INTRODUCTION:

Polycystic ovarian syndrome (PCOS) is one of the most common endocrinal disorders in women, among that 4-12% of women affecting at the reproductive age^1,². It is characterized by numerous cysts on the ovaries associated with hyperandrogenism, chronic anovulation, insulin insensitivity, and other metabolic abnormalities³. Hyperandrogenism and insulin resistance are the major pathological contributors to PCOS.

Insulin resistance not only contributes to the metabolic feature of PCOS but also a contributor of reproductive features through increased production of androgen in theca cells and increasing free androgen by inhibiting the hepatic synthesis of sex hormone-binding globulin (SHBG) in the liver⁴.

Metformin is one of the commonly prescribing insulin-sensitizing agents for PCOS women⁵. Apart from the hypoglycaemic effect, it also regularizes the menstrual irregularity, induces ovulation, and reduces the excessive androgen level. Metformin therapy has fewer side effects like hot flushes, bloating, mood swings, joint pain, and arthritis⁶. Due to these limitations, PCOS women are intense to use the alternate medication in association with traditional knowledge to improve the PCOS condition⁷.

Gymnema sylvestre (G. sylvestre) (Apocynaceae) is a well-known potent antidiabetic herb used in the Indian traditional system of medicine, such as Ayurveda, Siddha, Unani, and even in modern medicine. After chewing fresh leaves of G. sylvestre, one is unable to detect the sweet taste. The leaf extract was also reported to possess antidiabetic property in human and animal models⁸. Other reported activities are antipyretic, antioxidant, astringent, digestive, stomachic, laxative, liver tonic, cardiotonic and uterine tonic. The extract of G. sylvestre is used as a dietary supplement to reduce body weight, blood cholesterol, and triglyceride. These reported therapeutic effects are mainly due to its secondary metabolites triterpene saponins, gymnemic acids, acidic glycosides, and anthraquinones^9-13. Based on the reported hypoglycemic and hypolipidemic property of its secondary metabolites this study was designed to evaluate the therapeutic potential of Gymnema sylvestre leaf extract against letrozole-induced polycystic ovarian syndrome in rats.

MATERIALS AND METHODS:

Chemicals and reagents:

All the chemicals and reagents used in this study were of analytical grade. Letrozole was purchased from Sun Pharmaceutical Industry Limited, India. Metformin was purchased from Cipla Ltd., India. Carboxymethyl cellulose sodium (CMC) was from HIMEDIA, India. Other Biochemical and enzyme immunoassay kits were purchased from Thermo Fisher Scientific, India.

Plant material and extraction of Gymnema sylvestre:

The authenticated dried leaf powder of G. sylvestre was purchased from Genius Nature Herbs Pvt. Ltd., Coimbatore, India. Powdered material (500gm) was extracted with 95% ethanol in the Soxhlet apparatus at 75⁰C for 16 hrs. After filtration, the extract was evaporated under reduced pressure and controlled temperature (40⁰C) in a rotary evaporator until all solvent was removed to obtain a greenish-black jelly residue (yield = 18.2%w/w). The extract was then stored in an airtight container below 10⁰C until further use.

Figure 1. Fresh Leaves and dried leaves powder material of Gymnema Sylvestre

Animals:

Thirty colony inbred virgin female Albino Wistar Rats, weighing 180-250gm (12 weeks) were obtained from the Central Animal house facility of Swamy Vivekanandha College of Pharmacy, Namakkal – 637 205, Tamil Nadu, India. The animals were acclimated and housed in polypropylene cages at (23±2ºC), relative humidity of (60±10)%, and 12/12hr light/dark rhythm a week before the experimentation. They were fed with a standard pellet diet and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Swamy Vivekanandha College of Pharmacy, Namakkal-05. Care and use of laboratory animals were confirmed to CPCSEA guidelines. (IAEC Reference No: SVCP/IAEC/PG/1/03/2017), all the selected animals had at least two sequential estrous cycles that continued for four days. Estrous cycles were monitored by early morning vaginal smear sampling.

