Beneficial effect of polyherbal formulation in letrozole induced Polycystic ovarian syndrome (PCOS)

Abstract

Aim of the study

PCOS is an endocrine condition that results in enlarged ovaries with tiny cysts on the margins. The present study aims to investigate the beneficial effect of polyherbal formulation in Letrozole induced PCOS in female Albino Wistar rats.

Materials and methods

Acute toxicity study of the polyherbal formulation by administering a single dose (2000 mg/kg) was done in female Albino Wistar rats (20 weeks, 250 g) following OECD guideline 423. For PCOS induced study female Albino Wistar rats were divided into six groups (6 animals/group). Group I (control) was given 0.5% carboxymethylcellulose (CMC) suspension daily as vehicle control. Letrozole (1 mg/kg) was orally administered for 21 days in Group II to VI for induction of PCOS. After induction of PCOS, animals were treated with standard drug (Group III- Clomiphene citrate- 1 mg/kg) and polyherbal tablets (Group IV – 500 mg/kg, Group V- 750 mg/kg, and Group VI – 1000 mg/kg) up to 50 days. Vaginal smears were taken daily to check the estrous cycle. Body weight was measured weekly. Blood samples were withdrawn on 0,21 and 50 days for the determination of fasting blood glucose, lipid profile, LH, FSH, and hormonal levels.

Results

Administration of letrozole caused the abnormality in serum sex hormone profile, lipid profile, glucose, and the estrous cycle. The treatment with polyherbal formulation significantly decreased (P < 0.0001) the level of testosterone and improved estradiol and progesterone levels (P < 0.0001). There was a decrease in elevated glucose levels from 71.51 ± 0.15 mg/dl in disease induced group to 57.33 ± 1.90 mg/dl in treatment groups. The triglycerides level was normalized to 33.41 ± 1.81 mg/dl and HDL level was increased to 40.63 ± 1.35 mg/dl in treatment groups. The polyherbal formulation by exerting its beneficial effect also caused the disappearance of the cysts in the ovaries.

Conclusion

The polyherbal formulation was found to be effective in PCOS. The effect may be attributed to the individual herbs reported having a significant effect on the pathophysiology of letrozole induced PCOS.

Graphical abstract

Highlights

• •Studied logical combination of herbs as per traditional and modern concepts proved with therapeutic efficacy for PCOS.
• •Preclinical study of the polyherbal formulation reveals the restoration of distorted biochemical and hormonal parameters.
• •Treatment with polyherbal formulation regulated the estrous cycle and normalized histopathophysiological changes in the ovaries.
• •Polyherbal formulation can be considered to be a good alternative for prevention and therapeutic cure of PCOS.

List of abbreviations

PCOS

Polycystic ovarian syndrome

OECD

Organization for Economic Cooperation and Development

CMC

Carboxymethyl cellulose

COCP

Combined oral contraceptive pills

FSH

Follicle stimulating hormone

LH

Luteinizing hormone

IAEC

Institutional Animal Ethical Committee

CPCSEA

Committee for Purpose of Control and Supervision of Experiments on Animals

HDL:

High density lipoprotein

LDL:

Low density lipoprotein

ANOVA

Analysis of variance

NC

Normal control

DC

Disease control

SC

Standard control

TG1-500

Treatment group −1 (500 mg/kg)

TG2-750

Treatment group −2 (750 mg/kg)

TG3-1000

Treatment group −1 (1000 mg/kg)

1. Introduction

Polycystic ovarian syndrome (PCOS) is a hormonal disorder with several symptoms related to an imbalance of hormones, androgen excess, ovarian dysfunction, and polycystic ovarian morphology which affects women and girls in their reproductive period.¹ The typical clinical features of PCOS include hirsutism, irregular menstruation, chronic anovulation, and infertility. Chronic hyperandrogenism is associated with impaired hypothalamic-pituitary feedback, luteinizing hormone (LH) hypersecretion, premature granulosa cell luteinization, aberrant oocyte maturation, and premature arrest of activated primary follicles.² Women with PCOS also show the signs of insulin resistance and hyperinsulinemia and are at higher risk for early onset of type 2 diabetes and metabolic syndrome. Unrecognized or untreated PCOS is a risk factor for cardiovascular disease.³

