|Year : 2014 | Volume
| Issue : 1 | Page : 16-22
Phytochemical analysis and a study on the antiestrogenic antifertility effect of leaves of Piper betel in female albino rat
Department of Pharmacology, VSS Medical College and Hospital, Burla, Odisha, India
|Date of Web Publication||4-Feb-2015|
Department of Pharmacology, VSS Medical College and Hospital, Burla, Odisha
Source of Support: None, Conflict of Interest: None
Objective: To study the effect of graded doses of the aqueous and methanolic extract of the leaves of Piper betel (PB) Linn (PBL) on the estrous cycle of female albino rats.
Materials and Methods: Both the extracts were tested for their effect on the estrous cycle at three dose levels of 500, 1000 and 1500 mg/kg/day and the vaginal smears were examined daily microscopically for the different phases of the estrous cycle for a period of 30 days.
Result: The estrous cycle was irregular and prolonged in the treated groups indicating anestrus condition, which would result in infertility. Both types of the extract showed a significant decrease in the duration of proestrus and estrus with a prolonged diestrus at 1000 mg/kg/day and 1500 mg/kg/day doses as compared with control. However, no change was seen in the metestrus phase. The rats treated with PB showed a significant (P < 0.05), dose-dependent decrease in the estrus phase, in comparison to the control group, the effect was more with the methanolic extract. Large, cornified cells appeared after proestrus phase with decreased number of cornified cells. There was a significant reduction in the number of the estrous cycle, in the PBL treated group. Anestrus phase appeared in all the rats treated with the aqueous and methanolic PB extract, which was not observed in the control group. However, the aqueous extract at a dose of 500 mg/kg/day had no effect either on the estrous cycle or on its different phases. The observed effect of PB leaves could be due to the flavonoids and saponin contents, which also contributes to its antiestrogenic mechanism of action.
Conclusion: Both the aqueous and methanolic extract of PBL possesses antifertility effect in female albino rats.
Keywords: Anestrus phase, antiestrogenic, estrous cycle, phytoconstituents, Piper betel, vaginal smear
|How to cite this article:|
Biswal S. Phytochemical analysis and a study on the antiestrogenic antifertility effect of leaves of Piper betel in female albino rat. Ancient Sci Life 2014;34:16-22
| Introduction|| |
Fertility control has become a major concern for people of all walks of life in the developing countries. The commonly used conventional combined oral pills are usually associated with many untoward adverse effects, necessitating newer and indigenous drugs. Plant products have always been used because of their therapeutic potential, easy availability, economic reasons and lack of harmful side effects.  Many such plant products have been used as antifertility agents in folklore as well as in the traditional systems of medicine. 
Piper betel (PB) Linn (PBL) of family Piperaceae commonly known as the betel vine or paan is an important medicinal plant whose leaves are widely used as a mouth freshener and it is extensively grown in India, Sri Lanka, Malaysia, Thailand, Taiwan and other Southeast Asian countries. Comprising of about 10 genera and 2000 species, it is largely distributed in the tropical and subtropical regions of the world. Its leaves have traditionally been used for chewing purposes along with other condiments as part of the tradition, custom, or rituals. This practice is ancient and is prevalent from South Asia to the Pacific region.  Studies have revealed that its leaves possess antidiabetic, antiulcer, antifertility, cardiotonic, respiratory depressant and antihelminthic properties. ,, In Indian folk medicine, betel leaf is popularly used as an antiseptic and applied on wounds for its healing effects.
The fresh juice of betel leaves has been used in many ayurvedic preparations for their diverse pharmacological actions. They have been reported to possess antihistaminic, antioxidant, anti-inflammatory, antitumour, antimutagenic, radioprotective and immunomodulatory effects. ,,,,,, Studies have reported that expectant mothers who chewed betel quid, during their pregnancies had a significant increase in adverse outcomes in their newborns.  Though there are studies on the alcoholic extract of the leaf stalk of PB, there are no studies reporting the effect of aqueous or the methanolic extract of the whole PB leaves on any parameters of fertility.
Since a pregnancy requires the functioning of a normal estrous cycle, any interference with the estrous cycle would indicate the antifertility effect of a compound. Thus, the estrous cycle serves as a surrogate marker and has been frequently utilized in evaluating the antifertility effect of any agent.  Besides this, the estrous cycle of the female albino rat is very short as it lasts for a period of only 4-5 days, which makes it convenient to observe any changes that can be produced by any administered exogenous agent.
