INTRODUCTION:

Balanites aegyptiaca Del. also known as ‘desert date’ in English, a member of the family Zygophyllaceae, the one of the plant species of the common but neglected wild plant species of the dry land areas of Africa and South Asia.¹ The tree can grow 6-10 meters in height, is highly resistant to stresses such as sandstorms and heat waves, and grow with minimal available moisture. The tree has thick, tough glossy leaves, spiny branches and a double root system and produce date-like fruits. The plants grow extensively even neglected. One estimate is that more than 400,000 tons of Balanites fruit are produced Sudan alone.²It can successively grow in a marginal sand dune with saline and sewage water. Various parts of Balanites tree have been used for folk medicines in many regions of Africa and Asia.^2-5 Literature has revealed antifeedant, antidiabetic, molluscicide, anthelmintic and contraceptives activities in various Balanites extracts.^6-10 Balanites aegyptiaca is a small evergreen thorny tree found in drier parts of India. The bark, unripe fruits and leaves of this plant are reported to have anthelmintic, antifertility, purgative and antidysentric properties.^11-13

Phytochemical investigations on Balanites aegyptiaca yielded in the isolation of several classes of secondary metabolites, many of which expressed biological activities such as coumarins, flavonoids, saponins and tannins.¹⁴ The root and bark of Balanites aegyptiaca tree have several steroidal saponins, yamogenin and glycosides were isolated.¹⁵ Two furostanol glycosides and 6-methyl-diosgenin were obtained from the fruits.^16,17 More recently five new steroidal glycosides were isolated from root of the plant.¹⁸Aerial part of Balanites aegyptiaca showed significant anti-inflammatory and analgesic activity.¹⁹The antifeedant active saponins have been isolated from bark.⁷ Aqueous suspension of dried fruits of this plant is being used as abortifacient by local herbal healers. The present study was undertaken to find out the possible actions of bark of Balanites aegyptiaca for its antiimplantation activity.

MATERIAL AND MEHODS:

Plant materials

Balanites aegyptiaca bark Del. was collected in month of August 2008 from Satnavari Forest Region of Nagpur District. It was authenticated at Department of Botany, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur. The Balanites aegyptiaca bark were air dried in shade, under normal environmental conditions and then subjected to size reduction to get coarsed powder. The coarsed powder material was charged into the Soxhlet apparatus for defatting with petroleum ether (60-80) followed by extraction with methanol. Preliminary phytochemical study of methanolic extract of Balanites aegyptiaca bark showed the presence of saponins, flavonoids, glycosides, tannins, sugar and amino acids.²⁰

Animals

Wistar strain rats of either sex weighing between 200-250 g procured from Shree Farms, Bhandara were used for the present investigation. Animal Ethical Committee had approved the experimental protocol under the guidelines of CPCSEA, New Delhi. The rats were housed at controlled temperature (25±2ºC), relative humidity (65±10%), light and dark cycle (12:12h) and fed standard pellet food, water ad libitum. The initial body weight of each animal was recorded. The vaginal smear of the rats was studied microscopically for estrus cycle every morning. Only female rats with normal estrus cycle were selected for the antiimplantation activity.

Acute Toxicity Study

Toxicity study of methanolic extract of Balanites aegyptiaca bark Del. was carried out in mice according to OECD guidelines. Extract at different doses up to 3000mg/kg body weight, orally was administered and animals were observed for behavioral changes, any toxicity and mortality up to 48 h. There was no toxic reaction or mortality and found to safe. Based on acute toxicity result we have selected 150 mg/kg body weight and 300-mg/kg-body weight for antiimplantation activity.

