Anti-ovulatory and Abortifacient Potential of the Ethanolic Extract of Henna Roots in Rats

S Rani^*, R Manavalan and D Kilimozhi

Department of Pharmacy, Annamalai University, Annamalai Nagar - 608002, Tamil Nadu. India.

ABSTRACT

The rise in population in the developing world is over whelming and this intensifies the need for effective birth control measures. The synthetic agents available today for fertility control produce severe side effects. Such as hormonal imbalance, hypertension, increased risk of cancer and weight gain. Thus there is a need to replace these agents by safe and effective agents such as plants based contraceptive agents.

Therefore, the present study was under taken to explore the abotifacient and antiovulatory activity of ethanol extract of henna roots. Thus, the successive ethanolic extract showed promising strong abotifacient and antiovulatory activity was observed at dose level of 400mg/kg body weight. Histological studies were carried out to confirm this effect.

KEY WORDS: Abortifacient, ovulation, contraception.

INTRODUCTION

The henna plant Lawsonia inermis Linn is a fragrant shrub native to Asia and northern Africa. The word henna which means “to become queen” is indicative of something highly elegant. The plants extract or its purified compounds exhibit a variety of biological activities. Such as anticomplementary activity (Handa et al, 1997), dihydroorotate dehydrogenase inhibitory activity, (Knecht et al;2000), macrophage stimulating activity as result of stimulation of secretion of granulocyte macrophage colony stimulating factor (GM-CSF) (Wagner et al, 1988), antimicrobial activity (Malekzadeh, 1968; Abd-el-Malek et al, 1973), antisickling activity (Chang and Suzuka, 1982; Clarke et al, 1986), hepatoprotective activity (An-aad et al,1992), cytotoxic activity (Ali and Grever,1998), anti-inflammatory, antipyretic and analgesic activities (Ali et al, 1995). In the light of the previous activities, the study ours examined antiovulatory and abortifacient activities of the ethanolic extract of henna roots.

MATERIAL AND METHODS:

Plant collection and preparation of the extract:

The fresh henna roots were collected from Cuddalore district. Tamil Nadu. They were identified and authenticated by Dept of Botany, Annamalai University, Chidambaram. A Specimen sample of the same was preserved at the herbarium of the Department of Botany, Annamalai University with Voucher no 12456, for future reference.

The roots were isolated and chopped into small pieces. Next, they were dried under shade at room temperature for seven days. The dried roots were powdered, passed through sieve (coarse 10/44) and extracted with ethanol using soxhlet extractor (Shivalinagappa Het al, 2001). The combined extracts were concentrated of 40º C to obtain dark brownish yellow residue. The yield obtained from this process was found to be 25.5%w/w.

Experimental animals:

Colony bred virgin female adult Wister strain rats were used in all experiments. All animals were housed in standard cages in uniform conditions (12 hours dark and 12 hours light cycle) and at room temperature. Animals were fed on pellet and tap water ad libitum. Animal

Table 1: Effect of the ethanolic extract of henna roots on the duration of the different phases of the estrous cycle in rats.

Group	Treatment	Dose (mg/kg)	No. of days in proestrous	No. of days in estrous	No. of days in metestrous	No. of days in diestrous
1.	Control (1%Tween80)	--	2.062±0.026	3.095±0.038	3.923±0.032	5.215±.0066
2.	Ethanolic extract	200	7.065±0.055^b	1.233±0.044^b	1.602±0.070^b	5.135±0.036^b
3.	Ethanolic extract	400	7.085±0.035^a	0.056±0.223^a	0.740±0.015^a	6.322±0.077^a

Values in tables are represented in Mean ± SEM (n = 6)

a = P <0.02 is significant when compared with control groups, b = P<0.05 is significant when compared with control groups.

Table 2: Effect of the ethanolic extract of henna roots on the ovarian weight and cholesterol level.

Group	Treatment	Dose (mg/kg)	Ovarian weight in mg/100g body weight	Cholesterol level in ovary (mg/50mg)
1.	Control (1% Tween80)	---	40.357±0.096	0.378±0.032
2.	Ethanolic extract	200	30.393±0.135^b	1.058±0.017
3.	Ethanolic extract	400	34.188±0.062^a	0.465±0.011

Values in tables are represented in Mean ± SEM (n = 6)

a = P<0.02 is significant when compared with control groups, b = P<0.05 is significant when compared with control groups.

Table 3: Abortifacient effect of ethanolic root extract of henna on rats when fed orally between days 6 to day 15 of pregnancy.

