INTRODUCTION

Around the world, population management is a dangerous topic. One percent of pregnant women die from unexpected pregnancies or have abortions to prevent having unwanted children [1]. Although plants are a highly abundant source of phytoconstituents molecules, in the past 30 years most research efforts have been focused primarily on the development of antifertility drugs from the chemical source, with little emphasis given to the herbal source. New, efficient, and secure antifertility drugs can be discovered using a variety of herbal plants [2]. Synthetic medicines and steroidal contraceptives, which are used for fertility control, have several dangerous side effects, including hormonal imbalance, gastrointestinal distress, depression, hypertension, painful uterine contractions, and an increased risk of cancer [3]. As a result, we must use herbal contraceptive methods because they are affordable, secure, and have few adverse effects. Melia azedarach is one of the plants grown in different areas in India. Melia azedarach Linn, often known as mahaniba, belongs to the family Meliaceae. Melia azedarach, also known as the chinaberry tree, bead tree. The whole plant or its parts (leaves, roots& stem) are used for different medicinal purposes. and have a long history of use by indigenous and tribal people of different parts of India.  They have been used in this plant traditionally to treat dental pain, Piles, leprosy, itching, blood purifier, asthma, and cough [4]. Different investigations have found that this plant possesses anticancer, antiviral, antimalarial, and antibacterial activity [5]. Folklore in West Bengal suggests that Melia azedarach has antifertility properties. We, therefore, conducted this investigation to assess the estrous cycle study, antiimplantation action, and GC-MS analysis of the leaf of Melia azedarach methanolic extract.

MATERIALS AND METHODS

Drugs/chemicals

Ethinylestradiol (Infar India Ltd) was used standard drug for antifertility activity study. All other chemicals were obtained from Merck, Mumbai, India.

Collection of plant materials

The leaves of Melia azedarach were taken in September 2021 from Jamalpur in the Purbabardhaman District of West Bengal, India, and identified by a taxonomist from the Botanical Survey of India in Howrah, West Bengal. The Department of Pharmacognosy, Bengal School of Technology (A College of Pharmacy), Chuchura, Hooghly and West Bengal has received a voucher specimen (CNH/Tech.II/2022/27 dated 23/05/2022) for deposit. India.

Preparation of plant extract

Melia azedarach leaves (1.5 kg) was air dried, mechanically ground to a coarse powder, and then extracted for 48 hours in a Soxhlet extractor with 3 L of methanol. To produce a dark green solid mass (15%w/w), the extract was concentrated to dry in a Rota evaporator (Buchi type) at reduced pressure and moderate temperature (50-550C). A refrigerator was used to store the dry extract. For this investigation, a suspension of the dry extract made with Tween-80 (1% w/v) was distilled water.

Phytochemical test

The existence of several chemical components is qualitatively examined in the methanol extracts of Melia azedarach (leaf). The extract was subjected to phytochemical screening utilizing the following reagents and substances, and corresponding analyses are made.

Test for alkaloids

Hager’s test: Few ml of extract with 1 ml of Hager’s reagents were added. The presence of a creamy white precipitate indicated the presence of Alkaloids.

Wagner: There were added 1 ml of extract and 1 ml of Wagner's reagent. Alkaloids were present as indicated by the formation of a reddish brown precipitate.

Test for tannin

Lead acetate test: 1 ml of extract and 3 drops of lead sub-acetate solution were added. A creamy gelatinous precipitate indicated the presence of tannins.

Ferric chloride test: There were added 2 ml of the extract together with 2 ml of the 5% ferric chloride. The existence of tannins was indicated by the color green.

Test for glycosides

Borntrager’s test: A mixture of 2mL filtrated hydrolysate and 3mL chloroform was added. The chloroform layer was split after vigorous shaking. After adding a 10% ammonia solution, the existence of glycosides was detected by a pink-colored solution.

Test for steroids

Salkowski: Few ml of extract and few drops of conc. H₂SO₄ was mixed. (Shake well and allowed to stand). The presence of a red color (in the lower layer) indicated the presence of steroids.

Test for flavonoids

Zinc hydrochloride test: Few ml of extract was added with a pinch of zinc dust and after that conc. HCl along the side of the test tube was added. The appearance of magenta Colour indicated the presence of flavonoids.

Shinoda test: 5mL of alcohol was used to dissolve plant extract. A few drops of concentrated HCl and pieces of magnesium ribbon were introduced. Flavonoids were present when the solution was pink to crimson.

Test for terpenoids

Liebermann-burchard test: 2 ml of the extract, 2 ml of acetic acid, 2 ml of chloroform, and 1 ml of concentrated H2SO4 were added. Terpenoids were present because of their blue-green appearance.