Acute toxicity studies:

Acute oral toxicity of ethanolic leaf extract of G. sylvestre was carried out as per the OECD-423 guide line¹⁴. albino wister rats (n=3) were used for acute toxicity studies. They were fasted overnight and provided only water. The extract was administered at the starting dose level of 2000mg/kg body weight through gastric intubation and observed mortality and behavioral changes for 14 days. If mortality was observed in two out of three animals, the dose was assigned as a toxic dose. If mortality was observed in one animal, then the same test was repeated to confirm the toxic dose. If there is no mortality, one-tenth of the maximum acute toxicity tested dose was used as a low dose and one-fifth was selected as a higher dose for further pharmacological evaluation.

Experimental design and induction of PCOS:

The selected thirty rats with regular estrous were randomly divided into five groups each of six. All the animals except group-I were treated with Letrozole (1mg/kg b.w.) orally once daily for 21 days to induce PCOS¹⁵. Group-I served as vehicle control and receives only 1ml of 0.5% CMC orally. The induction of PCOS was primarily confirmed by measuring the irregularity of the menstrual cycle. From day 22^ndGroup-II served as PCOS disease control. Group-III received metformin (20mg/kg b.w. p.o.), Group-IV and V received ethanolic leaf extract of G. sylvestre 200 mg/kg and 400mg/kg body weight respectively through orally. The metformin and extract treatments were continued for further 28 days.

Vaginal smear observation:

Vaginal smear was prepared by collecting vaginal fluid by inserting the tip of the pipette with 10μL of normal saline into the rat vagina. This procedure was carried out every morning between 8.00 to 9.00 am. After collecting the vaginal fluid, the smear was prepared on a glass slide and stained with methylene blue (0.5% aqueous solution). The prepared slides were observed under the light microscope (40X objective lens magnification)¹⁶.

Oral glucose tolerance test (OGTT):

At the end of the study period, all the rats fasted for 12 hr, and OGTT was performed using an Accu Check Active glucometer (Roche Diagnostics Ltd). After 12 hr fasting, an initial blood glucose level was measured using the tail vein blood sample. Each rat received 0.5 ml of 400mg of glucose solution orally; 2 hr after administration of glucose solution, blood glucose level was monitored again¹⁷.

Serum biochemical and hormonal analysis:

After 28 days of drug treatment, all the rats were anesthetized with 90mg/kg ketamine and 10mg/kg xylazine i. p.¹⁸. The blood sample was drawn through retro-orbital plexus and the serum was separated after centrifugation without anticoagulants, at 3000rpm, for 10min. The serum lipid profile like high-density lipoprotein (HDL), low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL) cholesterol, and triglyceride were analyzed by auto analyzer kits standard techniques following the manufacturer’s instructions (Thermo Fisher Scientific, India). The serum LH, FSH, testosterone, estradiol, and progesterone were measured using an enzyme-linked immunosorbent assay standard kit according to the manufacturer’s instructions (Thermo Fisher Scientific, India)^15,19.

Histopathological examination of the ovary:

After collection of the blood sample, animals were sacrificed by an overdose of anesthesia. The left ovary from one rat of each group was removed and fixed in 10% neutral formal saline, embedded in paraffin wax, and then sectioned at 5-μm thickness. Sections were mounted and stained by the hematoxylin and eosin procedure. Some sections were stained by Masson’s trichrome and examined under a light microscope with 100 x magnification for histopathological changes¹⁵.

Statistical analysis:

Data were expressed as mean ± standard deviation (mean±SD) to determine significance. When rats were compared over time or within groups, one-way analysis of variance was performed, followed by post hoc Dunnett’s test using SPSS V.17. The values were considered statistically significant when P<0.05.

RESULT:

Effect of ethanolic extract of G. sylvestre on acute oral toxicity studies:

Acute toxicity study results showed that the ethanolic extract of G. sylvestre did not cause any toxic symptoms or mortality up to a dose level of 2000mg/kg and was considered safe. Hence 1/10^th and 1/5^th (200mg and 400 mg respectively) of the dose were selected for further pharmacological screening as submaximal and maximal doses.

Effect of ethanolic extract of G. sylvestre on estrus cyclicity in letrozole induced PCOS rats

Twenty-one days after letrozole treatment, they had an irregularity in estrus cyclicity. Significant (p<0.001) reduction in the estrus stage and concomitant increased in the diestrus duration when compared to the normal control (group I). After 28 days (namely, on day 50 of the study) of drug treatments, the increased diestrus duration was significantly (p<0.001) reduced in all the drug treatments (groups III- V) when compared to the PCOS control (group II) (Table -1).