The diagnosis and treatment of PCOS are controversial with challenges in defining individual components with the diagnostic criteria, significant clinical heterogenicity generating a range of phenotypes with or without obesity, ethnic differences, and variation in clinical features across the life course.⁴ Currently, the treatment of PCOS involves multicomponent lifestyle interventions like diet and exercise. Pharmacological treatment for PCOS includes combined oral contraceptive pills (COCP) and metformin. The COCP is recommended for the treatment of irregular menstruation and hyperandrogenism whereas metformin alone or in combination with COCP is recommended to manage weight and metabolic comorbidities.⁵

Aromatase is a key enzyme widely expressed in human tissues such as the placenta, ovary, and testis. It converts testosterone and androstenedione into estradiol and estrone respectively. In the development of PCOS, there is decreased aromatase activity in the ovary which is one of the pathophysiologic hypotheses.⁶

Chemical-based medications induce a women ovulatory cycle instead of allowing it to be restored to its natural healthy rhythm. The treatment of infertility without any side effects and allowing the body to cure naturally can be done by using natural, non-invasive, and non-chemical remedies. The herbal therapies are found to restore the normal rhythm of menstruation by balancing hormones and curing anovulation.⁷

According to Ayurveda, there is no specific condition to be recognized as PCOS. But various conditions related to PCOS is described in utero vaginal disorders (Yoni Vyapat) in Ayurveda. PCOS is known to have the involvement in bodily humor (Dosha), fundamental elements of the body (Dhatu), and subsidiary tissues (Upadhatu). Improper diet and food (Vishama ahara and vihara) cause reduced digestion and metabolism and an increase in fatty tissues (meda dhatu), phlegm (kapha), aggravates vitality and movement (vata prakopa), and production of undigested food (ama) which leads to incomplete metabolism and hormonal imbalance. This leads to amenorrhea (Anartava), oligomenorrhea (Ajaska), anovulation (Abeejata), and anovulation menstruation (Pushpaghni Revati).^8,9

PCOS is a complex disease occurring due to various etiological factors, a single drug cannot be used for its treatment. So, the concept of polyherbalism and synergism which is peculiar in Ayurveda is applied.^10,11 The selected herbs for the preparation of polyherbal tablets are Bauhinia variegata L. (BV), Trigonella foenum graceum L. (TFG), Berberis aristata DC (BA), Curcuma longa L. (CL), Commiphora myrrha (Nees) Engl. (CM) and Saraca asoka (Roxb.) Willd. (SA). Asokaristha is the classical ayurvedic formulation described in Ayurvedic text (Bhaisajyaratnavali, strirogadhikara) and used in menorrhagia (Asrighara) and excessive bleeding (Raktapitta).^12,13 Kanchnara guggulu – Sarangadharasamhita, Madhyamakhanda adhaya is used in the treatment of cystic swelling (granthi).^14,15 TGF (Shanbalileh) and CM (Baijahundana) are used in traditional Persian medicine for metabolic dysfunction in PCOS.¹⁶ CL and TFG also find their place as the traditional Chinese medicine used in women with PCOS.¹⁷ BA containing berberine is used traditionally as an Ayurvedic regime and in Chinese medicine for the treatment of ovarian cyst.^{17, 18, 19} Thus, taking into consideration the above traditional aspects, the drugs were selected for incorporation into the formulation. The polyherbal formulation is a new combination based on the traditional uses of the plants in the pathogenesis of the disease as per Ayurvedic concepts. The amount of drugs used in the formulation was decided based on the dravyaguna of the drug.¹²

TFG is an insulin sensitizer that is used to prevent diabetes and is also used to treat PCOS.^{20, 21, 22} SA is used to manage menorrhagia and other female reproductive abnormalities, which are also indications of PCOS.^{23, 24, 25, 26, 27} BV corrects the pathophysiology of PCOS by diminishing cysts and preventing them from becoming larger in the ovaries.^14,28 CM helps to regulate normal hormone levels and decreases morphological abnormalities in ovarian follicles.^{29, 30, 31} BA containing berberine as an active ingredient that treats PCOS women with clinical, metabolic, and reproductive issues.^{32, 33, 34} CL containing curcumin has been shown to help people with PCOS by rectifying abnormalities in their serum steroid, lipid, glucose, and glycosylated hemoglobin levels, as well as a decrease in antioxidant activity. It has anti-inflammatory properties in the granulosa layer of the corpus luteum.^{35, 36, 37}

2. Materials and methods

2.1. Preparation and evaluation of polyherbal tablet

The alcoholic extract of TFG (31 mg), CL (62 mg), and BA (31 mg) whereas hydroalcoholic (50:50) extract of SA (62 mg) and BV (62 mg) and CM (250 mg) purified in cow's urine was used for the preparation of polyherbal tablet (500 mg). Pre-formulation study of the powder blend and post-compression evaluation of tablet was done by various parameters like weight variation, friability, hardness, thickness, diameter, disintegration time, in-vitro dissolution, and accelerated stability study.

