Estrogenic Effect of Bambusa arundinacea, Trichosanthes dioica and Punica granatum on Rats

Many plants have been discovered to have estrogenic activity, implying that they contain estrogenic compounds. They contain phytoestrogens, which have biological activity similar to estrogen. The importance of phytoestrogens in human health has been revealed in numerous studies. Phytoestrogens bind to estrogenic receptors in a competitive manner, causing estrogen-responsive genes to be induced (Procházková et al., 2017). ‘Bans’ is the common name for Bambusa arundinacea (Poaceae). It has anthelmintic, antidiabetic and wound healing effects, as well as anti-inflammatory, antiulcer and antifertility properties. Its use in strangury and urinary discharge has been reported in a number of studies (Tripathi et al., 2013; Kumar et al., 2012; Wardhani et al., 2016). Almost every part of the plant has been shown to have medicinal value, such as the root (burnt root) for bleeding gums, the bark for skin eruptions and the leaf as an emmenagogue. Trichosanthes dioica Roxb is an Asian pacific tropical herb. Fruit contains the majority of the medicinal properties (Kumar et al., 2012). It’s been used for centuries to treat jaundice and hepatic diseases, as well as swollen liver and spleen (Khatua et al., 2016). In addition, it works as an antipyretic, diuretic, cardiotonic, laxative and antiulcer agent. Experiments have shown that it is successful in promoting fertility (Khan et al., 2020). The pomegranate, Punica granatum, of the Punicae family, has been found to contain estrone, a sex steroid (Peters et al., 1980). Many pharmacological properties, such as anti-microbial, anti-parasitic, anti-viral and anti-cancer activity, have been discovered in studies (Mertens-Talcott et al., 2006; Sreeja et al., 2012). Pomegranate juice has been found to contain flavonoids such as anthocyanins and phenols (Devi et al., 2017). The effects on fertility and estrogen-like effects have also been discovered in experiments (Bekoe et al., 2020).
       
Therefore, the current research used an uterotropic assay, vaginal cytology and vaginal opening calculation to assess the estrogenic effects of alcoholic extract of Bambusa arundinacea, Trichosanthes dioica and Punica granatum on wistar rats.

Study area
 
This experiment was conducted at Vivek College of Technical Education, Bijnor, Uttar Pradesh during June 2015 to December 2016.
 
Experimental animals
 
Impuberal female albino wistar rats, 21-day-old, with body weight of 45-50 g, were used.  Animals were housed four per cage, in a multiple rat rack. Temperature (21±2°C) and humidity (55±15%) were controlled and a 12 h light/dark cycle was maintained. Water and food were ad libitum. The experiment protocol was approved by the Institutional Animal Ethical Committee (IAEC) according to the regulation of committee for the purpose of control and supervision of experiments on animals (CPCSEA) and ethical norms was strictly followed during all experimental procedure (Ref. No. VCTE/07/2016 CPCSEA). All animals had been acclimatized for three days in the animal room prior the first treatment (Parhizkarsup et al., 2011).
 
Dose selection
 
Doses were selected on the basis of previous toxicity studies carried out for Bambusa arundinaceae (Nilsson et al., 2001) (Table 1), Trichosanthes dioica (Sharangouda et al., 2008) (Table 2) and for Punica granatum (Valadares et al., 2010) (Table 3). The doses selected were 200 mg/kg, 300 mg/kg and 400 mg/kg, p.o.
 

 

 

 
Preparation of doses
 
The plant extracts were prepared in distilled water using carboxymethylcellulose (CMC) as a suspending agent (1%). The standard drug was also prepared in CMC and given as suspension to the animals. Control group (Negative control group/Olive oil control group) here through with the help of orogadtric tube 0.2 ml of olive oil was given orally once in a days in between experimental time. Conjugated Equine Estrogen (CEE 0.2 mg/kg) was purchased from Pharmacy shop, Bijnor, India. CEE (Wyeth Montreal, Canada), prepared in a dosage (p.o.) of 0.2 mg/kg by dissolving it in distilled water (Malaivijitnond et al., 2006) and was used as a positive control for comparing with the Test groups. The Plant extracts of Bambusa arundinaceae, Trichosanthes dioica and Punica granatum in different doses (200 mg/kg, 300 mg/kg and 400 mg/kg body weight, orally) was dissolve in 0.2 ml of Olive oil and with the help of Orogastric tube was given by oral route (Goodman and Parker, 2008).
 