Thus keeping all the facts in view, this study was conducted with an objective of investigating the antifertility effect of both the aqueous and methanolic extract of the total leaf of PB, by observing their effect on the estrous cycle of female albino Wistar rats using the estrus cycle as a parameter of fertility.
| Materials and methods|| |
The leaves of the PB plant were collected from the Khurdha district of Odisha during the period from May 2012 to June 2012. The specimens were identified and authenticated by a Botanist of The Ravenshaw University, Cuttack, Odisha. The leaves were cleaned, shade dried and pulverized to a powder form. The powder was then passed through a sieve no. 40 and preserved in air-tight plastic containers till their use in preparation of their aqueous and methanolic extracts.
Drugs and chemicals
The following drugs and chemicals were used-aqueous and methanolic extracts of the leaves of PB, distilled water and dyes such as hematoxylin and eosin.
Preparation of the extracts
Aqueous extract (Maceration method)
A volume of 200 g of the powdered leaves were stirred with 1000 ml of double distilled water and left aside for 24 h. The mixture was filtered using a Whatmann no. 1 filter paper. The filtered solution was then dried at room temperature and the dry extract collected. The percentage yield of extract was found to be 7.1%.
Powdered leaves of PB were packed in a soxhlet apparatus, and continuous extraction was done using 70% methanol for 72 h. The extracts were then evaporated under reduced pressure to obtain solid masses. The final extract obtained amounted to about 5.58%.
All the extracts were stored in desiccators for further use. A freshly prepared suspension of the extract in distilled water was used for the study.
Phytochemical screening of the extracts
A preliminary phytochemical study (color reactions) of the plant extracts were performed using standard procedures. 
- Test for phenols: 2 ml of ferric chloride solution was added to 0.5 g of the extract. The presence of phenols was determined by a reddish brown coloration at the interface
- Salkowski test for terpenoids: 2 ml of chloroform was added to 0.5 g of the extract. Then 3 ml of concentrated sulfuric acid was carefully added along the sides of a test tube. A reddish brown coloration was seen at the interface indicating the presence of terpenoids
- Test for flavonoids: 5 ml of diluted ammonia solution was added to the extract followed by addition of 1 ml of concentrated sulfuric acid. A yellow color appeared that disappeared on standing, and this indicated the presence of flavonoids
- Test for saponins: When 5 ml of distilled water was added to 0.5 g of the extract and shaken vigorously, froth appeared. This stable froth was then mixed with 3 drops of olive oil and shaken vigorously. The formation of a stable emulsion denoted the presence of saponins.
- Test for tannins: 0.5 g of the extract was boiled with 10 ml of water for 5 min and then filtered. 2-3 drops of 0.1% ferric chloride was added to the filtrate and was observed for brownish green or a blue-black coloration to prove the presence of tannins
- Test for alkaloids: Ten ml of acid alcohol was added to 0.5 g of the extract that was then boiled and filtered. To 5 ml of the filtrate 2 ml of diluted ammonia was added followed by 5 ml of chloroform to extract the alkaloidal base present in the extract. The extracted alkaloidal base was then filtered, following which 10 ml of acetic acid was added and finally divided into two portions. To one part of the obtained extract, Mayer's reagent was added while Dragendorff's reagent was added to the second portion. The formation of cream with Mayer's reagent or reddish brown precipitate with Dragendorff's reagent was considered as a positive test for the presence of alkaloids
- Tests for steroid glycosides: 2 ml of meta dinitrobenzene solution was added to the 0.5 g of alcoholic solution of the drug containing a few drops of NaOH and heated, formation of violet color indicates the presence of keto-steroid.
Twelve weeks old young female albino rats of the Wistar strain weighing about 150-200 g and showing a regular estrous cycle of 4-5 days (for consecutive 5 cycles) were selected for the study. They were randomly divided into seven equal groups of six rats in each group. The rats were maintained under standard conditions in the departmental animal house as approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals. The temperature was maintained at around 23 ± 2°C and at a humidity of 50 ± 5%. They were provided with standard rat feed and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee of S.C.B. Medical College, Cuttack, Odisha prior to the commencement of the study.