Carbohydrate test

Molish test: Molish reagent, 2 ml of extract, and 2 ml of concentrated H2SO4 were all added. The crimson ring was a sign that carbohydrates were present.

Fehling’s test: Filtrate was combined with Fehling's solutions A and B in a volume of 1 ml each, and the mixture was then heated in a water bath before boiling. Carbohydrate presence was identified by a red precipitate.

Amino acid test

Millon’s test: A few drops of Millon's reagent were added to 2ml of extract. It did not produce a precipitate that was white and showed the presence of amino acids.

Ninhydrin test: I combined 2ml of the extract with 2 drops of the Ninhydrin solution. There was no purple-colored sol. of amino acids.

Fixed oil test

Saponification test: When heated for two hours, an extract containing a few drops of 0.5N alcoholic KOH and a drop of phenolphthalein caused the soap to develop, indicating the presence of fixed oil [6, 7].

GC-MS study:  The samples were examined by Institute for Analysis of Dairy, Food, and Cultures (IADFAC) Bangalore. For analysis, the chromatographic conditions listed below were used. The ion chamber temperature is 250 °C, the GC interface temperature is 250 °C, the front inlet temperature is 220 °C, the column is HP 5 Ms, the carrier gas is highly pure helium, the flow rate is 1 ml/min, the mass analyzer is the quadruple double-focusing mass analyzer, the analyzer is photon multiplier tube and electron impact ionization is 70 eV.

Determine the components: GC-MS mass spectrum interpretation was done with the help of the National Institute of Standards and Technology (NIST). The mass spectra of the unidentified chemical substance and those of its recognized constituents that are housed in the NIST database were compared.

Acute toxicity study

The acute toxicity investigation of the methanol extracts of Melia azedarach adhered to OECD guideline 420. (leaf) [8].

For this investigation, female, non-gestational rats weighing 150–200 g were employed. All animals were monitored for toxicities such as diarrhea, decreased appetite, hair erection and damage, lacrimation, convulsion, salivation, lethargy, paralysis, and mortality continuously for the first hour, intermittently for the following three hours, continuously for the following 24 hours, and periodically for 14 days.

Wister breed, colony-bred virgin female albino rats weighing 150–200g were employed in our antifertility study. In the animal house, rodent food and unlimited water were available to all the animals. The humidity was 55.5% and the temperature was 22.20C. The Institutional Animal Ethical Committee of Bengal School of Technology (A College of Pharmacy), Chuchura, Hooghly, West Bengal, accepted all animal experimentation protocols (Registration No.: 1726/CPCSEA/IAEC/2022-002).

All the experiments were performed & followed by the Committee for the Control and Supervision of Experimentation on Animals (CPCSEA).

Pharmacological activity study

• Estrous cycle study

Estrous cycle research used vaginal smears and the length of several estrous cycle phases. For the estrous cycle investigation, albino female rats from colonies were chosen because they had regular estrous cycles. Rats typically go through an estrous cycle every 4-5 days. phases of the estrous cycle are (i) estrous (cornified epithelial cells), (ii) metaestrous (cornified cells plus leucocytes), (iii) diestrous(leucocytes), and (iv) proestrous (epithelial cells). The above-mentioned animals were split into three groups, each with six animals, in order to evaluate the impact of methanol extracts on the estrous cycle. Only a vehicle was given to group (I) (Tween-80,1%). The methanolic extract was given to groups II and III at doses of 100 mg and 200 mg/kg body weight, respectively. Three typical estrous cycles were covered during the 15-day treatment period. Every morning, a vaginal smear from the experimental animals was examined, and the length of each phase of the cycle was recorded [9, 10].

• Anti-implantation activity studies

For antiimplantation action, virgin female albino rats from a colony were employed (150–200gm). Each day in the morning, the vaginal smears from each female rat were examined. For the experiment, only the rats with a healthy estrous cycle were chosen. The antifertility activity was measured following Khanna and Chowdhuary's descriptions [11]. In the evening of proestrous, the female rats were housed with male rats that were fertile in a 2:1 ratio, and the next day, the copulation evidence was reviewed. Rats with dense aggregates of spermatozoa or the copulation plug in their vaginal smears were separated, and the day was declared to be day 1 of pregnancy. These rats were then divided into 4 groups, each with six rats. The group (I) serves as the control and only got the vehicle (between 80, 1%). Groups III and IV got dosages of 100 mg and 200 mg b.w. of methanol extract, respectively, whereas Group (II) received ethinylestradiol as the conventional treatment at a dose of 0.45 mg/kg b.w. All therapies were administered orally. From day 1 through day 7 of pregnancy, the aforementioned therapies were administered, and on day 10, laparotomies were carried out under mild ether anesthesia while maintaining sterility. The number of implantation sites, corpora lutea in the ovary, and resorption sites in the uteri were all counted. The rats were allowed to mature after the abdominal incision was closed with sterile sutures under aseptic conditions. Calculations were made for early and anti-implant activity rates. The combined early abortifacient & antiimplantion activity of the studied materials results in% of antifertility activity. Below are the computation formulae [12].