Treatment protocol
 
Immature Uterotrophic animals were divided into five groups each containing six animals and one group of normal immature rats. Animals were fasted 18 hrs prior to dosing and 3-4 hours after administration of the plant extracts. The plant extracts were given at a dose of 200 mg/kg, 300 mg/kg and 400 mg/kg oral routes to the immature female animals for a period of 14 days.
 
Induction of uterotrophic activity in rats
 
Bilaterally ovariectomized, female immature female albino and wistar rats were randomly allotted to different groups. All test substances were administered for 14 days. The animals will be examined twice a day on 4^th and 5^th day for the presence of the vaginal opening. After noticing the vaginal opening, the vaginal cytology will be observed to examine the cornification of epithelium. Body weight will be recorded on daily basis. On presence of vaginal opening 14th day the animals will be sacrificed by over anesthesia. The uterus was collected and weighed. The percentage of vaginal opening, cornification and relative organ weight were calculated (Nazreen et al., 2011).

The ethanolic extracts of the Bambusa arundinaceae, Punica granatum and Trichosanthes dioica respectively caused increase in the weight of uterus. The results (Table 4-6) showed the estrogenic effect of B. arundinaceae Punica granatum and Trichosanthes dioica when tested in immature ovariectomized rats. In the control animals weight of uterus was changed while the administration of standard dose of ethinyl estradiol) (0.2 mg/kg) resulted in the significant increase in weights of uterus. In comparison to the control group all the 3 plants extracts showed promising results Fig (1-3).
 

 

 

 

 

 

 
Vaginal opening in female rats
 
On analysis of the vaginal opening of the female rats it was found that with increasing days of treatment the vaginal opening dose dependently decreased (Table 7). In the standard group the opening was to the max at day 7 i.e. 80%. The treatment of the extracts concised the muscles and resulted decrease in the vaginal opening (Fig 4a-8b).
 

 


Histopathology studies
 
The histopathology studies of Bambusa arundinaceae revealed promising results. In vaginal smear of ovariectomized rat from control group treated with normal saline for 7 days. A great number of leukocytes were observed (Fig 9). The Vaginal smear of immature rat treated with ethinyl estradiol (0.2 mg/kg) for 7 days cornified and nucleated epithelial cells were observed (Fig 10). The vaginal smear of immature rat treated with low dose (200 mg/kg) of Bambusa arundinaceae cornified, nucleated epithelial cells and leukocytes were observed (Fig 11). Vaginal smear of immature rat treated with medium dose (300 mg/kg) of Bambusa arundinaceae showed cornified, nucleated epithelial cells and leukocytes (Fig 12). The vaginal smear of ovariectomized rat treated with medium dose (400 mg/kg) of Bambusa arundinaceae cornified, nucleated epithelial cells and leukocytes are observed (Fig 13).
 

 

 

 

 

       
Administration of B. arundinaceae, Punica granatum and Trichosanthes dioica extract on immature ovariectomized rats results a significant increase of uterine weight. This might be due to the activation of estrogen responsiveness in the uterine wall. Another study found that uterine knockout mice showed significant results in terms of increasing of uterus after the application of estrogen due to estrogenic response in the immature uterus (Weihua et al., 2000). The bioactive molecules of the applied extract may have Estradiol-17β (E₂) like flavonoides which are responsible for the significant effect on the vaginal opening, development of cornified epithelial cells and an increase of uterine wet weight (Galand et al., 1971; Mäkelä et al.,  1999). Therefore, these are the most probable reason for various effects of B. arundinaceae, Punica granatum and Trichosanthes dioica extract as phytoestrogens on reproductive organs of rats.

In this study, it was aimed to evaluate the estrogenic activity of ethanolic extract of Bambusa arundinaceae, Punica granatum and Trichosanthes dioica, respectively. The uterotropic assay showed the dose-related increase in uterine wet weight after administration of 200 mg/kg, 300 mg/kg and 400 mg/kg b.w., p.o. both in immature and mature ovariectomized rats. The ethanolic extract of Bambusa arundinaceae, Punica granatum and Trichosanthes dioica, respectively at higher dose (400 mg/kg b.w., p.o.) showed only cornified epithelial cells. The vaginal opening also showed that EBA (400 mg/kg b.w., p.o.) was having significant estrogenic activity. Thus, it can be concluded that the ethanolic extract of Bambusa arundinaceae, Trichosanthes dioica and Punica granatum play an important role in estrogenic activity in female rats.

First author is highly grateful to Dr. Nisha Dutta and Dr. Avilekh Naryal, Department of Science and Pharmacology, for sharing their technical expertise. We are highly thankful to management of Vivek college of Technical Education, Bijnor, for the financial support to carry out this research work.

All authors declare that they have no conflict of interest.