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Evaluation of therapeutic action of Pisum sativum in management of polycystic ovarian syndrome and formulating its sustained release tablet
*Corresponding author: Aakash P. Chavda, M.Pharm Department of Pharmacology, School of Pharmacy, RK University, Rajkot, Gujarat, India. aakashchavda188@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Chavda AP, Ganatra TH. Evaluation of therapeutic action of Pisum sativum in management of polycystic ovarian syndrome and formulating its sustained release tablet. Am J Pharmacother Pharm Sci 2026:008.
Abstract
Objectives:
Polycystic ovarian syndrome (PCOS) is a multifactorial endocrine disorder marked by hormonal imbalance, anovulation, and polycystic ovarian morphology. Although clomiphene citrate is primarily prescribed for women with PCOS seeking fertility treatment, aligning with its established clinical indication for ovulation induction. This study aims to investigate the therapeutic potential of the methanolic extract of Pisum sativum seeds (MEPS) in a letrozole-induced PCOS rat model and to assess its bioactive composition and interaction with estrogen receptors.
Materials and Methods:
PCOS was experimentally induced using oral letrozole (1 mg/kg/day) for 21 days. Rats were subsequently treated with MEPS at doses of 200 and 400 mg/kg, and the standard group received clomiphene only for 15 days. Phytochemical constituents were identified via standard tests and thin-layer chromatography (TLC), and in silico docking assessed ligand binding to estrogen receptors. Antioxidant potential was evaluated using the 2,2-Diphenyl-1-Picrylhydrazyl assay. Biological assessments included hormonal assays (follicle-stimulating hormone, luteinizing hormone, estrogen, progesterone, and testosterone), insulin levels, hepatic enzyme activity, lipid profiling, body weight monitoring, estrous cycle analysis, and ovarian histopathology.
Results:
Phytochemical testing confirmed the presence of flavonoids and phenolics. TLC analysis indicated the presence of quercetin, catechin, and epicatechin. Docking studies demonstrated strong binding of these compounds with estrogen receptors. MEPS administration significantly improved hormonal balance, restored estrous cyclicity, reduced insulin and liver enzyme levels, and normalized ovarian architecture. The 400 mg/kg dose showed comparable outcomes to clomiphene citrate.
Conclusion:
MEPS effectively counteracted PCOS-related alterations in the rat model, likely through its phytoestrogenic and antioxidant mechanisms.
Keywords
Antioxidant activity
Herbal therapeutics
Letrozole-induced model
Molecular docking
Polycystic ovary syndrome
INTRODUCTION
PCOS, or polycystic ovarian syndrome, is a complex endocrinological condition that is characterized by elevated testosterone levels, irregular menstrual cycles, and/or small cystic patches on one or both ovaries. One of the main pathophysiological mechanisms of polycystic ovarian syndrome has been identified as an increased ratio of luteinizing hormone, also known as luteinizing hormone (LH), to follicle-stimulating hormone (FSH), along with an increased frequency of gonadotropin-releasing hormone secretion.[1-3] hyperandrogenism (elevated male hormones): High levels of androgen that result in symptoms including hirsuteness, skin conditions such as acne, and thinning of the scalp hair. Ovulatory dysfunction (irregular or absent cycles): Infrequent or missing ovulation results in irregular or non-existent menstrual cycles. Polycystic ovaries (multiple small follicles on ultrasound): Several cysts on the ovaries that can be seen with ultrasonography.[4-6]
Normal state
One dominant follicle forms and takes part in a single ovulation event during each menstrual cycle in women during the reproductive period. Primordial follicles are recruited to begin this complex process, and they are then stimulated to begin their developmental trajectory. In the ovarian environment, primary, secondary, tertiary, and finally Graafian follicles mature as a result of sequential follicular development.
Disease state
The physiological process of the growth of follicles is disturbed and does not proceed as it normally would in those who have PCOS. While the attraction and growth of smaller Graafian follicles are at the beginning, the selection of a predominant follicle that can ovulate is often impaired, resulting in the accumulation of multiple tiny Graafian follicles called cysts. There is some different pathophysiology of polycystic ovary syndrome: Primary ovarian pathophysiology, endocrine dysregulation: Ovarian hyperandrogenism, altered hypothalamic-pituitary regulation, insulin resistance and metabolic dysfunction, impaired ovarian steroidogenesis, genetic and epigenetic factors, inflammation, obesity and environmental toxicants, liver function is often affected in PCOS due to associated metabolic disturbances, and in this study, it was evaluated using biochemical markers such as serum glutamate pyruvate transaminase (SGPT) and serum glutamate oxaloacetate transaminase (SGOT) to assess hepatic involvement.[7,8]
Pisum sativum contains a number of active phytochemicals, including phenolic components such as coumaric acid, quinic acid, chlorogenic acid, and flavonoids such as quercetin, catechin, epicatechin, rutin, luteolin, apigenin, and kaempferol.[9,10] It has been demonstrated to have antioxidant activity, anti-inflammatory effect, anticancer properties, antihyperglycemic agent, and cardiovascular benefits. Due to the existence of several bioactive chemicals such as alkaloids, flavonoids, glycosides, isoflavones, phenols, saponins, and tannins, medicinal plants can be used as a source of innovative therapeutic agents.[11,12]
MATERIALS AND METHODS
Procurement of plant
The seeds of P. sativum used in the study were procured from a nearby grocery market in Rajkot, and the plant was taxonomically identified. After collecting, the seeds were peeled and dried in the sunlight. Once dried, they were ground into a fine powder.
