6 September 2009)

The aim of the present study was to assess the anti-fertility activity ofethanolic extracts of Tabernaemontana divaricata (TD) leaves in oestrogenicactivity models in immature female rats. Mature green leaves of TD werecollected and authenticated. Extractions of the dried leaves were carried outwith ethanol in a Soxhlet’s apparatus. For oestrogenic activity, the extractswere administered orally once daily at a dose of 200 and 400mgkg�1, andthe activity was compared with the standard drug ethinyl oestradiol(0.02mg). The extracts caused significant increase in uterine weightcompared to the control. The ethanolic extract exhibited oestrogenicactivity. The histological study of epithelium tissues with the 400mg of TDextract-treated animals showed increases in the height of the luminalepithelium and loose edematous stroma when compared with the 200mg ofTD extract-treated group of animals. However, this was better than thecontrol group of animals. Enhanced uterine weight and increase in theheight of luminal epithelium and histological characteristics suggest thatTD extract may be useful in anti-fertility therapy.

Keywords: Tabernaemontana divaricata; anti-fertility; oestrogenic activity;ethinyl oestradiol

1. Introduction

The quest for oral contraceptive agents that can control human fertility is asold as recorded history. Although a wide variety of synthetic contraceptive agents(Bygdeman, Christensen, Green, Zheng, & Lundstrom, 1983; Bygdeman,Danielsson, & Swahn, 1997) are available, these cannot be used continuously dueto their severe side effects (Sanchez, Tebar, Ruiz, & Padron, 1997; Vervest &Haspels, 1985). Hence people are looking back to the age-old tradition of usingherbal medicines, which have minimal side effects. India and in particular theWestern Ghat region has an enormous wealth of medicinal plants. Presently, a majorprogramme of systematic investigation into medicinal plants for their phytochemical,biological and pharmacological properties, including antifertility properties, was

*Corresponding author. Email: sachin225819@rediffmail.com

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2010 Taylor & Francis

DOI: 10.1080/14786410903314385

http://www.informaworld.com

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undertaken in our laboratory (Sreedhara, Pai, & Vaidya, 2001). As part of thisresearch programme, we present in this article the antifertility efficacy of the leavesof the plant Tabernaemontana divaricata (TD) – a glabrous, evergreen shrub, 1.8–2.4m in height, with silvery grey bark and milky latex. Its leaves are simple, opposite,elliptic or elliptic-lanceolate, smooth, glossy green, acuminate and have wavymargins, and flowers are white, sweetly fragrant in 1–8 flowered cymes at thebifurcations of the branches, and lobes of corolla overlapping right into the bud. It isused as a thermogenic, anodyne, astringent, vermifuge, odonalgia and in thetreatment of strangury, paralysis, arthralgia and melalgia (Warrier, Nambiar, &Ramankutty, 1996). The flower juice mixed with oil alleviates burning sensations,cures eye sores and skin diseases, and the leaf juice applied to wounds preventsinflammation and is used in opthalmia (Chatterjee & Pakrashi, 1995). The flowercontains dregamine, 20-epiervatamine, tabernaemontanine, vobasine, voacangine,voacamine, flavonoid aglycones, flavonol glycosides, isovoacristine, voaphylline-hydroxyindolenine, janetine (tetrahydrolivadine), N-methyl-voaphylline (hecubine)and kaempferol, and leaves contain dregamine, 20-epiervatamine, tabernaemonta-nine, vobasine, voacangine, voacamine, flavonoid aglycones, flavonol glycosides,isovoacristine, �-amyrin, lupeol and their acetates, �-sitosterol, coronaridine,apparicine, ervaticine (2-acyl indole derivative), ervatinine, hyderabadine, lahoricine,mehranine, stapfinine, voacristine, voharine and a dimeric alkaloid, conophylline 17-� oestradiol. A literature survey reveals that no systematic approach has been madeto study the antifertility activity of the leaves of this plant. Since the plant containsconophylline 17-� oestradiol, in the present work we have investigated theantifertility activity of the ethanolic extract of TD leaves against ethinyl oestradiol.

2. Materials and methods

2.1. Plant material and preparation of the extract

The leaves of TD were collected from Mandsaur, Madhya Pradesh. Prof H.S.Chattri (Principal of the Government PG College, Mandsaur, Madhya Pradesh)authenticated the plant. A voucher specimen (BRNCP/T/002/2006) has beendeposited at CDRI Lucknow. The material was air-dried under shade, pulverisedby a mechanical grinder and passed through a 40 mesh and then stored in airtightcontainers. The powdered leaves were extracted with ethanol (80% w/v) for 24 husing a Soxhlet extractor. This ethanolic extract was concentrated to dryness underreduced pressure and controlled temperature to yield solid masses that werecompletely free from solvents. The dried extract was subjected to preliminaryphytochemical tests.

2.2. Animals

Albino Wistar immature female rats of 21–23 days old, weighing 40–60 g, were usedin this study. They were procured from animal house of BR Nahata College ofPharmacy, Mandsaur, Madhya Pradesh. The rats were acclimatised for 10 daysunder laboratory conditions. They were housed in polypropylene cages andmaintained at 27�C� 2�C, relative humidity 65� 10%, under 12 h light/dark cycle.The animals were fed with a rodent pellet diet and water ad libitum. The Institutional

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Animal Ethical Committee approved the protocol (918/ac/05/CPCSEA/49/MPh/06)of this study.