Effect of ethanolic extract of G. sylvestre on oral glucose tolerance test in letrozole induced PCOS rats:

The results in table 2 indicated that both fasting and two hours after oral glucose changed blood glucose levels were significantly (P<0.001) higher in the PCOS control group (group II) when compared to normal control (group I). The drug treatments (groups III-V) significantly reduced the induced hyperglycemia in both cases when compared to the PCOS control.

Effect of ethanolic extract of G. sylvestre on lipid profile in letrozole induced PCOS rats:

Table 3 data represents that significant (P<0.001) elevation in serum VDL, VLDL cholesterol, and triglyceride levels, also accompanied by decreased HDL levels in the PCOS control (group II) when compared to the normal control (group I). All the treatments (group III-V) showed significant (P<0.001) improvement in the altered lipid profile when compared to the PCOS control group. The treatment of G.sylvestre showed dose-dependent improvement in the lipid profile.

Table1. Effect of ethanolic extract of G. sylvestre on estrus cyclicity in letrozole induced PCOS rats

Treatment	Proestrus		Estrus		Metestrus		Diestrus
Treatment	21^st day	50^th day	21^st day	50^th day	21^st day	50^th day	21^st day	50^th day
Group-I ( 0.5% CMC)	2.0±0.5	1.8±0.5	3.0±0.9	2.6±0.5	2.0±0.5	1.8±0.5	2.0±0.6	2.0±0.7
Group-II (Letrozole)	2.4±0.4	1.5±0.6	1.2±0.5^a***	1.2±0.5^a**	2.0±0.6	1.6±0.8	3.6±0.5^a*	5.2±0.8^a***
Group-III (Letrozole + Metformin)	1.8±0.6	1.6±0.5	1.4±0.6^a***	2.0±0.7	2.4±0.9	2.0±0.6	4.4±0.7^a**	2.8±0.8^b***
Group-IV (Letrozole + EEGS 200mg/kg)	2.1±0.7	1.8±0.6	1.4±0.5^a***	2.2±0.7	2.2±0.5	1.9±0.5	4.4± 0.9^a**	2.7±0.7^b***
Group-V (Letrozole + EEGS 400mg/kg)	2.0±0.8	1.7±0.6	1.2±0.5^a***	2.0±0.6	2.0±0.7	1.9±0.5	4.3± 0.8^a**	2.8± 0.6^b***

Values are expressed as mean ± SD, n=6. Comparisons were made between a- Group I vs II, III, IV, V; and b- Group II vs III, IV, V. Symbols represent statistical significance: ***P<0.001, **-P<0.01, *-P<0.05.

Table 2. Effect of ethanolic extract of G. sylvestre on oral glucose tolerance test in letrozole induced PCOS rats

Treatment	Altered Glucose Metabolism (mg/dl)
Treatment	0 h (fasting blood glucose)	2 h
Group-I ( 0.5% CMC)	103.17 ± 5.67	137.67 ± 14.18
Group-II (Letrozole)	183.00 ± 12.38^a***	275.50 ± 49.04^a***
Group-III (Letrozole + Metformin)	109.17 ± 7.13^b***	163.67 ± 27.35^b***
Group-IV (Letrozole + EEGS 200mg/kg)	108.52 ± 9.22^b***	169.35 ± 12.67^b***
Group-V (Letrozole + EEGS 400mg/kg)	106.48 ± 8.15^b***	162.77 ± 8.25^b***

Effect of ethanolic extract of G. sylvestre on hormonal profile in letrozole induced PCOS rats:

In table 4 data represented a significant (P<0.001) increased in serum levels of testosterone, luteinizing hormone, and decreased estradiol, progesterone, and follicle-stimulating hormone in PCOS control (group II) when compared to the normal control (group I). All the treatment groups (group III-V) showed a significant reversal effect on the hormonal profile as increased in serum estradiol, progesterone, and FSH level and decreases in testosterone and LH levels when compared to the PCOS control.