% abortifacient activity= (No of resorption/No of corpora luteum) × 100

% of antiimplantion activity = 100-(No of implantation/No of corpora luteum) × 100

% Total antifertility activity= % of antiimplantion activity +% abortifacient activity

Histology study

Ovarian tissue samples from both control and treatment animals were first fixed in Bouins solution for 24 hours, then dried with alcohol before being embedded in paraffin. For the histological analysis of the ovary, the paraffin block slices were cut at a 6 m thickness and stained with hematoxylin-eosin [13].

Analysis of statistics

One-way analysis of variance (ANOVA) and Dunnett's t-test was used to statistically examine the group differences. Statistics were deemed significant at p 0.05. The mean value SD is used to express all data.

RESULTS AND DISCUSSION

GC-MS analysis

Melia azedarach's methanolic extract's GC-MS chromatogram mentioned in Figure 1, which revealed forty peaks corresponding to the presence of forty bioactive chemicals. By contrasting the mass spectra of the unidentified compounds with known spectra kept in the NIST-11 collection, the unidentified compounds were described and identified in Table 1, the discovered compounds were displayed together with their retention time (RT), weight, molecular formula, molecular weight, peak area (%), and type of the compounds.

Table 1. Quantitative compounds identified from methanol extract of Melia azedarach (leaf) by GC-MS analysis.

The results showed that Preg-4-en-3-one, 17. alpha.-hydroxy-17.beta.-cyano-(1.35 %) Hexadecenoic acid methyl ester, (19.64 %) (, Undecanoic acid, 10-methyl-, methyl ester (0.93%) Tetradecanoic acid (0.90%), Tetradecanoic acid, 12-methyl-, methyl ester, (S) (2.33% ), n-Hexadecanoic acid (4.88%) were found as major compounds in the methanolic extract of Melia azedarach.

Acute oral toxicity test

Animals did not exhibit any changes in their motor activity, diarrhea, lacrimation, convulsions, or state of coma following a single oral administration of the maximum dose (2000 mg/kg). There were no observable alterations in the body, behavior, or nervous system. Additionally, no deaths were noted for the course of the observation period's full 14 days. Animals treated with methanolic extract tolerated doses up to 2000 mg/kg body weight without experiencing any ill effects. For our current investigation, we employ the extract at dosage levels of 100 mg and 200 mg/kg body weight. The acute oral toxicity mentioned in the Table 2.

Table 2. Effect of methanolic extract of Melia azedarach (Leaf) Duration of Different Phases of Estrous Cycle (Mean Duration ±SD in Days)

n= 6, One-way ANOVA followed by Dunnett’s test. ^*p<0.05when compared to the control group

The result of the methanolic extract of Melia azedarach (leaf) on the estrous cycle was shown in Table 2 and Figure 3 Rats treated (100mg and 200mg/kg body weight) with extract showed a significant increase (p < 0.05) in the duration of diestrous phase and a significant decrease (p <0.05) in the duration of metaestrous phase as compared to control. Different phases of estrous cycles showed in Figure 2.

Table 3. Postcoital anti-implantation activity of methanolic extract of Melia azedarach leaves (MEAN ± SD)

n= 6, One-way ANOVA followed by Dunnet’s test. ^*p<0.05when compared to control

Table 3 is displays the impact of the Melia azedarach (leaf) methanolic extract on antiimplantation action. According to the research, methanolic extract demonstrated substantial (p<0.05) antiimplantation action at doses of 100 mg and 200 mg/kg body weight. The outcomes demonstrated that the extract has an antifertility impact that is dosage dependent. Data revealed that extract-produced antifertility activity has dose-related responses that are more potent than those in the control group. In the rats treated with 100 mg and 200 mg/kg body weight of extract, respectively, the percentage of overall antifertility action was 83.25% and 90.36%. The antiimplantation activity of ethinylestradiol, which was used as the standard, was 98.18%. Figure 3 depicts a picture demonstrating antiimplantation activity.

The photograph of ovary control animals and treated animals' activity was shown in Figure 4.