Anti-implantation activity ^21-24

Proven fertile female rats of Wister strain weighing 200-250g were screened for 2-3 estrous cycles by examining the vaginal smears. The rats that showed normal cycles for two successive examinations were selected for study. The method of Khanna and Chaudhary was followed with necessary modifications; the rats in proestrous and estrous stages were caged with fertile male in the ratio 2:1. The following day vaginal smears were examined and the appearance of the sperms clusters in the smears was recorded as day 1 of pregnancy. Control animals in the Group-I received on Tween-80 (1%) + saline as vehicle only. Methanolic extracts of Balanites aegyptiaca bark at 150 mg/kg & 300mg/kg were administered orally to Group-II and Group-III in the form of suspension of dried powders with Tween-80 (1%) + saline respectively. On day 10 laprotomy was performed under light ether anesthesia to examined uteri for implant number and size. Then the abdomen was closed and rats were allowed to recover and deliver after full term pregnancy. Those rats, that not deliver, were laprotomised on the day 21 and uteri were examined for implantation sites. The fertility test was considered as positive if implantation sites were observed and negative in their absence. The born litters were observed for teratogenic abnormality, if any.

Biochemical studies

The present study besides evaluating the antiimplantation effect, also attempts to investigate some biochemical parameters, mainly blood glucose, cholesterol and triglyceride.

Statistical analysis

The values are expressed as Mean±SEM. ANOVA followed by Tukey-Krammer multiple compression test was performed to determine the difference between Mean and p<0.05 considered as statistically significant.

ResultS:

After treatment with Balanites aegyptiaca bark, body weights were reduced up to 21 days. Maximum reduction was observed in 300-mg/kg methanolic extract of Balanites aegyptiaca bark i.e.-5.42 % compared to 150 mg/kg methanolic extract of Balanites aegyptiaca bark i.e.3.98 %. The rise in body weight was + 4.05 % in control group of rats (table 1). Maximum percent inhibition or reduction of implantation on born litters was observed in methanolic extract (300 mg/kg body weight) and methanolic extract (150mg/kg body weight) of Balanites aegyptiaca bark. All rats delivered in control group (table 2). Biochemical changes observed in Balanites aegyptiaca bark extracts treated rats showed depletion in blood glucose, cholesterol and triglyceride levels as compared to Group-I i.e. control group (table 3). In Group-III i.e. methanolic extract (300mg/kg) of Balanites aegyptiaca bark, percent change observed was -7.38% in blood glucose, - 21.42 % in cholesterol, and –12.38% in triglyceride. In Group-II i.e. methanolic extract (150mg/kg) of Balanites aegyptiaca bark, percent change observed was –4.46 % in blood glucose, - 8.75% in cholesterol, and - 7.91% in triglyceride.

Table No. 1: Body weight changes in control as well as experimental female rats treated with methanolic extracts of Balanites aegyptiaca bark

Sr. No	Duration of treatment	Control (Wt. in gm)	Methanolic extract150mg/kg (Wt. in gm)	Methanolic extract300mg/kg (Wt. in gm)
1	Initial weight	242.5±7.04	246.83±4.56	245.83±2.91
2	7 days	245.66±7.24 (+1.30%)	244.5±4.73 (-0.94%)	242.16 ±2.82 (-1.49%)
3	14 days	250.00±6.83 (+3.09%)	242.5±4.89 (-1.75%)	238 ±2.74 (-3.18%)
4	21 days	252.33±6.93 (+4.05%)	237±4.59 (-3.98%)	232.5±2.63 (-5.42%)

Values are Mean ±SEM of six animals per group Test, Figure in parenthesis indicate percent change over control.

Table No. 2: Effect of methanolic extracts of Balanites aegyptiaca bark on reduction in pregnancy in rats

Gr. No.	Treatment	Dose Mg/kg (Body weight)	No. of rats pregnant/Treated	% Reduction in pregnancy	No. of implant in individual rats	No. of rats delivered (No. of pups)
I	Control	Tween-80+ Saline	6/6	0	13,12,9,7,8,10.	6(13,12,9,7,8,10)
II	Methanolic extract of B.a. bark	150	3/6	50	3,2,0,0,1,0.	3(3,2,0,0,1,0)
II	Methanolic extract of B.a. bark	300	0/6	100	0,0,0,0,0,0.	0(0,0,0,0,0,0,)

Table No. 3: Effect of methanolic extracts of Balanites aegyptiaca bark on blood glucose, cholesterol and triglycerites.