Table 3. Effect of ethanolic extract of G. sylvestre on lipid profile in letrozole induced PCOS rats

Treatment	HDL (mg/dl)	LDL (mg/dl)	VLDL (mg/dl)	TG (mg/dl)
Group-I (0.5% CMC)	34.33 ± 4.68	90.67 ± 8.94	46.67 ± 10.39	74.86 ± 9.97
Group-II (Letrozole)	15.67 ± 4.41^a***	148.17 ± 25.47^a***	90.33 ± 9.31^a***	165.33 ± 16.80^a***
Group-III (Letrozole + Metformin)	24.00 ± 4.34^{a** b*}	112.17 ± 17.11^{a* b***}	53.50 ± 12.05^b***	106.00 ± 19.87^{a* b*}
Group-IV (Letrozole + EEGS 200mg/kg)	27.20 ± 5.41^b***	102.40 ± 10.22^b***	55.46 ± 4.34^b***	83.14 ± 4.26^b***
Group-V (Letrozole + EEGS 400mg/kg)	28.82 ± 4.10^b***	99.47 ± 6.81^b***	52.45 ± 2.30 ^b***	78.20 ± 5.84^b***

Effect of ethanolic extract of G. sylvestre on ovarian histology:

Histopathology of normal control ovary showed well-developed follicles and corpus luteum. Letrozole induced PCOS control rats showed altered ovarian architecture such as undeveloped follicles, absence of corpus luteum, an increased number of follicular cysts. The treatment groups III and V showed normal healthy developing follicles, corpus luteum, and few follicular cysts. Group IV (G. sylvestre 200 mg/kg treatment) showed more follicular cysts and few developed follicle and absence of corpus luteum as compared to other treatments (group III & V)

Figure 2. Histopathological photomicroscophs of ovary sections (hematoxylin and eosin stain; magnificention 100x): (A) Normal control, (B) PCOS control group, (C) Letrozole + Metformin, (D) Letrozole + G. Sylvestre 200mg/kg, (E) Letrozole + G. Sylvestre 400mg/kg. FC: Follicular cyst; CL: corpus luteum; F: follicles.

DISCUSSION:

Apart from hyperandrogenism and anovulation, insulin resistance also plays a key role in the clinical aspect of PCOS. Insulin resistance, with compensatory hyperinsulinemia, plays a major role in the metabolic abnormalities associated with PCOS. The hyperinsulinemia in PCOS women results in increased production of androgen in ovaries²⁰. Gymnema sylvestre is one of the popular medications for hyperglycemia in the Indian traditional system of medicine and is also listed in the Indian pharmaceutical codex as a remedy for diabetes^21,22. Based on the insulin modulating mechanism of G. sylvestre we conducted this study to evaluate its effect on PCOS. Acute toxicity study with ethanolic leaf extract of G. sylvestre showed no evidence of toxicity or mortality and found to be safer up to the dose level of 2000mg/kg.

Letrozole-induced PCOS is one of the most reliable models which mimic the human PCOS clinical conditions such as hormonal imbalance, hyperglycemic conditions, and metabolic disturbances²³. In our study also PCOS was induced in rats with 21 days of treatment of letrozole (1mg/kg b. w.). The induction of PCOS was confirmed by measuring through vaginal smear, which demonstrated that irregularity of estrus cycle, such as increased diestrus duration and decreases in estrus duration. Estrus irregularity of letrozole is due to the altered steroidal hormone imbalance, circulatory hyperandrogenism, and excess intrauterine androgen-induced uterine changes²⁴. The treatments of metformin and G. sylvestre decrease the diestrus duration and estrus cycle irregularity. Besides, both doses of G. sylvestre showed the same effect. It indicates that G. sylvestre can normalize menstrual irregularity.

Hyperglycemia and impaired glucose tolerance is an important indicator on PCOS. The blood glucose level was significantly increased in PCOS rats. The hyperglycemia and impaired glucose tolerance in PCOS are mainly due to the elevated level of androgen²⁵. The standard treatment metformin significantly reduces the blood glucose level, compared with untreated PCOS rats. Remarkably, G. sylvestre treatments also significantly decrease the glucose level in nearer equal to the metformin treatment. Hence, the results of our study suggested that G. sylvestre reduces glucose tolerance in PCOS. It might be mediated by improving insulin secretion, potentiating the insulin-mediated uptake of glucose, and preventing excess glucose absorption in the intestine^26,27.

