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Indian J Pharmacol. 2010= Oct; 42(5): 306=E2=80=93311.

PMCID: = PMC2959215

Antiovulatory and abortifacient effects of Areca catechu (be= tel nut) in female rats

Jyot= i Shrestha, Tara Shanbhag, Smita Shenoy, Arul Amuthan, Krishnananda Prabhu,¹ Stuti Sharma,¹ Samik Banerjee,^{<=
/sup> and Sajala Kafle}

Department of Pharmacolog= y, Kasturba Medical College, Manipal, Manipal University, Karnataka, India<= /div>

¹Department of Biochemis= try, Kasturba Medical College, Manipal, Manipal University, Karnataka, Indi= a

Correspondence to: Dr. Tara Shanbh= ag E-mail: ni.oc.oohay@7gahbnahsarat

Author= information =E2=96=BA Article notes =E2=96=BA Copyright and License information= =E2=96=BA

This article has= been cited by other articles in PMC.

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Abstract

Objectives:

To study th= e antiovulatory and abortifacient effects of ethanolic extract of Areca= catechu in female rats.

Ma= terials and Methods:

For antiov= ulatory effect, ethanolic extract of A. catechu at 100 and 300 mg/= kg doses was administered orally for 15 days. Vaginal smears were examined= daily microscopically for estrus cycle. Rats were sacrificed on 16^{th<=
/sup> day. Ovarian weight, cholesterol estimation, and histopathological st=
udies were done. Abortifacient activity was studied in rats at 100 and 300=
mg/kg doses administered orally from 6^th to 15^th day=
of pregnancy. Rats were laparotomised on 19^th day. The number=
of implantation sites and live fetuses were observed in both horns of the=
uterus.}

Results:

The extract of A. catechu showed a= significant decrease in the duration of estrus at 100 mg/kg (P = =3D 0.015) and 300 mg/kg doses (P =3D 0.002) as compared with cont= rol. Metestrus phase was also significantly reduced at 100 mg/kg (P =3D 0.024) and 300 mg/kg doses (P =3D 0.002). There was a signif= icant increase in proestrus (P < 0.001) phase. However, diestru= s phase was unchanged. Histopathological study of the ovaries showed mainly= primordial, primary, and secondary follicles in the test groups as compare= d to control. There was also a significant (P =3D 0.002) decrease= in ovarian weight and a significant (P =3D 0.021) increase in ova= rian cholesterol level at 100 mg/kg dose. In the study to evaluate abortifa= cient effect, the mean percentage of abortion with 100 and 300 mg/kg doses= were 75.5% and 72.22%, respectively, which was significantly (P= =3D 0.008 and P =3D 0.006, respectively) increased when compared= with control.

Conclusi= on:

The ethanolic extract of A. catechu at doses of 100 and 300 mg/kg has antiovulatory and abort= ifacient effects.

Keywords: Abortifacient, Areca= catechu, antiovulatory, implantation sites, ovarian cholesterol

