Effect of the Ethanolic Extract of Gloriosa superba Linn (Langli) Roots on theReproductive System of Female Rats

Introduction

Various plants with estrogenic activity contain phytoestrogens. There are several reports describing the beneficial roles of phytoestrogens in protection from bowel, prostate, and breast cancers; reducing the risk of cardiovascular disease; relief from postmenopausal symptoms such as osteoporosis, etc. Phytoestrogens appear to have both estrogenic and antiestrogenic effects.^1,2

Plant-derived phytoestrogens are a group of heterogeneous polyphenolic compounds that are categorized into isoflavones, coumestans, and lignans. They either mimic endogenous estrogens and cause estrogenic effects or block estrogen receptors (ER) and cause antiestrogenic effects. Similar to estrogen agonists and antagonists, phytoestrogens can also be classified as selective estrogen receptor modulators (SERMs) that can be agonists or antagonists.^3,4

Progesterone plays a vital role in the maintenance of normal menstrual cycle in women. It is a major inhibitory factor in the luteal phase of the ovarian/menstrual cycle. Estradiol and progesterone are essential for regulating the feedback mechanism of the hypothalamic-pituitaryovarian axis. Progesterone inhibits the secretion of gonadotrophin-releasing hormone (GnRH) and luteinizing hormone (LH). This blockade of the LH surge has been observed in many species including the rat, ewe, monkey, etc. It prevents premature LH surges in women showing ovarian stimulation and is therefore, used in in vitro fertilization (IVF) regimes.⁵

Uterotonic agents cause uterine contractions. They are used in allopathic, as well as the traditional systems of medicine for their laxative, purgative, diarrheal, cathartic, abortifacient, and emmenagogic properties. Uterine stimulants are drugs that cause contractions in the uterus or increase the frequency and intensity of the uterine contractions and are therefore used to induce or augment labor. They are also used to reduce hemorrhage following a miscarriage, induce abortion, or prevent postpartum hemorrhage.⁶

Gloriosa superba Linn (family Liliaceae), commonly known as “Kalihari” (Hindi), Climbing-lily (English), Kallavi (Marathi) is a perennial, tuberous, climbing herb found in tropical Africa and many parts of Asia including India, Burma, Malaysia, and Sri Lanka. The tribes of Patalkot in Madhya Pradesh apply its rhizome extract over the navel and vagina to induce labor and facilitate normal delivery. Administration of 250 to 500 mg of the extract may induce abortion when given during the first trimester of pregnancy. Experimental research data also reveals uterotonic and abortifacient effects of the ethanolic extract of Gloriosa superba. ^6,7

The present study was undertaken to investigate the exact mechanism of abortifacient action of an alcoholic extract of Gloriosa superba. 

Materials and Methods

Plant material Tuberous roots of Gloriosa superba Linn were procured from the local areas of Kalaburagi in Karnataka, and authenticated by Rajasamarsen K Modi, Asst. Professor, Department of Botany, Govt. Degree College, Gulbarga. An herbal specimen (HGCG No-499) is stored in the department for future reference.

Animals

Immature female Wistar albino rats weighing 100-130 g were procured from M/s Sainath Agencies, Hyderabad (282/PO/Bt/S/2000/CPCSEA). They were housed in the animal house of HKES MTRIPS at a temperature of 25±1°C with 45 to 55% humidity and a 12-h light: 12-h dark cycle. The animals were fed on standard pellets and had access to water ad libitum. The Institutional Animal Ethics Committee (IAEC) approval was obtained before the initiation of the study (IAEC approval no.- HKES/ MTRIPS/IAEC/91/2017-19).

Extraction of plant material

Dried roots of Gloriosa superba Linn were pulverized to particle size (#40), and the powder was extracted with alcohol by hot percolation method at 40 °C for 48 h using the Soxhlet apparatus. The obtained extract was concentrated under vacuum with the help of a Rotary Flash evaporator, and dried.