The powdered seeds were subjected to extraction using a Soxhlet apparatus to isolate the phytoconstituents. The percentage yield of the extract was then calculated.
Soxhlet extraction
Figure 1 shows the procedure involves filling the circular-bottomed flask with the proper solvent and securing the filter paper-encapsulated sample within the thimble. The solvent enters the vapor phase after being heated to its boiling temperature. To return to its liquid condition, the vapor must climb through the device, pass through the thimble, and arrive at the condenser. The sample is then gradually submerged in the condensed solvent as it drops back into the thimble. The solution containing the extracted solutes is released back into the flask when it reaches the overflow threshold of the syphon. Until the target biomolecules are fully extracted, this cyclical process continues.[13]
- Soxhlet apparatus.
Phytochemicals
Alkaloid
Picric acid test: 2–3 mL filtrate + 3–4 drops of 2% picric acid solution, and a Light-yellow precipitate.
Flavonoids
Ferric chloride test: Extract aqueous solution + a few drops of 10% FeCl3 solution, and appear green color or precipitate. Lead acetate test: 1 mL plant extract + a few drops of 10% lead acetate solution, and a yellow precipitate.
Protein detection
Biuret test: 2 mL filtrate + 1 drop copper sulfate + potassium hydroxide (KOH) pellets, and appear pink color.
Phenolic compound
Lead acetate test: 3 mL plant extract + 3 mL of 10% lead acetate solution, resulting in a white precipitate.[14,15]
Thin layer chromatography (TLC)
TLC was utilized for identifying the chemical constituents quercetin and catechin from MEPS. The extract was mixed with methanol, spotted on silica-coated plates, and dried in the air.
The chromatographic apparatus was calibrated for an hour using Chloroform: Methanol Acid: Water (8:2:1 v/v/v) as the mobile phase for quercetin and Chloroform: Ethyl acetate: Glacial acetic acid (4:4:2) for catechin and epicatechin, respectively.
TLC plates have been developed for approximately 20 min, or until the solvent front reaches ±1 cm from the plate’s top. The TLC plate was dried at room temperature and then viewed under ultraviolet (UV) light at 254–366 nm. The Rf value was determined to be compared to the standard.[16,17]
In silico docking study
The compounds docked against the estrogen receptor using Auto Dock Vina software. Required software: (1) Auto Dock Vina, (2) Biovia Discovery Studio 2025, (3) Open Babel.
The steps involved in the docking are as follows
Conversion of refined enzyme into pdb format. Conversion of pdb format of the ligand into pdbqt format. Preparation of the grid box is done after setting the grid parameters. Docking process occurs by setting docking parameters. Viewing the docked conformation in docking software. Taking snapshots of the interactions for confirmation.[18]
In vitro model
2,2-diphenyl-1-picrylhydrazyl (DPPH) test
The DPPH assay is a commonly used technique to assess a substance’s antioxidant activity. DPPH is a persistent free radical having a deep violet color and a distinctive absorption at 517 nm in UV spectroscopy. We made use of a UV-visible spectrophotometer. First, we created a potent DPPH radical solution in methanol. Then, using foil to shield it from light, we diluted 0.24 mg in 100 mL of methanol in a flask. The absorbance values were fixed at 1.00 ± 0.200. Next, we combined 0.5 mL of the extract with 3 mL of the DPPH working solution, and another one is ascorbic acid as a standard solution, and let it sit in the dark for half an hour. The purple hue vanishes if an antioxidant is present. We just needed 0.5 mL of solvent to create a reference sample. Afterwards, the absorbance was measured at five different concentrations: 20, 40, 60, 80, and 100 µg/mL.[19]
In vivo model
Letrozole-induced PCOS model
Preclinical research on polycystic ovarian syndrome is anticipated to heavily rely on animal models, which provide useful platforms for assessing and validating possible treatment strategies. A thorough analysis of the scientific literature will guide the selection, development, and evaluation of many approaches for the creation of PCOS animal models.[20-22] All the experimental animals except the control group were orally administered letrozole at a dose of 1 mg/kg dissolved in 0.5% carboxymethylcellulose (CMC) once daily for 21 days. The control group received vehicle only (0.5% CMC) orally. Vaginal smears were collected daily and evaluated microscopically using a methylene blue stain to confirm the induction of PCOS. Clomiphene citrate is one of the common medications used in the treatment of PCOS, and so was utilized in the present study as a standard agent. Clomiphene citrate in CMC (1%) was administered orally to animals for 15 days. The test groups were treated with the extract in 1% CMC per the oral route for 15 days at predetermined dosages.[23]
Evaluation parameter
Weight
The weight of each animal was determined at the start of the study. Changes in the weight of the animal will be recorded between the start of the study and the end of the study.