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Introduction

Infertility= is one of the most common problems with significant medical, psychosocial,= and economic implications. There are various factors responsible for male= and female infertility. Environmental factors are also responsible like vo= latile organic solvents or silicones, biological agents (viruses, parasites= ), physical agents (radiation, exposure to electric shock, excessive vibrat= ion, or heat),[1] chemical dust, pesticides;= [2] alcohol,[3] smoking,[4]= caffeine consumption,[5,6] areca nut chewing,[7] etc. Chewing the mixture of areca nut and betel leaf= is a tradition, custom, or ritual, which dates back thousands of years fro= m South Asia to the Pacific region. It has now been established that areca= nut use causes variety of health hazards including precancerous lesions in= mouth. Use of areca nut is common among female population also. Areca nut= is most commonly chewed as a constituent of betel quid. The betel quid is= a mixture of betel leaf, areca nut, and slaked lime (calcium hydroxide).= The important constituents of areca nut are tannins (15%), gallic acids,= oily matter (fat 14%), gum and three main alkaloids arecoline (0.07%), are= caine (1%), and guracine, which have the property of vasoconstriction. Arec= aidine, guvacoline, guvacine, and choline are present in traces only. Pharm= acological actions of arecoline resembles that of muscarine and pilocarpine= . Dried areca nut has hepatoprotective,[8]= hypoglycemic,[9] astringent, vermifugal, si= alogogue,[10]= antibacterial, antioxidant, antiseptic, bronchostimulant, euphorient, and= wound-healing properties.[7] In addition, it has abortifacient, anti-implantation, and ant= ifertility activities.[11] Studies have revealed that areca nut in male albino rats of Wi= star strain causes morphofunctional changes such as stimulation of hormonog= enesis and disruption of spermatogenesis.[12] A survey of literature revealed that antif= ertility effect of areca nut has not been documented in female albino rats.= Hence, this study was undertaken to evaluate antiovulatory and abortifacie= nt effects of A. catechu in female albino rats.

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Materials and= Methods

Plant material= and extraction[13= ]

The nuts of Areca cat= echu were purchased from local market, chopped into small pieces, and= dried under the sun for few days. The dried chopped nuts were powdered and= defatted in petroleum ether. The residue was hot extracted in soxhlet appa= ratus (Tensil Glass Works, Bangalore, India) using 400 mL of 70% ethanol (Q= ualigens Fine Chemicals, Mumbai, India) for five cycles. This extract was= filtered, lyophilized, and concentrated over a water bath to obtain dried= extract. This extract was stored in a desiccator (Quality Traders, Eranaku= lum, Kerala). A suspension of the extract in 4% gum acacia (Nice Chemicals,= Cochin, India) was used for the study.

Acute toxicity study

Acute toxicity was done in female albino rats of Wistar strain weighing= 150=E2=80=93200 g. Rats were fasted overnight. They were divided into six= groups of two rats each. The alcoholic extract of betel nut was administer= ed through the feeding tube to each group in ascending and widely spaced do= ses, namely 10, 30, 100, 300, 1000, and 3000 mg/kg (p.o.). The rats were ob= served continuously for 2 h and then occasionally for further 4 h, and fina= lly overnight mortality was recorded.[14] No signs of toxicity were observed even with 30= 00 mg/kg of ethanolic extract of A. catechu. Therefore, two doses= of the extract were chosen, 300 mg/kg corresponding to one-tenth of the ma= ximum tolerated dose (3000 mg/kg) and the other was 100 mg/kg.

Animals

The experimental protocol was approved by Institutional Animal= Ethics Committee. The rats were maintained under standard conditions in an= imal house approved by the Committee for the purpose of Control and Supervi= sion of Experiments on Animals (CPCSEA). The temperature was 23 =C2=B1 2=C2= =B0C and humidity was 50 =C2=B1 5%. Mature, healthy female albino rats weig= hing 150=E2=80=93200 g were used. They were provided with standard rat feed= (Amrut lab animal feed, Pranav Agro Industries Ltd., Sangli, Maharashtra)= and water ad libitum.

<= h3>Estrus cycle

Phases of estrus cyc= le of each female rat used for study of antiovulatory and abortifacient eff= ects were determined by observing vaginal smear daily between 9 and 10 AM= continuously for 15 days. Vaginal smear was prepared by introducing a drop= of distilled water into the vagina with the help of a dropper, collecting= back and placing it on a clean slide after adding a drop of glycerin.[15] The prepared= smear was examined microscopically under low power for different types of= cells [Figu= re 1]. There are four phases in estrus cycle of rat. If majority= of cells are leucocytes, then it was labeled as in diestrus phase [Figure 1a]. Presence of large number of nucleated cells indicated proestrus phase= [Figure 1b= ]. Estrus phase was confirmed when the smear showed more than 50= % cornified epithelial cells [Figure 1c]. Metestrus phase was indicated by= the presence of many neutrophils and scattered squamous epithelial cells= in the smear[14] [Figure= 1d]. Rats exhibiting three consecutive regular estrus cycles we= re chosen for the study.