The polycystic ovarian syndrome is commonly associated with dyslipidemia such as low levels of HDL, high levels of LDL, VLDL, and triglycerides²⁸. The results of our study also showed a significant reduction in HDL cholesterol, but an elevated level of LDL, VLDL, and triglyceride in letrozole induced PCOS control compared to the normal control. The altered lipid profiles were significantly reversed by all the treatment groups. The treatment of G. sylvestre showed a numerically better dose-dependent effect when compared to the metformin standard treatment. It indicates that G. sylvestre leaf extract also has a better impact on the metabolic complication (dyslipidemia) of PCOS.

The altered concentrations of sex hormones and gonadotrophins are considered key indicators of PCOS. Administration of letrozole blocks the conversion of androgen substrates into estrogens by inhibiting aromatase. This leads to abnormally increased testosterone and reduced estradiol levels contribute to the pathogenesis of PCOS^{29, 30}. In this study, letrozole induced rats showed elevated levels of testosterone, luteinizing hormone, but low levels of estradiol, progesterone, and follicle-stimulating hormone. The administration of G. sylvestre significantly up-regulating the estradiol, progesterone, and FSH concentration and down-regulates the testosterone and LH, which could promote follicular development, and induce ovulation. In comparison with treatments metformin treatment also producing remarkable effect but the treatment of G. sylvestre showed numerically better dose-dependent effects.

Ovarian histopathological reports confirmed that letrozole induced ovarian dysfunction, where ovaries of PCOS rats showed many follicular cysts and atretic follicles and absence of corpus luteum. These altered abnormalities were reverted by the treatments metformin and G. sylvestre, evidenced by the presence of different stages of mature follicles with corpus luteum indicating ovulation and regular estrous cycle³¹. When comparing the treatment metformin and G. sylvestre low dose (200mg/kg) treated groups showed the minimum number of follicular cysts, histological structure of ovaries treated with G. sylvestre high dose (400mg/kg) showed almost nearer ovarian histology with null follicular cysts, which proved that the supplementation of G. sylvestre possesses significant effect in the management of PCOS.

Based on our study results, G. sylvestre showed a beneficial effect on PCOS rats. It could be proposed that G. sylvestre act through regularize the menstrual irregularity and promote ovulation by correcting the hormonal irregularity and promoting follicular growth. These beneficial effects of G. sylvestre on PCOS could be mediated through its insulin resistance modulating property by improving glucose utilization. However, more mechanistic studies are needed to further explore this mechanism.

CONCLUSION:

In conclusion administration of Gymnema sylvestre regularizes the estrus cyclicity, reduces the elevated blood glucose level, improves lipid profile and hormonal irregularity, and prevents ovarian damage in PCOS rats after 28 days of treatment. Hence Gymnema sylvestre might be considered as an alternative remedy to treat reproductive and metabolic complications in PCOS women.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are acknowledged to the Chancellor, Vinayaka Mission’s Research Foundation (Deemed to be University), Salem for providing necessary support for writing this article.

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Received on 28.06.2021 Modified on 08.11.2021

Research J. Pharm. and Tech 2023; 16(1):385-390.

DOI: 10.52711/0974-360X.2023.00066

Treatment	Testosterone (ng/dl)	Estradiol (pg/ml)	Progesterone (ng/dl)	LH (ng/ml)	FSH (ng/ml)
Group-I ( 0.5% CMC)	72.20 ± 11.19	68.00 ± 5.79	41.20 ± 6.91	2.22 ± 0.50	81.2 ± 9.01
Group-II (Letrozole)	109.40 ± 11.31^a***	31.60 ± 7.13^a***	14.40 ± 4.51^a***	8.68 ± 0.62^a***	43.80 ± 5.31^a***
Group-III (Letrozole + Metformin)	85.80 ± 10.87^{a* b***}	48.40 ± 5.37^{a* b*}	30.80 ± 3.27^{a* b*}	3.48 ± 0.55^{a b*}	75.20 ± 7.80^b***
Group-IV (Letrozole + EEGS 200mg/kg)	80.30 ± 9.75^b***	58.40 ± 4.53^b***	34.20 ± 2.88^b***	3.06 ± 0.43^b***	71.32 ± 5.23^b***
Group-V (Letrozole + EEGS 400mg/kg)	78.40± 10.24^b***	64.20 ± 6.96^b***	37.60 ± 4.09^b***	2.94 ± 0.38^b***	74.80 ± 6.46^b***