Vaginal smears
Microscopic examination of cellular compositions within vaginal smears had historically served as a reliable method for delineating the distinct phases of the estrous cycle in laboratory rodents, particularly rats and mice. This technique was instrumental in evaluating the functional integrity of the hypothalamic–pituitary–ovarian axis, thereby offering a robust index of reproductive status.[24,25]
Hormonal serum assay
The chemiluminescent immunoassay (CISA) and enzyme-linked immunosorbent assay techniques were used to measure the levels of many hormones in the blood, including progesterone, testosterone, and estrogens. Among them, the CISA method was widely used for hormonal assays in female rats to measure reproductive hormones such as estrogen, progesterone, FSH, LH, and prolactin. There are limited instruments and requirements in the institute, so all biochemical analyses were performed by external, certified diagnostic laboratories rather than in-house.[26]
Histopathology of organs
After blood sampling, all animals were euthanized as per the different group times of endpoints. The ovaries were excised, cleaned, and weighed. The ovaries were then stored in 10% formalin and used for further histopathological examination.
Carbon dioxide (CO2) inhalation method
CO2 induces hypoxia, leading to loss of consciousness followed by respiratory and cardiac arrest.
Procedure
Animals (commonly rodents) are placed in a transparent euthanasia chamber.
Compressed CO2 gas from a cylinder is introduced gradually into the chamber. Flow rate: 20–30% of the chamber volume per minute. Gradual filling prevents distress caused by sudden exposure.
The gas is continued for 1–2 min after respiratory arrest.
Death is confirmed by the absence of respiration, heartbeat, and reflexes.
Optionally, a secondary method (such as cervical dislocation or exsanguination) may be used to ensure death.[5]
Statistical analysis
The results were expressed as mean ± standard error of the mean. The statistical analysis was carried out using GraphPad Prism (PRISM) version 8 software. The data of all parameters were analyzed by means of one-way analysis of variance (ANOVA) followed by Tukey’s test using a computer-based program for comparisons.
Statistical comparison: Each group (n = 6), each value represents the mean ± standard error of the mean. One-way ANOVA followed by Tukey’s test was performed. ns denotes non-significant, *denotes P < 0.05 **denotes P < 0.01, ***denotes P < 0.001 and **** denotes P < 0.0001.
Formulation and evaluation of sustained-release tablet of MEPS
Materials
Hydroxypropyl methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone K30, talc, magnesium stearate (MS) (Pharmaceutics Lab, School of Pharmacy, RK University, Rajkot, Gujarat, India), and isopropyl alcohol (Pharmacological Lab, School of Pharmacy, RK University, Rajkot, India) were all the ingredients and excipients obtained from the department’s laboratory.
P. sativum seed extract was used as the natural active pharmaceutical component in the formulation of the sustained-release matrix tablets. To regulate the release of drugs over a long period of time, hydroxypropyl methylcellulose, also known as (HPMC K15), was used as the sustained-release polymer. To add bulk and enhance tablet compressible properties, microcrystalline cellulose was employed as a diluent. During the process of granulation, polyvinylpyrrolidone K30 (PVP K30) was used as a binder to improve the granule. Talc served as a glidant to enhance the particle flow characteristics, and MS was added as an adhesive to reduce friction during tablet compression. To enable wet granulation and consistent mixing of the formulation ingredients, isopropyl alcohol was used as the granulating fluid.[27]
Procedure
All formulation ingredients HPMC K15M, Microcrystalline cellulose, PVP K30, MS, Talc, including the P. sativum seed extract, sustained-release polymer (HPMC K15), and excipients, were first passed through a 40-mesh sieve and weighed accurately. The sieved materials were blended thoroughly in a mortar or suitable blender to achieve a uniform powder mixture. Separately, the binder (PVP K30) was dissolved in isopropyl alcohol to prepare a 10% binding solution, which was gradually poured into the powder while mixing to produce a wet mass. This wet mass was then passed through a 16 sieve to obtain granules and dried in a hot air oven maintained at 40–50°C. The dried granules were then sieved through a 20 mesh to ensure uniformity, and then after gentle mixing with lubricants and glidants such as MS and talc. A tablet compression machine fitted with punches is then used to compress the prepared granules into tablets.[28]
Evaluation parameter
Weight variation
Twenty pills were weighed separately using a digital scale to conduct the weight variation test. To evaluate the average weight and total weight.
Hardness
A Monsanto hardness tester was used to measure tablet hardness. A fixed jaw and a sliding jaw were used to hold each tablet. The tablet was compressed progressively until it broke by turning the screw knob. The mechanical strength of the tablet was estimated by measuring the force needed to shatter it, which was expressed in kg/cm2.
Friability
The tablets were completely dedusted before testing. After carefully weighing the tablet sample, the tablets were placed in the drum. The drum was then rotated 100 times at 25 revolutions per minute. After rotation, the tablets were removed, dedusted again, weighed, and the percentage of friability was calculated.
Thickness
A caliper was utilized to evaluate uniformity.