Figure 1

Va= ginal smear of rat showing different phases of estrus cycle: (a) Diestrus,= (b) proestrus, (c) estrus, and (d) metestrus.

Antiovulatory effect

This was studied by observing the effect of= A. catechu on the duration of phases of estrus cycle, estimation= of ovarian weight and cholesterol. The rats with three regular estrus cycl= es were divided into three groups of six each. Rats were grouped in such a= way that all the groups were in estrus phase at the start of treatment. Ra= ts in Group I received 2 mL of 4% gum acacia (p.o. daily) for 15 days and= served as control. Groups II and III received ethanolic extract of A.= catechu (p.o. orally) at 100 and 300 mg/kg body weight, respectively,= for 15 days. Daily vaginal smears were observed in the morning between 9= and 10 AM. The number of days spent in each phase by each rat was calculat= ed. Then average number of days spent in each phase by each group was calcu= lated. At the end of the study, i.e. on the 16^th day, 24 h after= the last dose, all the rats were killed by cervical dislocation.[16] Ovaries were diss= ected out, freed from extra deposition, and weighed. Right ovary from each= rat was processed for cholesterol estimation and another for histopatholog= ical study.[17= ]

Cholesterol estimation

Ovarian tissue was weighed and hom= ogenized in cold saline to get uniform suspension (1:20 ratio that is 20 = =C2=B5L of saline was added per milligram of tissue). Cholesterol was estim= ated using manual kit supplied by Aspen Laboratory Pvt. Ltd., Delhi. Values= were expressed as microgram/milligram of ovary.

Abortifacient effect

Rats exhibiting three consecutive regular estrus cycles were chosen= for the study. The female rats in proestrus phase were mated with male rat= s of known fertility in the ratio of 2:1 in the evening. Vaginal smears wer= e examined in the following morning for the presence of sperms to confirm= mating. The pregnancy rate was 100%. Female rats exhibiting thick clumps= of spermatozoa in the vaginal smear were chosen for the study and that day= was considered as day one of pregnancy. Eighteen pregnant rats were divide= d into three groups of six each. Group I served as a control, which receive= d 2mL of 4% gum acacia (p.o. daily). Groups II and III received ethanolic= extract of A. catechu (p.o. daily) at 100 and 300 mg/kg body weig= ht, respectively, from 6^th to 15^th day of pregnancy= which is the period of organogenesis. On 19^th day of pregnancy,= all the rats were laparotomised under light ether anesthesia. The number= of implantation sites and live fetuses were noted in both horns of the ute= rus.[16] The= observations of the drug-treated groups were compared with control group.<= /p>

Percentage of abortion was calculated by the formula:

Percentage=E2=80=83of=E2=80=83abortion=E2=80=83=3D=E2=80=83(Number=E2=80=83of=E2=80=83implantatio= ns=E2=80=83=E2=88=92=E2=80=83Number=E2=80=83of=E2=80=83live=E2=80=83= fetuses<= span style=3D"font-family: MathJax_Main;">)=E2=80= =83=C3=97=E2=80=83100= Number=E2=80=83of=E2=80=83implantations<= span style=3D"display: inline-block; width: 0px; height: 4.002em;"><= /span>= =

$$

Statistical analysis

Re= sults were expressed as mean with standard deviation for data following nor= mal distribution and median with interquartile range for skewed data. P= < 0.05 was considered statistically significant. The differences= between experimental groups were compared using one-way ANOVA followed by= Tukey=E2=80=99s post hoc test and also Kruskal=E2=80=93Wallis H-test followed by Mann=E2=80=93Whitney test with Bonferroni correcti= on for skewed data.