Preparation of dried ethanolic extract and standard solution doses

The dried plant extract was freshly suspended in gum acacia (2% w/w) with a few drops of Tween 80 for administration during the experiment. The 20 mg/kg and 40 mg/kg oral doses were prepared from the stock solution (10 µg/mL) containing 10 µg of β-estradiol in 1 mL of olive oil. β-estradiol (1 μg/rat/day) was used as the standard.

Evaluation of EyGS for estrogenic activity

The immature female Wistar rats were ovariectomized using 5% ketamine hydrochloride (80 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) under sterile conditions. After recovery, the animals were divided into 4 groups comprising 6 animals each. Group I (control) was administered with the vehicle, i.e. gum acacia (2% w/v p.o.). Group II animals were subcutaneously administered with β-estradiol (1 μg/rat/day) suspended in olive oil, which served as a positive control. Group III and IV animals received EyGS orally at 20 mg/kg and 40 mg/kg, respectively.

All the groups were treated for seven consecutive days. The final body weight, and the vaginal opening and cornification were assessed on the 8th day. At the end of the experiment, blood was withdrawn from the retroorbital plexus (ROP) and processed for the estimation of estrogen, alkaline phosphatase, and total cholesterol levels. The animals were then sacrificed using anesthesia. The uteri were cut and taken out, cleaned to remove the surrounding tissues, blotted on filter paper, and weighed quickly on an electronic balance. The uterine horns of the control and treated animals were stored in 10% formalin solution for 24 h, and histopathology was carried out.⁸

Evaluation of EyGS for progestogenic activity

Bilateral ovariectomy was done in immature female Wistar rats (100-130 g) and after recovery, the animals were allocated to 4 groups containing 6 animals each. Group I (control) was treated with the vehicle (distilled water, 10 mL/kg); Groups II and III were administered EyGS at 20 mg/kg and 40 mg/kg p.o., respectively; and Group IV received hydroxyprogesterone caproate (0.04 mg/animal, s.c.). The total duration of treatment was 14 days. Each animal in all the groups was treated with 0.5 μg estradiol s.c. once daily for 4 days, followed by treatment with vehicle, EyGS (20 and 40 mg/kg p.o.), and hydroxyprogesterone (0.04 mg/animal s.c.) as per the group as described earlier, for 9 days. On the fifth day of treatment with progesterone, vehicle, and EyGS, the uterine horns of all the animals were exposed, and 1.0 mg histamine dihydrochloride was injected into the lumen of the left horn while the other horn (control) was injected with distilled water (vehicle). All the animals were sacrificed at the end of the experiment. Blood was withdrawn from the ROP of all the animals in each group before sacrificing them. Both the uterine horns were cut, taken out, and weighed. The degree of deciduoma formation was evaluated based on the percentage increase in the weight of the histamine-injected uterine horn compared to the control horn. The uterine horns of the control and treated animals were stored in 10% formalin solution for 24h, and histopathology was performed.⁹

Histological assessment of the uterus

The uterine horns stored in 10% formalin were grossed and fixed again in 10% formalin. The specimens were then dehydrated in 70, 90, and 100% alcohol for 2 h each and later immersed in xylene three times for 1 h each to facilitate clearing of the tissue. The tissue thus treated was infiltrated twice for 1 hour each, with paraffin wax. Paraffin blocks containing the tissue were then cut into 3-5 μm thick sections using a microtome and mounted on glass slides coated with egg albumin/glycerin. The sections were stained with hematoxylin and eosin stains and subjected to microscopic examination.¹⁰

Serum estrogen and progesterone estimation

The serum was separated from blood by centrifugation. Estrogen was analyzed by chemiluminescent immunoassay (CLIA) using Immulite, a fully automated immunoassay analyzer, USA.

