Letrozole tablets were obtained from Sun Pharmaceutical Ltd, Bengaluru, India. All consumables were provided by Hi Media (India) and Merck (India).

Roots of A. racemosus and seeds of Vitex negundo were collected from a local Ayurvedic Pharmaceutical Shop (Midnapore, West Bengal) and ground into powder form. Each set of 100 g powdered herbs was dissolved in 1000 mL distilled water and continuously stirred for 2 days before filtration. The filtrates were concentrated using a rotary evaporator. Collected concentrates were air-dried and stored for the experiment. The total yield of aqueous extracts of A. racemosus and V. negundo were 17.23 g and 13.73 g, respectively. Roots and seeds were used according to previous reports 21, 23.

A. racemosus and V. negundo were administered at a dose of 250 mg/kg BW according to the findings of a previous in vitro screening (data not shown) and published reports 21, 23.

Female albino rats (n=24), weighing 100–120 g and aged 6–8 weeks, were acclimatized for 8 days at 25 °C and 50–70% humidity. Animals were procured from an authorized provider (M/S Chakraborty Enterprise; Regd no. 1443/PO/Bt/s/11/CPCSEA; India) and kept in polycarbonate cages with free access to food and water.

The Animal Ethics Committee of Vidyasagar University approved the experiment with ethical clearance numbers VU/IAEC/CPCSEA/19/7/2022 (A. racemosus) and VU/IAEC/CPCSEA/17/7/2022 (V. negundo). The study complied with CPCSEA (India) norms and EU Directive guidelines (2010/63/EU) for laboratory animal care.

Letrozole (1.0 mg/kg BW) was diluted in 0.5% carboxymethyl cellulose (CMC) and administered orally for 21 consecutive days to induce PCOS 20. Twenty-four Wistar rats were divided into four groups:

• Control Group: 0.5% CMC orally for 21 days;
• PCOS Group: letrozole (1.0 mg/kg BW) orally for 21 days 24, 25;
• ARA Group: letrozole (1.0 mg/kg BW) orally for 21 days + A. racemosus aqueous extract (250 mg/kg) orally for the next 21 days 23;
• VNA Group: letrozole (1.0 mg/kg BW) orally for 21 days + V. negundo aqueous extract (250 mg/kg) orally for the next 21 days 21.

The experiment lasted 42 days. Rats were monitored for body weight, estrous cycle, and discomfort signs. On day 43, rats were sacrificed using Ketamine/HCl (80 mg/kg BW, intraperitoneal) and Xylazine (8 mg/kg BW, intraperitoneal) according to CPCSEA guidelines. Serum and organs were stored at -20 °C for further analysis.

Vaginal cytology was performed throughout the study. 100 μL normal saline was used to collect vaginal fluid using a sterile dropper. The fluid was transferred to glass slides, air-dried, stained with Leishman stain, and observed under a light microscope.

Body weight was monitored regularly. Reproductive organ weights were measured post-sacrifice.

Serum was separated by centrifugation at 2,500 rpm for 6 min. Glucose, triglycerides, HDL-C, and LDL-C were measured using kit assays per manufacturer instructions.

ELISA kits were used to assess serum estradiol, ESR1, and 3βHSD (ovarian tissue-specific) following manufacturer protocols. Readings were taken spectrophotometrically at 450 nm.

Ovarian tissue (50 mg/mL) was homogenized in ice-cold lysis buffer (pH 8.4) and centrifuged at 10,000 g for 15 min at 4 °C. The supernatant (50 μL) was mixed with 10 mM pyrogallol and 50 mM Tris-HCl (pH 8.4) to measure SOD absorbance change at 420 nm. Catalase was measured with the remaining supernatant mixed with H₂O₂, noting absorbance change at 240 nm.

SOD and catalase expression were analyzed using 12% and 8% native gels, respectively. Ovarian tissue (50 mg) was homogenized in chilled PBS (1.0 mol/L, pH 7.4) and centrifuged at 10,000 g for 15 min at 4 °C. Protein extract (50 μg) was applied to native PAGE. The 12% gel was incubated in the dark with 28 mM TEMED, 2.3 mM NBT, and 28 mM riboflavin for SOD visualization 26. The 8% gel was treated with 0.003% H₂O₂ and then exposed to 2% potassium ferricyanide and 2% ferric chloride to generate achromatic catalase bands 26. Densitometry was determined using ImageJ software 1.54, NIH, USA.

Ovaries were fixed in formalin, dehydrated in increasing ethanol concentrations, cleared with xylene, embedded in paraffin, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin and observed under a light microscope.

Data were analyzed using ANOVA followed by post hoc Tukey’s test with SPSS 16.0 software to determine statistical significance between groups.