Sr. No.	Biochemical parameters (mg/100ml)	Control	*Methanolic extract of B.a.* bark (150mg/kg)**	*Methanolic extract of B.a.* bark (300mg/kg)**
1	Blood glucose	77.31±1.04	73.86±0.23** (-4.46%)	71.6±0.53***(-7.38%)
2	Cholesterol	70.61±0.19	64.43±0.79***(-8.75%)	55.48±0.57***(-21.42%)
3	Triglyceride	156.78±1.06	144.37±2.15***(-7.91%)	137.36±0.72***(-12.38%)

*p<0.05, ***p<0.001. **p<0.01 when compared with control group.

DISCUSSION:

In this present study methanolic extracts of Balanites aegyptiaca bark evaluated for its antiimplantation activity. In this antiimplatation model, we determined the body weight, percent pregnancy inhibition (implatation) and biochemical parameters. We found that decreases in body weight after extract treated group and also percent inhibition of pregnancy (implantation) increases as the dose of methanolic extracts of Balanites aegyptiaca bark increases.

In biochemical parameters such as blood glucose, cholesterol and triglyceride showed the decreased in levels as compared to control group. Since the cholesterol is the precursor of the steroidogenesis of ovarian endocrine tissues. These decreases in cholesterol level can cause diminution of ovarian steroidogenesis.

It is well known fact that for implantation and sustenance of pregnancy, exact equilibrium of secretion estrogen and progesterone is necessary. Any imbalance in the level of these hormones can cause antiimplantation or induce abortion.²⁵

Presence of chemical constituents in Balanites aegyptiaca bark triterpeniod sapogenin, diosgenin, yamogenin, furanocoumarin, bergapten and (+) mermesin, β-sitosterol, balanitin 1, balanitin 2, balanitin 3 and balanitol these constituents might be responsible for its antiimplantation activity.

The present experimental findings suggest that, methanolic extract of Balanites aegyptiaca bark has antiimplantation activity, hence its antiimplantation action responsible for its antifertility activity. Further detailed study using different animal species to establish its antifertility activity and also undersigned cellular mechanism of action.

REFERENCES:

1. Hall JB, Walker DH, Balanites aegyptiaca Del. A monograph. School of agricultural and RForest Science.Banger: University of Wales; 1991.p.1-12.

2. Mohamed AM, Wolf D, Spicess WE. Recovery and characterization of Balanites aegyptiaca Del. Kernels proteins: Effect of defatting, air classification, wet sieving and aquous ethanol treatment on solubility, digestability, amino acid composition and sapogenin content. Nahrung 2000; 44:7-12.

3. Iwu MM, Handbook of African Medicinal Plants. CRC Press, Kamel MS, Ohtani K, Kurokawa T, Assef MH, El-Shannawany MA, Ali AA, Kasai R, Ishibhasi S, Tanaka O. Studies on Balanites aegyptiaca fruits, an antidiabetic Egyptian Folk medicine. Phytochemistry 1991, 31:3565-3569

4. Neuwinger HD. African Ethanobotany: Poisons and Drugs.1^st SA: Chapman and Hall publication; 1994. p.884-890.

5. Mohamed AM, Wolf W, Spicess WE. Physical, Morphological and chemical characteristics .oil recovery and fatty acid decomposition of Balanites aegyptiaca Del. Kernels. Plant Foods Hum Nutr. 2002; 57:179-189.

6. Liu HW, Nakanishi K. The structure of Balanites: Potent molluscides isolated from Balanites aegyptiaca.Tetrahedron. 1982; 38:513-519.