Dissolution rate test
The dissolution study was conducted by placing a single tablet (n = 6) as placed in each vessel in a dissolution vessel containing 900 mL of dissolution medium, such as distilled water, 0.1 N hydrochloric acid, or phosphate buffer (pH 6.8), maintained at a temperature of 37 ± 0.5°C. The test was carried out using USP Apparatus II (paddle method) operated at a speed between 50 and 100 rpm. At time intervals of 1, 2, 4, 6, 8, 10, 12 h, samples of 5–10 mL were withdrawn from the medium. These samples were filtered, if necessary, and analyzed using UV-visible spectrophotometry to determine the drug concentration at each time point (Ct).[29-31]
RESULTS
Phytochemical screening
Alkaloids
Picric acid test: - Yellow observed, test positive.
Flavonoids
Ferric chloride test: Green precipitate observed, test positive. Lead acetate test: Yellow precipitate observed, test positive.
Protein detection
Biuret test: Pink color observed, test positive.
Phenolic compound
Lead acetate test: White precipitate observed, test positive.
Optimization of the mobile phase of the TLC system
Figure 2 shows a sample analysis of TLC; in the method optimization, a composition of Chloroform: Ethyl acetate: Glacial acetic acid (4:4:2) was performed as a mobile phase. TLC analysis of MEPS of Rf value was found to be 0.30, and previously reported standard value of catechin and epicatechin Rf value to be 0.2–0.4.
- Thin layer chromatography analysis of extract of catechin and epicatechin.
Figure 3 shows a sample analysis of TLC; in the method optimization, a composition of Chloroform: Methanol acid: Water (8:2:1) was performed as a mobile phase. TLC analysis of MEPS of Rf value was found to be 0.63, and the previously reported standard value of quercetin Rf value was 0.48–0.65.
- Thin layer chromatography analysis of extract of quercetin.
Optimization of in silico computational system
Figures 4-6 show that the binding affinity of epicatechin, catechin, and quercetin to the estrogen receptor, and then show that the enthalpy was 7.1, 6.6, and 7.8 kcal/mol, respectively. The above-mentioned binding affinity shows that the phytoconstituent of the extract was able to manage the polycystic ovary syndrome disease.
- Epicatechin bind with estrogen receptor.
- Catechin bind with estrogen receptor.
- Quercetin bind with estrogen receptor.
DPPH radical scavenging inhibition as in vitro models
As shown in Table 1, in this parameter, we checked the % inhibition of all groups. As a standard (Ascorbic acid) was used, and the test group contained the extract of P. sativum in five different concentrations. The above five different concentrations, like 20, 40, 60, 80, 100 ppm concentrations solutions were prepared in methanol, and the DPPH solution was added to find out % of inhibition.
| Concentration (µg/mL) | Inhibition of extract (%) | Inhibition of standard (%) |
|---|---|---|
| 20 | 36.51 | 62.36 |
| 40 | 40.73 | 68.53 |
| 60 | 45.08 | 74.01 |
| 80 | 48.36 | 79.03 |
| 100 | 52.61 | 83.2 |
As shown in Figure 7, there was a significant and gradual increase in % inhibition in the standard control group as compared to the test groups. The finding of this model suggests that there was a significant increase in % inhibition in the standard control compared to that of the test extract.
- 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging inhibition graph.
Effect of letrozole induced in PCOS by identification of vaginal smear
Figure 8 shows the normal group vaginal smear, where we can identify different phases such as proestrus (nucleotide cornified epithelial cell), estrus (nucleotide and A nucleotide epithelial cell), metestrus (cornified epithelial cell and leukocytes), and diestrus (leukocytes).
- (a-d) Normal control phases (a) proestrus, (b) estrus, (c) metestrus, (d) diestrus. Stained cells (10x), Giemsa stain.
Figure 9 shows disease group vaginal smear where we can identify phase diestrus (leukocytes) only because of giving oral letrozole 1 mg/kg for 21 days for inducing polycystic ovary syndrome.
- (a-d) Disease control phases (only diestrus). Stained cells (10x), Giemsa stain.
Figure 10 shows after inducing disease in rat standard drug as clomiphene 1 mg/kg for 15 days given to rat to compare normal group and standard group and standard group vaginal smear where we can identify different phase such as proestrus (nucleotide cornified epithelial cell), estrus (nucleotide and A nucleotide epithelial cell), metestrus (cornified epithelial cell and leukocytes), and diestrus (leukocytes).
- Standard control phases (a) diestrus, (b) metestrus, (c) estrus, (d) proestrus. Stained cells (10x), Giemsa stain.
Figure 11 shows after inducing disease in rat test drug as MEPS (seed) 200 mg/kg for 15 days to rat to compare normal group and Test 1 group and Test 1 group vaginal smear where we can identify different phase such as proestrus (nucleotide cornified epithelial cell), estrus (nucleotide and a nucleotide epithelial cell), metestrus (cornified epithelial cell and leukocytes), and diestrus (leukocytes).
- (a-d) Test 1 control phases (a) diestrus, (b) metestrus, (c) estrus, (d) proestrus. Stained cells (10x), Giemsa stain.