Fertility studies: Effect on the estrous cycle
The short length of the estrous cycle of rats makes them ideal models for fertility studies. The estrous cycle in a female albino rat is characterized by four phases, proestrus, estrus, metestrus and diestrus that occur in a cyclical pattern every 4-5 days. These stages can be identified microscopically using the vaginal smear method as described by Goldman et al.  The smears can be prepared by introducing a drop of distilled water into the vagina with the help of a dropper, collecting it and placing it on a clean slide after adding a drop of glycerin. The smears are then stained with hematoxylin and eosin and observed under high power microscope. If the majority of the cells in the smear are leucocytes, then the rat is said to be in the diestrus phase while presence of a large number of nucleated cells suggests the proestrus phase. In the estrus phase, there are >50% of cornified epithelial cells in the smear while the metestrus phase is indicated by the presence of plenty of neutrophils and scattered squamous epithelial cells.  Nucleated epithelial cells are relatively constant in number and hence are not taken into account in determining the different phases. Thus, the three types of cells recognized in a vaginal smear would be epithelial, cornified and leucocytes cells. In this study, the proportion of the different cells were taken into account to determine the different phases of the estrous cycle.  Vaginal smears showing <30% cornified epithelial cells are usually denoted as the anestrus phase.
The significance of these phases is that they correlate with the levels of the circulating ovarian hormones, estrogen and progesterone which reach a peak level during proestrus, intermediate on metestrus and are lowest on the day of estrus. On the other hand during the diestrus phase the circulating estrogen levels are at their intermediate levels while progesterone levels are low. The diestrus index was calculated as: Number of days in diestrus/total duration of treatment in days multiplied by 100.
Experimental design of the study
The selected rats exhibiting three consecutive regular estrous cycles were administered drugs as follows:
- Group 1: Served as control and received 2 ml of distilled water
- Group 2-4: Aqueous extract of PB leaves in a dose of 500 mg/kg/day, 1000 mg/kg/day and 1500 mg/kg/day
- Group 5-7: Methanolic extract of PB leaves in a dose of 500 mg/kg/day, 1000 mg/kg/day and 1500 mg/kg/day.
All the drugs were suspended in 2 ml of distilled water and given orally at the same time of the day that is, between 9.00 am and 10.00 am every day, for a total period of 30 days. The control group received the vehicle in the same dose volume of 2 ml. From the very next day of the drug treatment, the vaginal smears of the treated as well as the control groups were obtained and observed under the microscope so as to correlate the observed changes with the different stages of the estrous cycle. These observations were made for 30 days so as to observe the changes in six complete estrous cycles. The number of the estrous cycles occurring within the study period as well as the average period of each stage of the estrous cycle was then computed.
Change in the body weight and hematoglogical parameters like white blood cells (WBCs), red blood cells (RBCs), hemoglobin content and hematocrit values were measured. Blood glucose, serum cholesterol were also measured in each group at the end of the study period.
All the observations were expressed as mean ± standard error of the mean. The tests for significance was done using the one-way ANOVA test followed by post hoc Tukey Kramer multiple comparison tests using commercial scientific 2D graphing and statistics software published by GraphPad Software, Inc., a privately held California corporation (version 5) and a P < 0.05 was considered to be statistically significant.
| Observations and results|| |
0Phytochemical screening of extracts
Phytochemical screening of the extracts revealed the presence of alkaloids, flavonoids, tannins, sterols, phenols, terpenoids and saponins [Table 1]. The aqueous extract was found to possess high concentrations of alkaloids, flavonoids, glycosides and tannins, while the methanolic extract had high concentration of alkaloids, sterols, phenols, tannins and flavonoids, with moderate concentration of saponins and terpenoids.
Effect of aqueous extract of Piper betel on estrous cycle
The animals of the control groups exhibited a normal estrous cycle for the number of cycles in this group was about 5.82 ± 0.22 days within the study period, with its different phases changing over from proestrus (5.03 ± 0.08 days), estrus (8.01 ± 0.05 days), metestrus (5.87 ± 0.12 days) to diestrus (11.57 ± 0.07) and with a diestrus index of 38.57%, all of which were within normal limits [Table 2].
