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|Year : 2009 | Volume
| Issue : 6 | Page : 273--277
Antiovulatory and estrogenic activity of Plumbago rosea leaves in female albino rats
E Sheeja, SB Joshi, DC Jain
Department of Pharmacognosy, B. R. Nahata College of Pharmacy and Research Center, Mhow Neemuch Road, Mandsaur - 458 001, Madhya Pradesh, India
Department of Pharmacognosy, B. R. Nahata College of Pharmacy and Research Center, Mhow Neemuch Road, Mandsaur - 458 001, Madhya Pradesh
Objective : To evaluate the effect of petroleum ether (60-80), chloroform, acetone, ethanol and aqueous extracts of Plumbago rosea leaves on the estrous cycle and to identify the estrogenic activity of active acetone and ethanol extracts in female albino rats.
Methods : Plant extracts were tested for their effect on the estrous cycle at two dose levels: 200 and 400 mg/kg, respectively. The effective acetone and ethanol extracts were further studied on estrogenic activity in rats. Histological studies of the uterus were carried out to confirm their estrogenic activity.
Results : The acetone and ethanol extracts were most effective in interrupting the normal estrous cycle of the rats (P<0.05, <0.01, <0.001). These later exhibited prolonged diestrous stage of the estrous cycle with consequent temporary inhibition of ovulation. The antiovulatory activity was reversible on discontinuation of treatment. Both the extracts showed significant estrogenic and antiestrogenic activity.
Conclusion : The acetone and ethanolic extracts of P. rosea leaves have an antifertility activity.
|How to cite this article:|
Sheeja E, Joshi S B, Jain D C. Antiovulatory and estrogenic activity of Plumbago rosea leaves in female albino rats.Indian J Pharmacol 2009;41:273-277
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Sheeja E, Joshi S B, Jain D C. Antiovulatory and estrogenic activity of Plumbago rosea leaves in female albino rats. Indian J Pharmacol [serial online] 2009 [cited 2022 May 26 ];41:273-277
Available from: https://www.ijp-online.com/text.asp?2009/41/6/273/59927
Population control is of immense importance for individual and national welfare. Although a variety of synthetic contraceptive agents are available, their use is associated with severe side-effects.  Hence, an approach was pursued to identify new antifertility agents from natural sources. Numerous indigenous drugs have been explained in folkloric Indian medicine for the management of various reproduction-related purposes. However, so far, no single plant is available that can be developed into a potent antifertility agent. 
Plumbago rosea L. (Plumbaginaceae), commonly known as Rakta Chitrak,  grows in the wild and in abundance in India. Traditionally, it is used in inflammatory disorders, skin diseases,  gastric acidity, constipation, abdominal pain  and as an abortifacient. ,, The roots of the plant have been reported to possess antitumor  and antiatherogenic  activities. The active constituents reported in this plant are plumbagin,  hydroxy-1,4-napthaquinone, sitosterol glycoside, fatty alcohol and tannins. 
So far, during the literature survey, it was found that the roots of this plant have been used for their antifertility and uterine activity.  The present study was carried out to evaluate the antifertility effect of P. rosea leaves. The uprooting of the plant could be avoided in case the antifertility action was observed.
Materials and Methods
The leaves of P. rosea L. were collected from Kanyakumari district, T.N., and positively identified by Dr. H. S. Chatree, Botanist, Govt. Arts and Science College, Mandsaur, M.P. Voucher specimen (P/002/2006/BRNCOP) was deposited in the herbarium of the Department of Pharmacognosy, BRNCP, Mandsaur.
The leaves of the plant were shade dried and powdered. The powdered material was extracted using petroleum ether (60-80 0 ) for 72 h and successively extracted with chloroform, acetone, ethanol and water for 72 h each in a soxhlet apparatus. The extracts were evaporated under reduced pressure to obtain solid masses and the percentage yield of the extracts was found to be 2.32%, 2.05%, 1.5%, 4.57% and 25.6%, respectively.
In order to determine the presence of alkaloids, glycosides, flavones, tannins, terpenes, sterols, saponins, fats and sugars, a preliminary phytochemical study (color reactions) with leaf extracts was performed. 
Female albino rats (Wistar strain weighing 150-200 g) were used for antiovulatory activity and immature female rats (Wistar strain), 21-23 days old, were used for estrogenic activity. The animals were housed in standard environmental conditions of temperature (21 ± 2 0 C), humidity (55 ± 10%) and a 12-h light-dark cycle. Rats were supplied with standard pellet diet and water ad libitum. The animals were acclimatized to laboratory hygienic conditions for 10 days before starting the experiment. Animal study was performed in the Division of Pharmacology, B R Nahata College of Pharmacy, Mandsaur, with due permission from the Institutional Animal Ethics Committee (Reg no. 918/ac/05/CPCSEA).