Treatment	Dose (mg/kg)	Average No. of foetus	Average No. of rats aborted	Abortion in %
Control (1% Tween80)	-	9.83 ± 1.25	6/0	0%
Ethanolic extract	200	6.17 ± 0.87^b	6/0	0%
Ethanolic extract	400	-	6/6	100%

Values in tables are represented in Mean ± SEM (n = 6), b = P<0.05 when compared with control.

were handled in this study as per the International Guidelines for Handling Experimental Animals (OPPT guidelines).

Antiovulatory activity:

Vaginal smear from each rat was examined dally for 15 days, and those rats exhibited three regular cycles ⁽¹²⁾ were included in the study. The selected were divided into three groups of six animals each. Drugs and vehicle were started in estrous phase and administrated orally; daily for 15-day treatment was to cover three regular estrous cycles. Group I received vehicle only (1% tween 80 p.o daily) and served as control. Groups II and group III received ethanolic extract 200 and 400mg/kg respectively. The 15-day treatment was to cover three regular estrous cycles. Vaginal smear from each animal was observed every morning between 7 to 10 am. On 16^thday, 24 hrs after the last treatment, the animals from each group were sacrificed. Ovaries and uteri were dissected out, freed from extra deposition and weighed on sensitive balance (Precisa, XB series). One other ovary was fixed in 10% formalin buffer for histological study.

Abortifacient activity:

Proven female Wistar rats weighing between 145 to 200g were selected and left over night with males of proven fertility in ratio of 2.1 in the proestrous phase day 1 at the pregnancy was confirmed by the presence of spermatozoa in the vaginal smear. The pregnant rats divided into three groups of animals each. Group I received vehicle only (1% tween 80 per oral daily) and served as control. Group II and group III received ethanolic extract at 200 and 400mg/kg per orally daily respectively. The extracts were administrated orally through gastric gavages from the 6^th to 15^th day of pregnancy (period of organogenesis). The animals were laprotomised after cervical dislocation of the on 19^th day of pregnancy. Both horns at the uterus were observed for the number of implantation sites, resorptions, and dead and alive fetuses. (12), (13), (14).

Statistical Analysis:

The data was analyzed using one way ANOVA followed by the Tukey-Kramer multiple comparison post-test. A P value < 0.05 was considered to be statistically significant.

RESULT:

Dose fixation:

Dose fixation was carried out by staircase method on Swiss mice (80 - 100gm).All the extracts were mixed in 1% of tween 80 and dissolved in distilled water, and they were administrated to mice orally by means intra gastric catheter. It was observed that none of the animal was found to be lethal even at the dose of 4000mg/kg body weight. Therefore, we selected 1/10^th& 1/20^thof the dose was selected for the study.

Ethanolic extract at 200mg and 400mg/kg caused a significant decreased in the duration of the estrous and metestrous phases, no change in the duration diestrous phase, and significant increase in the duration of the proestrous phase, compared with control group. (Table1)

Ethanolic extract at 200mg and 400mg/kg significantly decreased the weight of ovaries, compared with control group (Table-2). An increase in preovulatory follicles and atretic follicles was seen in the treated groups. A highly significant increase in ovarian cholesterol level was observed at 200mg /kg compared with control group. At 400 mg/kg ethanolic extract showed a highly significant abortifacient effect. At 200mg/kg, ethnolic extract did not show any abortifacient activity.

DISCUSSION:

Many crude extracts and active principles derived from medicinal plants were evaluated for their anti fertility effects in animal models. Many changes occur in the ovary during the estrous cycle. During the maturation of pre ovulatory follicles, ovulation taken place under the combined and balanced influence of ovarian and extra ovarian hormone. Imbalance in this hormone leads to irregulatory in the ovarian functions and duration of the estrous cycle. (16, 17)

The estrous cycle in the rats treated with extract (200 and 400mg/kg) showed a decrease in the duration estrous and metestrous phases. It was also characterized by a prolongation of proestrous phase. The prolongation of proestrous phase indicates that maturation of the follicle in the preovulatory phase was delayed, leading to non-maturation of graiffian follicle Non-availability of matured graffian follicle was indicated by reduction in the estrous and metaestrous phases. Therefore ovulation was inhibited. The result was further supported by our histopathological studies in which the transverse section of the ovary showed the presence of primary or developing follicles. The presence of increased atretic follicles in the rats treated with ethanolic extract, compared with control rats, indicates that the extract promotes the degeneration of preovulatory follicles. Cholesterol is the precursor for the steroidogenesis of ovarian endocrine tissues. The significant increase in ovarian cholesterol in the treated groups (200mg/kg) indicates that cholesterol is not used for steroidogenesis. But 400mg/kg dose did not show a similar effect indicating that it occurs only lower dose. Abortion refers to the premature expulsion of the products of conception from the uterus. Abortion may be to maternal exposure to chemicals, which can disrupt pregnancy cause detachment of the embryo (Medeiros RM et al, 2000). Ethanolic extract at 400mg/kg showed 100% abortificient activity.