7. Iwu MM, Handbook of African Medicinal Plants. CRC Press,Boca Raton,FL, Jain DC, Tripathi AK. Insect feeding detergent activity of some saponin glycosides. Phytother Res. 1991; 5:139-141.

8. Kamel MS, Ohtani K, Kurokawa T, Assef MH, El-Shannawany MA, Ali AA, et.al. Studies on Balanites aegyptiaca fruits, an antidiabetic Egyptian Folk medicine. Chem Pharm Bull. 1991; 31:1229-1233.

9. Ibrahim AM. Anthelmintic activity of some Sudanese medicinal plants. Phytother Res. 1992; 6:155-157.

10. Rao MV, Shah KD, Rajani M. Contraceptive efficacy of Balanites roxburgii pericarp extract in male mice (Mus Muscullus).Phytother Res. 1992; 6:469-471.

11. Kirtikar KR, Basu BD. Indian Medicinal Plants, 1. 2^nd ed. New Delhi: International Book Distributors; 2005. p. 512-515.

12. Chopra RN, Nayer SL, Chopra IC. Glossary of Indian Medical Plants (1).1^st ed. New Delhi: Council of Scientific and Industrial Research; 1956. p.32.

13. Rastogi RP, Mehrotra BN. Compendium of Indian Medicinal Plants (4).1^st ed. New Delhi: Central Drug Research Institute, Lacknow and Publication and Information Directorate; 1985-1989. p. 96.

14. Sarkar SD, Bartholomew B, Nash Rj. Alkoloids from Balanites aegyptiaca. Fitoterapia. 2000; 71:328-330.

15. Pettit GR, Doubek DL, Numata A, Takahasi C, Fujiki R, et. al. Isolation and structure of cytostatic saponins from African medicinal plant Balanites aegyptiaca. J Nat Prods. 1991; 54:1491-1502.

16. Honsy M, Khalifa T, Calis I, Wright AD, Sticher O, Balanitosides: A furastanol glycosides and 6-methyl-diosgenin from Balanites aegyptiaca. Phytochemistry. 1992; 31:3565-3569.

17. Kamel MS.A Furostanol saponin from fruits of Balanites aegyptiaca. Phytochemistry. 1998; 48(4): 755-757.

18. Farid H, Hasliger E, Kuneri O. New glycoside from Balanites aegyptiaca. Helvetica Chim Acta. 2002; 85:1019-1026.

19. Gour K, Nema RK, Kori ML, Sharma CS, Singh V. Anti-inflammatory and analgesic activity of Balanites aegyptiaca in experimental animal models. Inter J Green Pharm. Oct-Dec 2008; 212-217.

20. Khandelwal KR. Practical Pharmacognosy. 11^th ed. Pune: Nirali Prakashan; 2004.p.149-156.

21. Khanna U, Chaudhury RR. Antifertility screening of plants part-I:Investigation of Butea monosperma (lam) Kutze. Indian J Med Res. October 1968; 56(10): 1575-1580.

22. Koneri R, Sarswati CD, Balaraman R, Ajeesha EA. Antiovulatory and abotifacient Potential of ethanolic extract of roots of momordica cymbalaria Fenzl in rats. Indian J Pharmacol. 2007; 39(20): 90-96.

23. Vaidya VP, Padmashali B, Vagdevi HM, Satyanarayana ND.Antifertility efficasy of Balanites roxburgii in female rats. Indian J Pharm Sci. 2006; 68(3): 347-351.

24. Kothawade PS, Thakare VN, Dhote VV. Deshpande AD. Antifertility activity of ethanolic extracts of Allium cepa Linn. in rats. Inter J Pharma Tech Res. Jan-March 2009; 1(1):73-78.

25. Pcychoyos A. Recent Research on egg implantation CIBA Foundation study group.

Received on 09.02.2011 Modified on 19.03.2011

Research J. Pharm. and Tech. 5(2): Feb. 2012; Page 288-290