2.3. Acute toxicity study

The acute toxicity for the ethanolic extract determined in albino mice weremaintained under standard conditions. The animals were fasted overnight prior tothe experiment and the fixed dose method was adopted as per OECD Guideline No.420; (Annexure-2d) of CPCSEA (Veeraraghavan, 2003).

2.4. Oestrogenic activity on immature female rats

Immature female rats were divided into four groups of six animals each. The groupswere treated as follows:

Group I – Control (saline solution) p.o.Group II – Reference standard (ethinyl oestradiol 0.02mg kg�1) p.o.Group III – Ethanolic leaves extract of TD (200mgkg�1, p.o.)Group IV – Ethanolic leaves extract of TD (400mgkg�1, p.o.).

The treatment was given for six days and 24 h after the last dose. All the animals weresacrificed by decapitation and the uteri were dissected out, cleared off the adhesivetissue, blotted on filter paper and weighed quickly on a sensitive balance. The tissueswere fixed in Bouin’s fixative for 24 h, dehydrated in alcohol and embeddedin paraffin. The paraffin blocks were sectioned at 6 mm and stained withhaemotoxylene–eosin solution (H and E Stain) for histological observations(Jagadish & Rana, 2002). The uteri were processed for histopathological study onthe seventh day.

2.5. Statistical analysis

The data are expressed as mean� SEM. The differences among means were analysedby ANOVA. A value of p5 0.05 was considered statistically significant.

3. Results

Preliminary phytochemical analysis of the ethanolic extract revealed the presence ofcarbohydrates, steroids, glycosides, flavonoids, tannins and alkaloids. The extractsignificantly increased the weights of uteri (Table 1). Histopathological studiesshowed normal architecture of the uterus in vehicle-treated rats. It indicates thesurface epithelium with no secretory activity (Figure 1). Ethanolic extract-treatedgroups showed an increase in the height of the luminal epithelium, as well as looseand edematous stroma with stimulated uterine glands (Figures 2 and 3).

4. Discussion

Tabernaemontana divaricata is a traditional plant used for family planning. It wasfound that this plant has a variety of phytochemical constituents, which havethe multiplicity of pharmacological actions. Previous literature studies reported the

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presence of flavonoids, alkaloids, phytosterols, amino acids, conophylline 17-�oestradiol, etc. in TD. The present preliminary phytochemical investigation on leafextracts reveals the presence of carbohydrates, steroids, glycosides, flavonoids,alkaloids and tannins in the ethanolic extract. The ethanolic extract of TD exhibited

Figure 1. Showing the section of uterus indicating surface epithelium with no secretoryactivity (control group).

Figure 2. Showing the section of uterus indicating an increase in the height of the luminalepithelium (ethanolic 200mg kg�1).

Table 1. Effect of TD leaf extracts on uterine weight of immature female rats.

Group Extracts/drug Dose (mgkg�1 p.o.) Uterine weight (mg)

I Control (vehicle) – 214.25� 22.79II Ethinyl oestradiol (standard) 0.02 283.12� 23.50*III Ethanolic extract 200 323.25� 08.78***IV Ethanolic extract 400 351.57� 11.22**

Notes: Values are mean� SEM (n¼ 6). *p5 0.05, **p5 0.01, ***p5 0.001 as compared tothe control group.

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significant oestrogenic activity, as indicated by an increase in uterine weight as

compared to the control group of rats.It is a well-known fact that oestrogenic substances inhibit pregnancy by

suppressing the level of both follicular stimulating hormone (FSH) and luteinising

hormone (LH), which in turn prevent the implantation. Oestrogen and progesterone

are the hormones responsible for histological and functional modifications of the

female genital tract. The exogenous administration of physiological doses of

oestrogen, in sexually immature rats, stimulated histoarchitecture of the uterus.

According to Laurence and Bacharach (1964), any compound possessing oestrogenic

activity may exhibit antifertility activity; they act by suppressing gonadotrophin

secretion, with the consequent inhibition of ovulation. In immature female rats,

ethanolic extracts exhibited definite oestrogenic activity. As supported by histological

evidence, the oestrogenic effect of an ethanolic extract at a dose of 400mgkg�1 may be

due to the increased height of the luminal epithelium with hyperplasia, loose and

oedematous stroma with stimulated uterine glands. Isoflavones, along with

coumentans (also flavonoids) and lignans, belong to a class of substances known as

non-steroidal phytoestrogens, which produce infertility in animals (Evans, 2001).

According to Miksicek (1993), several commonly occurring flavonoids mimic

the biological effects of 17-�-oestradiol by the virtue of their ability to bind to and

activate the nuclear oestrogen receptor.

5. Conclusion

The present study demonstrated that an ethanolic extract of TD leaves has

the antifertility activity. Increase in uterine weight, increase in the height of the

luminal epithelium and loose and edematous stroma with stimulated uterine glands

support the need for further evaluation of TD leaves.

Figure 3. Showing the section of uterus indicating an increase in the height of the luminalepithelium, loose and edematous stroma with stimulated uterine glands (ethanolic400mg kg�1).

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