Letrozole-induced PCOS group showed alteration in estrous cycle followed by continuous diestrus phase. The vehicle-treated control group had typical estrous patterns. ARA and VNA groups showed longer estrus phase and decreased duration of the diestrus phase compared with the PCOS group (Fig. 1A and 1B).

Figure 1.

A significantly higher body weight was observed in PCOS rats compared to the control group (Table 1). ARA and VNA supplementation resulted in significantly lower body weight in PCOS-induced rats (Table 1). No significant change in ovarian weight was observed compared to the control. Uterine weight was significantly lower in PCOS rats compared to the control. Supplementation with ARA and VNA significantly attenuated the uterine weight of PCOS rats (Table 1, Fig. 2).

Table 1.

Groups	Body weight (gm)		Organo-somatic indices (gm%)
	Initial body weight	Final body weight	Ovary in pair	Uterus
CON	110.33±0.98a	135.50±2.69a	0.048±0.008a	0.405±0.030a
PCOS	110.50±0.76a	150.16±2.28b	0.043±0.002a	0.171±0.020b
ARA	111.16±1.07a	141.00±1.29ac	0.031±0.003a	0.318±0.020ac
VNA	112.50±1.60a	146.83±1.22bc	0.035±0.004a	0.277±0.010cd

Data are presented as mean±SE (N=6). Groups with different superscripts (^a,b,c,d) in a column indicate statistically significant differences at p<0.05. Statistical analysis was performed with ANOVA followed by Post hoc Tukey’s test. CON=Control group; PCOS=Polycystic ovary syndrome model group; ARA=Asparagus racemosus aqueous extract group; VNA=Vitex negundo aqueous extract group.

Figure 2.

Significantly higher levels of blood glucose and dyslipidemia were observed in the PCOS group compared to the control group. ARA and VNA post-treatment significantly lowered serum glucose, LDL, and triglycerides (Table 2).

Table 2.

Groups	GLUCOSE	LDL	HDL	TG
CON	128.35±3.53a	25.61±1.44a	20.61±1.08a	56.75±1.71a
PCOS	201.01±7.56b	43.56±2.02b	9.69±1.30b	80.76±2.90b
ARA	171.60±6.96c	30.19±3.34ac	13.49±1.91ab	65.72±2.56ac
VNA	173.00±6.40cd	31.77±3.88acd	12.405±2.67bc	61.70±2.62acd

Data are presented as mean±SE (N=6). Groups with different superscripts (^a,b,c,d) in a column indicate statistically significant differences at p<0.05. Statistical analysis was performed with ANOVA followed by Post hoc Tukey’s test. CON=Control group; PCOS=Polycystic ovary syndrome model group; ARA=Asparagus racemosus aqueous extract group; VNA=Vitex negundo aqueous extract group.

A significant suppression of estradiol level and weak signaling of ESR1 were observed in the PCOS group compared to the control. Decreased concentration of steroidogenic enzyme 3βHSD was also observed in the PCOS group. Post-treatment with ARA and VNA significantly upregulated estradiol and ESR1, whereas a non-significant increase in 3βHSD was noted following treatment (Fig. 3).

Figure 3.

Pyrogallol autoxidation and H₂O₂ test revealed significantly higher SOD and catalase activity in PCOS compared to the other groups (Fig. 4A). The ARA and VNA groups had non-significantly different antioxidant enzyme activity compared to the control (Fig. 4A). Native gel expression showed higher band density of these antioxidant enzymes in the PCOS group and lower density in ARA and VNA groups (Fig. 4B).

Figure 4.

Light-microscopic images (10x and 40x) of ovary sections of the letrozole-treated group (Fig. 5, Table 3) displayed multiple cystic follicles characterized by a diminished granulosa cell layer and enhanced follicular antrum. The ARA and VNA groups had a lower number of observable cystic follicles compared with PCOS rats (Fig. 5, Table 3).

Table 3.

Groups	Primary follicles	Secondary follicles	Tertiary follicles	Corpus luteum	Cystic follicles
CON	20.33±0.53a	12.16±0.24a	6.33±0.20a	10.83±0.64a	0
PCOS	7.16±0.24b	1.16±0.19b	0.83±0.12b	1.50±0.17b	6.83±0.24b
ARA	13.50±0.37c	3.83±0.12c	2.33±0.08c	4.50±0.17c	3.33±0.13c
VNA	15.33±0.40cd	4.66±0.17cd	1.83±0.12bc	5.83±0.19d	2.83±0.12cd

Data are presented as mean±SE (N=6). Groups with different superscripts (^{a, b, c, d}) in a column indicate statistically significant differences at p<0.05. Statistical analysis was performed with ANOVA followed by Post hoc Tukey’s test.

CON = Control group; PCOS = Polycystic ovary syndrome model group; ARA = Asparagus racemosus aqueous extract group; VNA = Vitex negundo aqueous extract group.

Figure 5.