Serum alkaline phosphatase and cholesterol estimation

The serum alkaline phosphatase was estimated by P-Nitrophenyl phosphate-kinetic method using an Autoanalyzer (Hitachi, Model No-912) and kits (Roche, USA). The serum cholesterol was estimated by Fuji Dri-Chem NX500 fully automatic analyzer.

Uterotonic activity of ethyl alcohol extract (EyGS) in rat uterus (in vitro)

Adult female Wistar rats (120 to 200 g) were primed with 100 µg of estradiol benzoate 24 h prior to commencement of the experiment. Rats in the estrous phase were selected and killed by cervical dislocation, and the uterus was removed. One horn of the uterus was suspended in an organ bath containing De Jalon’s Ringer’s solution. The tissue was stabilized for 45 min and then washed at least 4 times with fresh De Jalon’s Ringer’s solution. The dose responses were recorded with oxytocin (0.05 mL, 0.1 mL 0.2 mL, 0.4 mL, and 0.8 mL) and EyGS (0.1 mL, 0.2 mL, 0.4 mL, 0.8 mL, 1.6 mL, 3.2 mL, and 6.4 mL) solutions with repeated washing after each dose until the ceiling effect was observed. The response obtained due to EyGS was compared with the standard response.⁹

Statistical analysis

Data was analyzed by one-way ANOVA followed by Dunnett’s test and expressed as mean ± S.E.M., and statistical analyses were carried out using GraphPad Prism software, San Diego California USA, www. graphpad.com. P value <0.05 was considered significant. 

Results

Effect of EyGS on vaginal cornification, and uterotrophic assay

All the female rats treated with EyGS at 20 mg/kg and 40 mg/kg dose levels showed vaginal opening and absence of vaginal cornification. The presence of cornified cells in the vagina were observed only in the β-estradiol group. EyGS at both the doses mentioned did not show any significant increase in the body and uterine weights of ovariectomized rats compared to the control group. Only β-estradiol group showed a significant (p<0.01) increase in these parameters in ovariectomized rats when compared to the control. The evaluation of morphometrical indices of the uterus showed that the β-estradiol group demonstrated a significant increase in the diameter of the uterus (p<0.01), as well as the diameter, thickness, and height of endometrium (p<0.001) in ovariectomized rats compared to the control whereas EyGS did not produce any significant increase (Table 1).

Effect on biochemical parameters

EyGS showed significant (p<0.0001) nonestrogenic potential by decreasing the level of serum estrogen at 20 mg/kg and 40 mg/kg dose levels in immature, ovariectomized rats in comparison to control. Furthermore, the extract showed a significant (p<0.0001) decrease in the serum cholesterol at 40 mg/kg dose level compared to control. The nonestrogenic potential of the extract was confirmed by a significant (p<0.0001) increase in the serum alkaline phosphatase at 40 mg/kg (Table 1).

Effect on histopathology of uterus

The histopathological changes in the uteri are summarized in Fig 1. The uterus of the control group was small, inactive, and showed a fissure lumen with compact stromal cells. The thin perimetrium layer and circular and longitudinal muscle layers of the myometrium with few small endometrial glands were seen. Treatment with estrogen demonstrated marked endometrial proliferation showing various endometrial glands, lumen with multiple folds, an increase in the thickness of the endometrium and myometrium, epithelial cells with leukocytes, and inflammation. Both the doses of EyGS exhibited no proliferation of endometrium but showed slit-like lumen and compact stromal cells with few small endometrial glands. Endometrial and myometrial hypoplasia without inflammation were also noted at both doses of EyGS.