The aqueous extract of PB leaf in a dose of 500 mg/kg/day had no significant effect either on the number of estrous cycle (5.79 ± 0.34 days) or on any of its phases in the female Wistar rats as compared to the control within the study period. The change in the diestrus index was also not significant.
Whereas the same extract in a dose of 1000 mg/kg/day and 1500 mg/kg/day showed a significant dose-dependent decrease in the duration of proestrus (3.41 ± 0.01 days and 2.91 ± 0.01 days), estrus (5.85 ± 0.34 days and 3.48 ± 0.04 days) with an increase in the duration of diestrus (15.73 ± 0.03 days and 18.03 ± 0.02 days) as compared to that in the control rats. On the other hand, there was no significant change observed in the metestrus phase which was 5.85 ± 0.08 days and 5.89 ± 0.01 days in a dose of 1000 and 1500 mg/kg/day respectively. Further there was a significant reduction in the total number of cycles as evident by 3.87 ± 0.43 days and 3.21 ± 0.41 days at doses of 1000 mg/kg/day and 1500 mg/kg/day respectively which were significantly less as compared to the control group (5.82 ± 0.22 days). The increase in the diestrus index at doses of 1000 and 1500 mg/kg/day was dose-dependent and highly significant.
Effect of methanolic extract of Piper betel on estrous cycle
The methanolic extract at all dose levels showed a significant dose-dependent decrease in the duration of proestrus, estrus with an increase in the duration of diestrus in comparison to the control group [Table 2]. On the other hand, there was no change in the metestrus phases even at a high dose of 1500 mg/kg/day. Treatment of rats with methanolic extracts resulted in significant lesser number of cycles (3.77 ± 0.01 days at 500 mg/kg/day, 2.89 ± 0.39 days at 1000 mg/kg/day and 2.67 ± 0.74 days at 1500 mg/kg/day) suggesting that the extract prolonged the duration of the estrous cycle. Estrous cycle of treated animals was lengthened with persistent diestrus. The vaginal smear showed a pattern of prolonged diestrus in the treated rats at doses of 1500 mg/kg/day with appearance of large cornified cells which were very few in number and the treated group finally became anestrus. There was also a prolonged increase in the diestrus index at all dose levels which were dose-dependent and highly significant. However, vaginal bleeding was not observed in any of the treated animals.
Effect of aqueous and methanol extract of Piper betel on other parameters
All rats fed with PB had no significant change in the body weight as compared to the control rats [Table 3]. The hematological parameters WBCs, RBCs, hemoglobin content, and hematocrit values were also found to be within normal range [Table 3]. This suggested that the ethanol plant extract of PB had no toxic effect on the normal physiology of the rat. Aqueous extract at a dose of 500 mg/kg/day had no significant effect on the biochemical parameters, but in a dose of 1500 mg/kg/day showed a significant decline in glucose levels (180.04 ± 9.98 from basal values of 199.67 ± 10.78) with elevated cholesterol levels (152 ± 21.78 from basal values of 136.38 ± 22.67). Alcoholic extract at doses of 500 mg/kg/day and 1500 mg/kg/day also showed a significant decline in glucose levels (135.6 ± 9.07 from basal values of 158.98 ± 10.01 and 90.07 ± 7.01 from basal values of 122.09 ± 7.01 respectively) with elevated cholesterol levels (93.76 ± 21.90 from basal values 60.89 of ± 19.89 and 148.7 ± 23.89 from 110.08 ± 21.78 respectively). This probably indicates a nonutilization pattern of the serum cholesterol, thereby suggesting an associated decrease in steroidal hormone synthesis in the PB treated rat.
| Discussion|| |
As evident from the present study, administration of the extracts of the leaves of PB to female albino rats resulted in a significant dose-dependent alteration in the duration of the different phases of the estrous cycle. The normal periodicity of the cycle was replaced by a distorted picture where there were a reduced number of cycles, prolonged diestrus along with shortened duration of the proestrus and estrus phase. Since estrus phase is the only phase in which the animals mate, its continued reduction and absence at high doses in the test animals as evident from our study could possibly suggest some antifertility effect of the leaves of the PB plant.
The pattern of the normal estrous cycle and the shift in the different stages in rats are mainly governed by the synthesis and levels of the various ovarian hormones, which, in turn, is regulated by the secretion of gonadotropins and hypothalamic-releasing factor from the pituitary.  The cyclic changes that occur in the female reproductive tract of the rat are depicted in the picture which shows that the level of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) begins to rise just after the diestrus phase. 