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Results

Acute toxicity studies

Mortality and behavioral changes were not observed in the acute to= xicity study till the dose of 3000 mg/kg. Therefore, two doses (100 and 300= mg/kg) were selected for the study.

Antiovulatory effect

Th= e number of days the rats spent in proestrus phase in groups II an= d III was significantly (P < 0.001) higher as compared with the= control group. There was also a significant decrease in the median duratio= n of the estrus at 100 mg/kg (P =3D 0.015) and 300 mg/kg= doses (P =3D 0.002) as compared to the control. Metestrus phase was also significantly reduced at 100 (P =3D 0.024) and 30= 0 mg/kg doses (P =3D 0.002) as compared to the control. However,= there was no significant change in the duration of the diestrus= phase between the groups as shown in Table 1.
Table 1
Effect of et= hanolic extract of A. catechu on the duration of the different pha= ses of estrus cycle in rats

Effect on ovarian weight (right ovary)

In drug-treated groups (100 and 300 mg/kg doses),= there was a significant decrease (P =3D 0.002) in ovarian weight= as compared with the control as indicated in Table 2.

Table 2
Effect= of ethanolic extract of A. catechu on ovarian weight and choleste= rol level in rats

Effect on ovarian cholesterol level (right ovary)

The mean ovarian cholesterol level in group= II (100 mg/kg dose) showed a significant increase (P =3D 0.021)= when compared with the control group, but there was no significant change= in cholesterol level in group III (300 mg/kg dose) as shown in Table 2.

Effect on histopathological chan= ges (left ovary)
Section of the ova= ries in the control group showed primordial, primary, graafian, ruptured fo= llicles, and corpus luteum in the ovarian stroma along with blood vessels,= adjacent fibrous areas with mature adipose tissue [Figure 2a]. In group= II, ovarian sections showed only primordial, primary and secondary follicl= es but were devoid of graafian and ruptured follicles [Figure 2b]. In group= III, 50% of the ovarian sections had histopathological features same as in= control group and remaining 50% were devoid of graafian follicles, rupture= d follicles, and corpus luteum.
<= span>(a) Section of rat ovary in control group showing mature graafian foll= icle and developing follicles. (b) Section of rat ovary in group II showing= primordial and primary developing follicles.

Abortifacient effect<= /h3>
The mean percentage of abortion wit= h 100 and 300 mg/kg doses were significantly increased (P =3D 0.00= 8 and P =3D 0.006, respectively) when compared with the control gr= oup as indicated in Tab= le 3. There was atrophy and a decrease in the number of fetuses= in the drug- treated groups [Figures [F= igures3b3b and =E2=80=8Band3c3c].
Table 3
= Abortifacient effect of ethanolic extract of A. catechu in= rats

Figure 3
Ut= erus with the fetuses and implantation sites: (a) control, (b) 100 mg/kg of= A. catechu, and (c) 300 mg/kg of A. catechu.

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Discussion

The= effect of ethanolic extract of A. catechu on the duration of diff= erent phases of estrus cycle, ovarian weight, cholesterol level, and histop= athology were studied in rats. During estrus cycle many physiological, bioc= hemical, morphological, and histological changes occur in the ovaries. Foll= icular growth and ovulation are regulated by endocrine (follicle stimulatin= g hormone, luteinizing hormone, and prolactin) and ovarian hormones (such= as progestin, estrogen, and androgen). Ovarian hormones are produced by di= fferent cell types of the ovary like granulosa cells of the mature follicle= s and the corpus luteum.[18] Imbalance in these hormones leads to irregularity in the ova= rian functions and duration of the estrus cycle.[19]