Effect of EyGS on deciduoma formation and serum progesterone

At 40 mg/kg, EyGS caused a significant (p<0.01) decrease whereas at 20 mg/kg, it showed a nonsignificant, dose-dependent decrease in the decidual weight of ovariectomized rats compared to control. Only hydroxyprogesterone caproate brought about a significant (p<0.01) increase in the decidual weight of the uterus of ovariectomized female rats compared to control. The extract showed (p<0.0001) nonprogestogenic potential by significantly decreasing the serum progesterone at both doses in immature female rats compared to control (Table 2). The histological changes in the uteri are shown in Fig 2. The uterus of the control group was small and inactive and showed a fissure lumen and compact stromal cells with few round endometrial glands. The progesterone-treated group demonstrated pronounced secretory changes such as secretion in the lumen, edematous or decidual stroma, many tortuous endometrial glands, and formation of vacuoles in cells showing spinal arterioles. Both doses of EyGS showed lumen without secretion, compact stroma, round endometrial cells, and absence of spinal arterioles. The histopathology confirmed the nonprogestogenic activity of EyGS.

Dose response effect of oxytocin and EyGS on isolated rat uterus (in vitro)

Oxytocin produced dose-dependent contractions of estrogen-primed nonpregnant uterus. Similarly,contractions were also induced by EyGS, but the intensity of the contractions was less when compared to that produced by oxytocin (Fig 3).

Discussion

An excess of estrogen can cause breast, endometrial, ovarian, and prostate cancer, and its deficiency can result in menopausal symptoms, cardiovascular disease, and osteoporosis. The major causes of estrogen deficiency in females are menopause and ovariectomy.¹¹

Typical estrogenic compounds possess the ability to increase the uterine weight and fluid retention, leading to ballooning of the uterus. They have also been noted to cause opening of the vagina and showed the presence of cornified cells in the vaginal smear of immature female rats.¹² Estrogens also exert an effect on lipid metabolism by slightly elevating the serum triglyceride and estradiol, and reducing the cholesterol levels.¹³ The metabolic and reproductive characteristics in mature and immature rats can be manipulated by the ingestion of a phytoestrogenic substance, which produces effects similar to those of gonadal steroids.¹⁴

An elevated level of progesterone during pregnancy plays a vital role in maintaining the pregnancy and acts as a key factor in implant formation.¹⁵ Estrogen is required for the initiation of synthesis of progesterone receptors, and progesterone is an absolute requirement for sustained decidualization, the absence of which results in embryo resorption.¹⁶

Previous studies show that that all the different extracts (ether, chloroform, and ethanolic) of Gloriosa superba possess abortifacient activity, out of which ethanolic extract (EyGS) is the most potent.⁷ Therefore, we attempted to study the effect of EyGS on estrogenic and progestogenic activity. The results revealed that the extract-treated rats did not show any increase in the body, uterine, and decidual weights in bilaterally ovariectomized immature rats at both the doses (20 and 40 mg/kg) in the uterotrophic assay. These results are in concurrence with the previous studies.⁹

The deficiency of estrogen caused by ovariectomy, as well as menopause, produces bone resorption and rapid bone loss, resulting in osteoporosis. A study by Garnero and Delmas indicated that the serum ALP level, which is associated with bone formation, is increased in osteoporosis^17. Our results are in concurrence with the findings in earlier studies. 

We analyzed the uterotonic activity of EyGS to find out the exact mechanism behind its abortifacient activity. Most of the plants containing phytoestrogens produce uterine contractions similar to the oxytocin that is produced in the posterior lobe of the pituitary gland, which stimulates the uterus, causing strong contractions and inducing labor.¹⁸ If plants with oxytocic potential are used during the first months of pregnancy, they can induce abortion.¹⁹ Our study revealed that the EyGS produces 80% contractions of the uterus compared to oxytocin. Thus, its anti-implantation and early abortifacient potential is possibly due to its oxytocic property.

The results of this study demonstrate antiestrogenic, antiprogestogenic, and 80% spasmogenic activity of EyGS compared to oxytocin (Fig 3), which is in line with similar studies of researchers.⁹

Conclusion

EyGS possesses potent antifertility activity that can be attributed to its spasmogenic (oxytocic) property. The study proves the use of Gloriosa superba in folk medicine for inducing labor at the time of delivery and abortion during the first trimester.