The release of these gonadotrophins results in follicular maturation and secretion of estrogen during the subsequent proestrus phase. Hence the proestrus phase indicates the start of the follicular phase where there is a surge in the levels of estrogen that commences in the start, peaks around the middle and then finally falls toward the end of the proestrus phase.  Similar to that in humans, this estrogen peak is followed by a surge in the LH levels during the mid of proestrus that triggers the onset of ovulation. Thus, ovulation is said to occur from the beginning of proestrus to the end of the estrus phase.  The progesterone levels also start to increase during the proestrus and peaks during ovulation. The estrus phase which permits mating in the animal usually occurs on day 3 of the cycle (based on vaginal cytology) and lasts for 12-14 h.  Finally, all the hormone levels return to their baseline values.
Thus, the reduction in the number of the estrous cycle along with a reduction in proestrus and the estrus phase as evident from our study corresponds to a reduction in the time during which ovulation occurs and hence a reduction in the rate of ovulation resulting in reduced fertility. Consequent to the reduction in the frequency of ovulation, there is either no availability of the matured Graafian follicles or nonmaturation of secondary follicles due to probable inhibition of ovulation by the extract.
The prolongation of the diestrus phase by the extract indicates that it possibly interferes with the actions of FSH, LH, estrogen and progesterone that are responsible for the control of the estrus cycle in female rats through its action on the pituitary-gonadal axis which might have resulted in diminished gonadotrophin release, in turn leading to reduced estrogen levels affecting ovarian cyclicity and estrus cycle. This hence suggests the antiestrogenic nature of the leaf extract which might be due to the presence of phytoestrogens in the leaf.
Normal estrus cycle is essential for ovulation, implantation and subsequent sustenance of pregnancy, which is in turn is dependent upon the coordinated action of estrogen and progesterone where both estrogen and progesterone levels rise in proestrus and decline at the end of estrus. Since perfect balances in their levels are highly essential for fertility, any disturbance in their levels can result in deranged ovarian functions that can affect the estrus cycle and thereby fertility. Shortened proestrus and estrus suggest that PBL extract might have interfered with the synthesis of hormones that might have brought about changes in the various phases of the estrus cycle and the number of cycles. In addition, such hormonal imbalances can also hinder the transport of spermatozoon as well as its subsequent passage in the female reproductive tract. 
Cholesterol serves as the basis for the production of all steroid hormones including cortisol, testosterone, progesterone and estrogen. In females, synthesis of estrogens starts I n theca interna cells in the ovary, by the synthesis of androstenedione from cholesterol. Androstenedione is a precursor for both androgens such as testosterone as well as estrogen. This compound crosses the theca interna cells, into the surrounding granulosa cells, where it gets converted either immediately into estrone or into testosterone and then finally into estradiol in an additional step. 
Since cholesterol is the parent molecule from which all ovarian steroid hormones are formed, the altered parameters associated with an increase in the serum cholesterol levels may be indicative of diminished utilization of cholesterol for steroidal hormone synthesis and thus might also result in reduced circulating levels of estrogen hinting at an antiestrogenic property of the extract.
According to literature flavonoids and saponins are known to exhibit antifertility activity. , The inhibitory effect of steroidal saponins on the estrous cycle has also been reported.  Preliminary phytochemical studies have indicated the presence of tannins, flavonoids, saponins, triterpenoids and naphthoquinone in the leaf extract that could be the contributing factors leading to the antifertility activity of the extract. These observations hence prove the antifertility activity of the extract that might be due to its antiestrogenic property. However, the exact mode of action is not clear.
Some studies on the contrary have suggested that when rats were administered the stalk of the PB leaf extract during the diestrus phase the estrous cycle was neither altered nor was any estrogenic or antiestrogenic effect observed. 
| Conclusion|| |
Both the aqueous and the methanolic extract of PB possess antifertility and antiestrogonic activity in female albino rats. However, further studies are required to be carried out to ascertain the mechanism involved in the biological activities of these compounds. The extracts of this plant can be further explored for its potential use as a contraceptive.
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[Table 1], [Table 2], [Table 3]