From the results of this investigation it may be concluded that henna root extract a highly significant decrease in the duration of estrous and metaestrous phase and increase the duration of the proestrotus phase was seen. In addition, highly significant decrease in ovarian weight and increase the cholesterol level, when compared with the control group was noted. This finding indicates the extract of henna roots produces inhibition of ovulation. The result of administration of extract to the pregnant rats during organogenesis shows that the extract is abortificent only at higher dose of 400mg/kg. So all this observation suggests that the henna roots extract produced antiovulatory and abortificient activity. Further studies is necessary to confirm the potent active components for the observed effect and exact mechanism of action, work on the semi purified total extract is in progress to validate the present findings.

REFERENCES:

1. Handa G.Kapil A., Sharma S., and Singh J. (1997), Lawnermis acid a new anticomplementary triterpenoid from Lawsonia inermis seeds. Indian J. Chem., Sect. B.36, 252-256.

2. Knecht W., Henseling J., and Loffler M. (2000), Kinetics of inhibition of human and rat dihydroorotate dehydrogenase by atovaquone, lawsone derivatives, brequinar sodium and polyporic acid. Chem. Biol. Interact. 124, 61-76.

3. Wagner H., Kreher B., and Jurcic K. (1988). In vitro stimulation of human granulocytes and lymphocytes by pico- and femtogram quantities of cytostatic agents. Arzneim. Forsch. 38, 273-275.

4. Malekzadeh F. (1968), antimicrobial activity of Lawsonia inermis L. Applied Microbiol 16, 663-664.

5. Abd-el- Malek Y.A., El-Leithy M.A., Reda F.A., and Khalil M. (1973). Antimicrobial principles in leaves of Lawsonia inermis L. Zentralbl. Bakteriol. Parasitenkd Infektionskr Hyg. 128, 61-67.

6. Chang H., and suzuka S.E., (1982). Lawsone (2-OH-1,4- naphthoquinone) derived from the henna plant increases the oxygen affinity of sickle cell blood. Bio-chem. Biophys. Res. Commun. 107, 602-608.

7. Clarke D.T., Jones G.R., and Martin M.M (1986), The anti – sickling drug lawsone (2-OH-1,4 – naphtha-quinone) Protects sickled cells against membrane damage, Biochem. Biophys, Res. Commun.139, 780 – 786.

8. Anaad K.K., Singh B., Chand D., and Chandon B.K. (1992). An evaluation of Lawsonia alba extract as hepatoprotective agent. Planta Med. 58, 22-25.

9. Ali M., and Grever M.R. (1998). A cytotoxic naphthoquinone from Lawsonia inermis. Fitoterapia LXIX ( 2 ) ,1810-1813.

10. Ali B H, Bashir A.K., and Tanira M.O. (1995), anti-inflammatory, antipyretic, and analgesic effects of Lawsonia inermis L (Henna) in rats. Pharmacology 51, 356-63.

11. Shivalinagappa. H., Satyanaryan. ND., and Purohit. MG. (2001). Antimplantation and pregnancy interruption efficacy of Rivea hypocrateriformis in the rat. J Ethnopharmacol; 74: 245-9.

12. -60. Hafez ES. (1970). Reproduction in breeding techniques for laboratory animals. Philadelphia: Leaand febiger.

13. Medeiros RM., Gorniak SL., and Guerra JL. (2000). Fetotoxicity and Reproductive effects of monocrotaline in pregnant rats. J Ethnopharmacol; 69: 181-8.

14. Feranada CG. Almedia., and Lone P. Lemonica (2000). The toxic effects of Coleus barbatus B on the different periods of pregnancy in rats. J Ethnopharmacol; 73: 53

15. Nath D., Sethi N., Singh RK., and Jain AK. (1992). Commonly used Indian abortifacient plants with special reference to teratologic effects in rats. J. Ethnopharmacol; 36: 147-54.

16. Prakash AO., and Mathur R. (1979). Studies on oestrous cycle of the albino rats: Response to Embelia ribes extracts. Planta Medica; 36: 131-41.

17. Shivalinagppa H., Satyanarayan ND., Purohit MG., Sharanabasappa A., and Patil SB. (2002). Effect of ethanol extract of Rivea hypocrateriformis on the estrous cycle of the rat. J Ethnopharmacol; 82: 11-7.

18. Circosta C., Sanogo E., and Occhiuto F. (2001). Effects of Calotropis procera on oestrous cycle and on oestrogenic functionality in rats. Farmaco; 56: 373-8.

Received on 08.08.2009

Accepted on 16.08.2009

Research J. Pharmacology and Pharmacodynamics 2009; 1(1): 18-20