Figure 12 shows after inducing disease in rats test drug as MEPS (seed) 400 mg/kg for 15 days to rat to compare the normal group and Test 2 group, and Test 2 group vaginal smear, where we can identify different phases such as proestrus (nucleotide cornified epithelial cell), estrus (nucleotide and A nucleotide epithelial cell), metestrus (cornified epithelial cell and leukocytes), and diestrus (leukocytes).
- (a-d) Test 2 control phases (a) diestrus, (b) metestrus, (c) estrus, (d) proestrus. Stained cells (10x), Giemsa stain.
Effects of MEPS on body weight level in letrozole-induced PCOS
Figure 13 shows that after administration of letrozole (1 mg/kg, p.o., 21 days), the weight significantly increased in weight when compared to the weight from 0 days to –36 days. The disease control group of animals also showed increased weight (36.00 ± 6.47 g), which was found to be statistically significant compared to the normal control group (13.12 ± 7.37 g).
- Body weight. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. **denotes P < 0.01, ***denotes P < 0.001 and ****denotes P < 0.0001.
Treatment with clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole-treated rats resulted in a change in body weight (29.05 ± 8.98 g) compared to the disease control group of weight of animals on 36 days (36.00 ± 6.47 g).
Treatment with Test 1 group (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg. p.o., 15 days) of MEPS led to a change in body weight (32.00 ± 7.90 g) and (27 ± 12.86 g), respectively, as compared to the disease control (36.00 ± 6.47 g).
Effects of MEPS on FSH level in letrozole-induced PCOS
Figure 14 shows that at the end of 21 days of Letrozole (1 mg/kg) treatment, there was a significant decrease in serum FSH level in female rats of the disease control group (2.13 ± 0.25 ng/dL) as compared to the normal control group (8.01 ± 029 ng/dL) of animals.
- Follicle-stimulating hormonal (FSH) level. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ****denotes P < 0.0001.
After treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats, there was a significant increase in serum FSH level (6.48 ± 0.11 ng/dL) as compared to the disease control group (2.13 ± 0.25 ng/dL).
Treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 day) of MEPS led to significantly increase in serum FSH level (5.31 ± 0.13 ng/dL) and (5.91 ± 0.10 ng/dL), respectively, as compared to disease control (2.13 ± 0.25 ng/dL).
Effects of MEPS on LH level in letrozole-induced PCOS
Figure 15 shows that at the end of 21 days of letrozole (1 mg/kg) treatment, there was a significant increase in serum LH level in female rats of the disease control group (8.73 ± 0.15 mIU/mL) as compared to the normal control group (5.78 ± 0.10 mIU/mL) of animals.
- Luteinizing hormone (LH) level. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. **** denotes P < 0.0001.
After treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats, a significant decrease in serum LH level (5.43 ± 0.15 mIU/mL) was observed as compared to the disease control group (8.73 ± 0.15 mIU/mL).
Treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 days) of MEPS led to significantly decreased serum LH levels (6.03 ± 0.80 mIU/mL) and (5.46 ± 0.14 mIU/mL), respectively, as compared to the disease control (8.73 ± 0.15 mIU/mL).
Effects of MEPS on testosterone level in letrozole-induced PCOS
Figure 16 shows that at the end of 21 days with letrozole (1 mg/kg) treatment, there was a significantly increased serum testosterone in female rats of the disease control group (228.46 ± 1.58 pg/mL) as compared to the normal control group of animals (92.96 ± 2.36 pg/mL).
- Testosterone level. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ***denotes P < 0.001 and ****denotes P < 0.0001.
Treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats resulted in a significant decrease in serum testosterone (114.60 ± 2.05 pg/mL) as compared to the disease control group (228.46 ± 1.58 pg/mL).
Treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 days) of MEPS led to significantly decreased in serum testosterone level (133.45 ± 1.27 pg/mL) and (125 ± 1.88 pg/mL), respectively, as compared to disease control group (228.46 ± 1.58 pg/mL).
Effects of MEPS on estrogen level in letrozole-induced PCOS
Figure 17 shows that at the end of 21 days with letrozole (1 mg/kg) treatment, there was a significantly decreased serum estrogen in female rats of the disease control group (2.25 ± 0.10 pg/mL) as compared to the normal control group of animals (9.21 ± 0.15 pg/mL).
- Estrogen level. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ****denotes P < 0.0001.
Treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats resulted in significantly increased serum estrogen (9.46 ± 0.11 pg/mL) as compared to the disease control group (2.25 ± 0.10 pg/mL).
Treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 day) of MEPS led to significantly increased in serum estrogen level (7.25 ± 0.13 pg/mL) and (8.26 ± 0.12 pg/mL), respectively, as compare to disease control group (2.25 ± 0.10 pg/mL).
Effects of MEPS on progesterone level in letrozole-induced PCOS
Figure 18 shows that at the end of 21 days with letrozole (1 mg/kg) treatment, there was a significantly decreased serum progesterone in female rats of the disease control group (15.56 ± 0.60 ng/mL) as compared to the normal control group of animals (30.98 ± 0.83 ng/mL).
- Progesterone level. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ***denotes P < 0.001 and ****denotes P < 0.0001.
Treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats resulted in significantly increased serum progesterone (29.98 ± 0.96 ng/mL) as compared to the disease control group (15.56 ± 0.60 ng/mL).
Treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 day) of MEPS led to significantly increased in serum progesterone level (24.11 ± 0.54 ng/mL) and (27.35 ± 0.58 ng/mL), respectively, as compared to disease control group (15.56 ± 0.60 ng/mL).
Effects of MEPS on serum liver profile SGPT, and SGOT level in letrozole-induced PCOS
Figures 19 and 20 show that at the end of 21 days with letrozole (1 mg/kg) treatment, there was a significantly increased serum SGPT (84.11 ± 1.35 U/L) and SGOT (100.76 ± 2.09 U/L) in female rats of the disease control group and as compared to normal control group of animals SGPT (39.10 ± 1.88 U/L) and SGOT (68.15 ± 1.97 U/L).
- Serum glutamate pyruvate transaminase (SGPT) level. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ***denotes P < 0.001 and ****denotes P < 0.0001.
- Serum glutamate oxaloacetate transaminase (SGOT) level. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ***denotes P < 0.001 and ****denotes P < 0.0001.
Treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats resulted in significantly decreased serum SGPT (43.31 ± 1.44 U/L) and SGOT (76.03 ± 1.96) as compared to the disease control group SGPT (84.11 ± 1.35U/L) and SGOT (100.76 ± 2.09 U/L).
Figure 19 treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 days) of MEPS led to slightly decreased in serum SGPT level (63.95 ± 1.58 U/L) and (52.48 ± 1.36 U/L), respectively, as compared to the disease control group (84.11 ± 1.35 U/L).
Figure 20 treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 days) of MEPS led to significantly decreased in serum SGOT level (87.70 ± 1.76 U/L) and (83.46 ± 1.45 U/L), respectively, as compared to disease control group (100.76 ± 2.09 U/L).
Effects of MEPS on insulin level in letrozole-induced PCOS
Figure 21 shows that at the end of 21 days with letrozole (1 mg/kg) treatment, there was a significantly increased insulin level in female rats of the disease control group (20.46 ± 0.44 mIU/mL) as compared to the normal control group of animals (13.21 ± 1.06 mIU/mL).
- Insulin level. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ns denotes non significant, **denotes P < 0.01, P < 0.001 and ****denotes P < 0.0001.
Treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats resulted in a significantly decreased insulin level (14.81 ± 0.34 mIU/mL) as compared to the disease control group (13.21 ± 1.06 mIU/mL).
Treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 days) of MEPS led to a significant decrease in insulin level (18.26 ± 0.59 mIU/mL) and (16.35 ± 0.52 mIU/mL), respectively, as compared to the disease control group (13.21 ± 1.06 mIU/mL).
Effects of MEPS on triglyceride in letrozole-induced PCOS
Figure 22 shows that at the end of 21 days with letrozole (1 mg/kg) treatment, there was a significantly increased serum triglyceride level in female rats of the disease control group (115.26 ± 1.68 mg/dL) as compared to the normal control group of animals (85.21 ± 1.79 mg/dL).
- Triglyceride. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ****denotes P < 0.0001.
Treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats resulted in a decrease in serum triglyceride (88.51 ± 1.26 mg/dL) as compared to the disease control group (115.26 ± 1.68 mg/dL).
Treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 day) of MEPS led to show that decreased in serum triglyceride level (98.68 ± 1.76 mg/dL) and (93.76 ± 1.27 mg/dL), respectively, as compared to disease control group (115.26 ± 1.68 mg/dL).
Effects of MEPS on low-density lipoprotein (LDL) cholesterol level in letrozole-induced PCOS
Figure 23 shows that at the end of 21 days with letrozole (1 mg/kg) treatment, there was a significantly increased serum cholesterol level in female rats of the disease control group (151.66 ± 3.15 mg/dL) as compared to the normal control group of animals (112.16 ± 3.10 mg/dL).
- Cholesterol. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ns denotes non significant, ***denotes P < 0.001 and ****denotes P < 0.0001.
Treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats resulted in a decrease in serum cholesterol (130.08 ± 2.88 mg/dL) as compared to the disease control group (151.66 ± 3.15 mg/dL).
Treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 day) of MEPS led to show that decreased in serum cholesterol level (142.43 ± 1.85 mg/dL) and (120.33 ± 1.45 mg/dL), respectively, as compared to disease control group (151.66 ± 3.15 mg/dL).
Effects of MEPS on LDL level in letrozole-induced PCOS
Figure 24 shows that at the end of 21 days with letrozole (1 mg/kg) treatment, there was a significantly increased serum LDL level in female rats of the disease control group (90.30 ± 1.38 mg/dL) as compared to the normal control group of animals (61.50 ± 1.87 mg/dL).
- Low-density lipoprotein (LDL) level. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ns denotes non significant, ***denotes P < 0.001 and ****denotes P < 0.0001.
Treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats resulted show that decreased in serum LDL (70.56 ± 1.25 mg/dL) as compared to disease control group (90.30 ± 1.38 mg/dL) treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 day) of MEPS led to show that decreased in serum LDL level (84.58 ± 1.22 mg/dL) and (72.11 ± 1.14 mg/dL), respectively, as compare to disease control group (90.30 ± 1.38 mg/dL).
Effects of MEPS on high-density lipoprotein (HDL) level in letrozole-induced PCOS
Figure 25 shows that at the end of 21 days with letrozole (1 mg/kg) treatment, there was a significantly decreased serum HDL level in female rats of the disease control group (22.20 ± 0.38 mg/dL) as compared to the normal control group of animals (28.58 ± 0.56 mg/dL).
- High-density lipoprotein (HDL) level. MEPS: Methanolic extract of pisum savitum, PCOS: Polycystic ovarian syndrome, STD: Sexually transmitted disease. ns denotes non significant, **denotes P < 0.01, ***denotes P < 0.001 and ****denotes P < 0.0001.
Treatment with standard clomiphene citrate (1 mg/kg, p.o., 15 days) in letrozole treated rats resulted show that increased in serum HDL (26.28 ± 0.59 mg/dL) as compared to disease control group (22.20 ± 0.38 mg/dL) treatment group Test 1 (200 mg/kg, p.o., 15 days) and Test 2 (400 mg/kg, p.o., 15 day) of MEPS led to show that slightly increased in serum HDL level (22.48 ± 0.60 mg/dL) and (24.56 ± 0. 45 mg/dL), respectively, as compare to disease control group (22.20 ± 0.38 mg/dL).
Ovarian histopathology of PCOS-induced rats
Human polycystic ovaries and letrozole-induced PCOS ovaries shared several histopathological characteristics. There were several subcapsular cysts in the group that received letrozole to trigger the condition.
Figure 26 shows that, in contrast to the normal control group, which had primary follicles and secondary follicles surrounded by a granulose cell layer, as well as the existence of corpus luteum, PCOS-induced groups showed destruction of granulosa cells and the number of follicular cysts in the ovary.
- Histopathology of ovary’s. (Giemsa stain, 10x).
After 15 days of treatment with clomiphene citrate (1 mg/kg po), MEPS (200 mg/kg) and (400 mg/kg) groups, respectively, the ovarian tissue recovered with reduced follicular cysts and improved with primary and secondary follicle structure, as well as mature follicles with oocytes free of cell defects and developed antral follicles.
Histopathology of a normal control ovary having a normal antral follicle with corpus luteum, primary follicle, and secondary follicle with theca cell (×10).
Histopathology of the letrozole-induced PCOS group with cystic degenerating follicles. Antral follicle cyst with a number of small cysts forms and damages the theca and granulose cells (×10).
Histopathology of the ovary of the disease control group of rats treated with clomiphene citrate (1 mg/kg, p.o. 15 days) showing the presence of developing antral follicles and corpus luteum, and Graafian follicles (×10).
Histopathology of MEPS (200 mg/kg) shows the presence of developing secondary follicles, and some follicular cysts are present in female rats (×10).
Histopathology of MEPS (400 mg/kg) showing the presence of developing antral follicles and corpus luteum with recovered ovarian function of female rats (×10).
Evaluation results of tablet formulation
The sustained-release tablets developed were subjected to a series of quality control evaluations, all of which met the established pharmacopeial standards. As shown in Table 2, the pills consistently weighed between 588 and 605 mg, which is within the permitted ±5% limit for tablets weighing more than 250 mg. The hardness test resulted in a value of 5.5 ± 0.4 kg/cm2, which is within the permissible range of 4–8 kg/cm2 and indicates sufficient tablet strength for handling and transportation. The tablets have an average thickness of 4.2 ± 0.1 mm, and their uniformity in size. Friability score of 0.62%, it was found that it’s in the limit of greater than. Figure 27 and Table 3 Dissolution test results show that the extract was released gradually, and at the beginning 11.8% in the 1st h, and gradually increasing to 89.3% by the 12th h. Graph indicates the over time drug release prolong time, like 12 h in phosphate buffer solution, which indicates the drug formulation transparency and stability. The effectiveness of the tablet formulation in prolonging medication availability over time is denoted by this sustained drug release profile.
- Dissolution profile of Pisum sativum sustained release (SR) tablet.
| Test | Result | Limit/specification |
|---|---|---|
| Weight Variation | 588–605 mg | ±5% for>250 mg tablets |
| Hardness | 5.5±0.4 kg/cm2 | 4–8 kg/cm2 |
| Thickness | 4.2±0.1 mm | Uniform |
| Friability (%) | 0.62 | <1 |
| Time (h) | Cumulative drug release (%) |
|---|---|
| 1 | 11.8 |
| 2 | 26.5 |
| 4 | 43.3 |
| 6 | 60.3 |
| 8 | 72.3 |
| 10 | 83.5 |
| 12 | 89.3 |
DISCUSSION
In polycystic ovarian syndrome aberrant hormonal alterations, such as low levels of progesterone and estrogen, high levels of testosterone, and aberrant levels of gonadotropin, cause menstrual irregularities in polycystic ovarian syndrome, including oligomenorrhea and amenorrhea, hyperandrogenism, anovulation, and insulin resistance.