Acute toxicity studies
The acute toxicity test of the extracts was determined according to the Organization for Economic Co-operation and Development guidelines no. 420. Female Wistar rats (150-180 g) were used for this study. After the sighting study, a starting dose of 2,000 mg/kg (p.o.) of the test samples were given to various extract groups containing five animals in each groups. The treated animals were monitored for 14 days for mortality and general behavior.
Experiments were carried out in female Wistar rats weighing (150-200 g). The vaginal smear of each rat was examined daily between 9-10 A.M for 15 days to select the animals showing regular cycles (4-5 days).  The selected rats were divided into 11 groups of six animals each. The extracts were administered orally for five days to cover one regular estrous cycle. Group I received vehicle (1% Tween 80, p.o. daily) and served as control. Groups II to XI received petroleum ether, chloroform, acetone, ethanol and aqueous extracts of P. rosea leaves at 200 and 400 mg/kg body weight. Vaginal smear from each animal was observed every morning between 9-10 A.M for five days of treatment and subsequently for 15 days.
Estrogenic and antiestrogenic activity
The extracts with antiovulatory activity were further studied for the estrogenic and antiestrogenic activity.  Immature female Wistar strain rats, 25-30 days old, weighing between 35 and 45 g, were divided into 10 groups of six rats each. The first group served as control and received vehicle only (1% Tween 80). The second group received ethinyl estradiol (standard) in distilled water, 0.02 mg/kg body weight. The third, fourth, fifth and sixth groups received acetone and ethanolic extracts of P. rosea leaves at two dose levels, 200 and 400 mg/kg body weight, respectively. The groups VII to X received ethinyl estradiol in addition to a test dose of acetone and ethanolic extract of the plant at the same dose. All the above treatments were given for three days (p.o.). On the fourth day, the rats were sacrificed by decapitation, the uteri dissected out and the surrounding tissues were removed. The uteri were blotted on filter papers and weighed quickly on a sensitive balance and fixed in Bouin's fluid for 24 h. The paraffin-embedded tissues were cut at 6 µm and stained with hematoxylin-eosin solution for histological observations.
The data were statistically analyzed and expressed as mean±SEM. Statistical analysis of the variance between control and experimental values was performed by Student's t-test.
The phytochemical screening of different extracts of P. rosea leaves revealed the presence of various constituents, as shown in [Table 1].
Acute toxicity studies
No mortality and behavioral changes were observed in the treated groups up to 2,000 mg/kg body weight. The 400 mg/kg dose was chosen as maximum dose for further experiments.
Effect of extract on the estrous cycle of rats
The present study revealed that the acetone and ethanol extracts of P. rosea leaves showed an antifertility effect. Treatment of rats with acetone and ethanolic extracts for five days prolonged the estrous cycle significantly (P Estrogenic and antiestrogenic activity
The effect of both acetone and ethanolic extracts of P. rosea leaves on immature rat uterus is shown in [Table 3]. Oral administration of the extracts at 200 and 400 mg/kg body weight caused a significant increase in the uterine weight in immature rats (PP. rosea leaves exhibited significant (PP 
The extracts with the antiovulatory activity were further studied for their estrogenic and antiestrogenic activity. These extracts also exhibited estrogenic activity as shown by the significant increase in the diameter of the uterus, uterine weight and thickness of the endometrial epithelium when compared with the control. It was also observed that the acetone and ethanol extracts suppressed the action of ethinyl estradiol when administered together. The extracts showed a significant estrogen-like activity when given alone but, with ethinyl estradiol, they exhibited a slight antiestrogenic nature. This indicates that the extract acted as a competitive antagonist to the more potent ethinyl estradiol. 
Preliminary phytochemical studies indicated the presence of tannins, flavonoids, triterpenoids and napthaquinone in the acetone extract and the ethanol extract showed the presence of carbohydrates, glycosides, tannins, flavonoids and saponins. According to the literatures, flavonoids and plumbagin (napthaquinone) are known to exhibit antifertility activity. ,, In our study, it is not solely the presence of napthaquinones or flavonoids that produced the antifertility activity because the petroleum ether extract contains napthaquinones and was devoid of any activity. On the other hand, irrespective of the absence of napthaquinones, the ethanol extract displayed significant activity when compared with controls, indicating that flavonoids could be responsible for the activity. The synergism produced by napthaquinones along with flavonoid could be the reason for the enhanced activity of the acetone extract when compared with that of the ethanol extract.
The results of the present study indicate that the acetone and ethanolic extracts of P. rosea leaves have significant antifertility activity. The leaves of this plant could be used to induce abortion. The extracts of this plant can be further explored for contraceptive use.
The authors are thankful to the Director, BRNCOP, for providing all the necessary facilities to carry out this research work.
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