In our study, the number of days the rats of= groups II and III spent in the estrus cycle showed a significant decrease= in the median duration of estrus and metestrus phases (a= s compared to the control group). Reduction in the estrus and = metestrus phases indicates nonavailability of the matured graafian fol= licles or nonmaturation of secondary follicles. Therefore, ovulation was in= hibited. There was an increase in the mean number of days the rats spent in= proestrus phase in groups II and III, which was significant as co= mpared with the control group. The prolongation of the proestrus= phase indicates that maturation of the follicles in the preovulatory phase= was delayed leading to nonmaturation of graafian follicles. This was due= to nonavailability of estrogen produced by the granulosa cells which is es= sential for the maturation and differentiation of the ovarian follicles, or= imbalance in the endogenous steroid, protein, and hormones. This result wa= s further supported by the histopathological studies [Figure 2b], in which= the transverse section of the ovaries in the drug-treated groups showed th= e presence of mainly primordial follicles, primary follicles, and secondary= follicles in the ovarian stroma along with dilated blood vessels, fibrous,= and adipose tissues. In the control group, ovarian sections showed in addi= tion to above, graafian follicles, ruptured follicles, and corpus luteum,= which indicates that ovulation has taken place and there was no hormonal= imbalance [= Figure 2a].

Ovary consists of aggregation of= three endocrine tissues, the stroma, the follicle, and corpus luteum. The= weight of these tissues constitutes the net weight of the ovary. During th= e estrus cycle, the weight of the ovarian tissue increases under the influe= nce of gonadotrophic and steroidal hormones. There was a significant decrea= se in the weight of ovaries in groups II and III as compared with the contr= ol group, which indicates a decrease in the activity of the stroma, the fol= licle, and the corpus luteum in the ovary. This decrease may be due to the= nonavailability of gonadotrophic or steroidal hormones or both.[20]

Cholesterol is the precurso= r for the steroidogenesis of ovarian endocrine tissues, estrogens, progesti= ns, and androgens. The significant increase in ovarian cholesterol in group= II when compared with the control group, indicates that cholesterol is not= utilized for steroidogenesis.[20] There= was no significant increase in the ovarian cholesterol level in group III= as compared to control group.

Insuff= icient progesterone secretion by the corpus luteum or placenta, also termed= a luteal phase defect, has been suggested to cause abortion.[21] Therefore, this could= be a possible cause for the significant increase in percentage of abortion= and abortifacient effect seen in drug-treated groups. Habit of chewing bet= el nut may cause abortion or disturb normal gestation. Hence, awareness nee= ds to be developed among female populations about the health hazards of bet= el nut.

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Conclusion

All th= e parameters in this study showed that the alcoholic extract of A. cate= chu has antiovulatory and abortifacient effects.

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Footnotes

Source of Support: Nil

Conflict of Interest: None declared.

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References

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2. Hage ML, Frazier LM. New York: Reproductive Hazards of the Workpla= ce; 1998. In normal reproductive and developmental biology; p. 3.
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= 5. Bolumar F, Olsen J, Rebagliato M, Bisan= ti L. Caffeine intake and delayed conception: A European multicenter study= on infertility and subfecundity. European Study Group on Infertility Subfe= cundity. Am J Epidemiol. 1997;145:324=E2=80=9334. [PubMed]
6. Sowers MR, Crawford S, McConnell DS, Randolph JF, Jr,= Gold EB, Wilkin ML, et al. Selected diet and lifestyle factors are associ= ated with estrogen metabolites in a multiracial/ethnic population of women.= J Nutr. 2006;136:1588=E2=80=9395. [= PubMed]
7. Azeez S, Amudhan S, Adiga S, Vasanthakumar A, Rao N, Udupa AL, Sana= th H. Wound healing profile of Areca catechu extracts on different= wound models in wistar rats. Kuwait Med= J. 2007;39:48=E2=80=9352.

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14. Ghosh MN.= 3rd ed. Kolkata: Hilton and Company; 2005. Fundamentals of Experimental= Pharmacology; pp. 193=E2=80=934.

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17. Sharma JD, Sharma L, Yadav P. Antifertility efficacy of Piper bete= l Linn.(Petiole) on female albino rats. Asian J Exp Sci. 2007;21:145=E2= =80=9350.

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