The present investigation shows that letrozole-induced PCOS in Albino Wistar rats may be effectively treated with the MEPS. Menstrual abnormalities, including oligomenorrhea and amenorrhea, hyperandrogenism, anovulation, insulin resistance, and dermatological signs such as acanthosis nigricans, are indicators of PCOS, a complicated endocrine condition. Hormonal imbalances, such as reduced levels of progesterone and estrogen, increased levels of testosterone, and changed gonadotropin levels, are the cause of these symptoms. The pathological characteristics of PCOS are immediately addressed by MEPS, which is abundant in naturally occurring estrogen-producing substances such as epicatechin, quercetin, and catechin. It has also shown a variety of pharmacological actions, including anti-inflammatory, estrogenic, and anti-hyperlipidemia properties. MEPS’s involvement in encouraging ovulation and general reproductive health is demonstrated by the normalization of the estrous cycle, avoidance of ovarian dysfunction, and improvement of hormonal balance that resulted from the therapy. One potential mechanism of action through phytoestrogenic activity is suggested by the extract’s apparent ability to influence endocrine activities in a way that corrects the abnormalities produced by PCOS. The findings not only provide insight into the potential of P. sativum as a natural remedy for PCOS but also open avenues for its use as a complementary therapy alongside conventional treatment approaches.
According to the current study, albino Wistar rats with letrozole-induced PCOS may benefit from treatment with MEPS at doses of 200 and 400 mg/kg. In contrast to the disease-induced rat group, which has a large number of ovaries and cysts, histopathological analysis demonstrates that MEPS therapy aids in improvements in ovarian shape and function. Increased testosterone levels and abnormal LH/FSH ratios, which are commonly seen in PCOS, were among the hormones and ovarian function that were shown to be improved by the 400 mg/kg dosage. Crucially, MEPS at 400 mg/kg was shown to be equally effective as the usual course of therapy with clomiphene citrate, a medication that is frequently recommended to induce ovulation. The formation and improvement of main and secondary follicles for ovulation and hormone regularity are demonstrated by histopathology, which indicates that both therapies were successful in causing ovulation and restoring anovulatory disorders. However, because of its natural origin and diverse pharmacological activities, MEPS provides extra advantages. The extract’s phytoestrogens, which may promote ovarian health and hormone balance, including epicatechin, quercetin, and catechin, are probably responsible for these benefits. The results not only shed light on P. sativum’s potential as a natural treatment for PCOS, but they also pave the way for its application as a supplemental therapy in addition to traditional therapeutic modalities. All of these adjustments prevented ovarian cell malfunction and brought the estrus cycle back to normal. The presence of phytoestrogens (quercetin, catechin, and epicatechin) may be the cause of this pharmacological effect. This finding suggests that MEPS has a promising role for treating pathological abnormalities in the polycystic ovarian syndrome condition. For the treatment, MEPS may offer a comprehensive substitute or adjunct therapy than clomiphene citrate, which may have restricted utility because of possible adverse effects or patient resistance. It is a viable option for more research because of its capacity to influence several components of the syndrome, such as oxidative stress, lipid metabolism, hormonal imbalance, and ovarian dysfunction.
Several limitations of the present study should be acknowledged. All biochemical and histopathological analyses were performed by external, certified diagnostic laboratories rather than in-house; we therefore relied on laboratory reports rather than direct control of assay platforms or raw data processing, which may introduce inter-laboratory variation. Second, the study used a single animal model or a longer follow-up to assess the durability of effects. Third, the sample size per group was limited and may reduce power to detect small-to-moderate effects on hormonal endpoints and ovarian histology. Finally, there is no direct evidence or research to compare the data and results to that limited evidence for the study.
Finding current and baseline data can help create future therapeutic advantages of P. sativum (seed) as an adjuvant therapy or as a stand-alone treatment for improved PCOS control. All things considered, this study provides a solid foundation for MEPS as a cutting-edge botanical strategy for PCOS management. It is imperative that future research concentrate on clinical validation, molecular processes, and active component separation to translate these findings into potential therapeutic applications for women suffering from PCOS.
CONCLUSION
The present study demonstrates that the MEPS, particularly at 400 mg/kg, effectively alleviates letrozole-induced PCOS in Albino Wistar rats. MEPS showed comparable efficacy to clomiphene citrate in restoring ovulation, normalizing hormonal imbalance, and improving ovarian histology. Its beneficial effects are attributed to the presence of phytoestrogens such as quercetin, catechin, and epicatechin, which offer estrogenic, anti-inflammatory, and anti-hyperlipidemia properties. MEPS provide a promising, natural alternative or adjunct to standard therapies for PCOS, with fewer side effects. These findings establish a basis for further research on MEPS in clinical applications for PCOS management.
Ethical approval:
The research/study was approved by the Institutional Review Board at RK University, Rajkot, number RKCP/COI/Re/24/145, dated January 11, 2025.
Declaration of patient consent:
Patient’s consent not required as there are no patients in this study.
Conflicts of interest:
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript, and no images were manipulated using AI.
Financial support and sponsorship: None.
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