2.2. Acute toxicity study

Acute toxicity study of the polyherbal formulation was performed following OECD guidelines 423.

2.2.1. Experimental animals

The animals for the acute toxicity study were approved by the Institutional Animal Ethics Committee (IAEC) of Parul Institute of Pharmacy & Research, approval no. 984/2019-09. Female Albino Wistar rats (6 females) of age 20 weeks and weight 200–250 g were used for the study.

2.2.2. Experimental design

The rats were acclimatized to their surroundings 5 days before the experiment by separating them from the rest of the animals and allotting different cages to them. The rats were identified by color marking and provided with rat feed and water ad libitum. Before dosing, the rats were weighed. The polyherbal tablet (500 mg) was crushed and suspended in CMC (0.5%) solution. The single-dose for the administration of polyherbal formulation (1.5 ml/animal) (2000 mg/kg) was administered. Following administration of a single dose of a polyherbal tablet, animals were observed for the clinical symptoms for 30 min, at the hourly intervals for the next 24 h and thereafter for total 14 days. The animals were observed for signs of convulsions, tremors, circling, depression, excitement, and mortality.

After 14th day, the animals were euthanized, and blood and all the vital organs like the heart, liver, kidney, ovaries, lungs, and brain were isolated, cleared from fat and stored in formalin solution. All the organs were fixed in paraffin wax and a histopathological study was carried out for the organs.³⁸

2.3. Letrozole induced PCOS study

2.3.1. Experimental animals

Adult female Albino Wistar rats (200–250 g) were employed for the study. Animals were allowed to acclimatize for two weeks. Throughout the study all animals were caged in polypropylene cages and maintained in a controlled environment of (22 ± 3 °C) temperature, (55 ± 5%) humidity and a 12 h light/dark cycle. Animals were fed with a standard diet and water ad libitum. The study was approved by the Institutional Animal Ethical Committee (IAEC) for the use of animals and care of the animals was carried out as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) with protocol no. 984/2019-09.

2.3.2. Drugs and reagents

Letrozole was purchased from Triveni Interchem Pvt. Ltd, Vapi, Gujarat, India. Clomiphene citrate was obtained as a gift sample from Shimoga chemicals, Maharashtra. All the chemicals were of analytical grade.

2.3.3. PCOS induction

All the experimental animals except the control group were orally administered with letrozole at a dose of 1 mg/kg dissolved in 0.5% CMC daily for a period of 21 days. Control group received vehicle only (0.5% CMC). Vaginal smears were collected daily and evaluated microscopically using crystal violet stain to confirm the induction of PCOS. The disease was confirmed by the irregularity of estrous cycle.³⁹

2.3.4. Study design

The study consisted of 36 female Albino Wistar rats equally divided into six groups designated as Group I (served as a control group), Group II (served as PCOS induced group), Group III (served as a standard group) and Group IV, V and VI (served as treatment groups).

Following letrozole administration, the standard group was administered with clomiphene citrate (active ingredient) at a dose of 1 mg/kg in 0.5% CMC. Treatment groups IV, V and VI were administered with polyherbal formulation with the dose of 500 mg/kg (low dose), 750 mg/kg (medium dose) and 1000 mg/kg (high dose) body weight respectively in 0.5% CMC for 28 days.

As per the study model, letrozole was given for 21 days. After 21 days, drug treatment was started to observe the changes in the estrous phase. In the evaluation of PCOS, regularization of the cycle is one of the important parameters for accessing the efficacy of the formulation. In humans, for regularization of the menstrual cycle treatment is required for 3–4 months i.e., 3–4 cycles. In rats, one cycle is of five days so the study was designed to cover four cycles and hence treatment was given for 28 days.⁴⁰

The body weight of all animals was measured at the beginning and weekly intervals throughout the study. Vaginal smears were collected daily to confirm the phase of the estrous cycle. Serum glucose, total cholesterol, HDL, LDL, triglycerides, LH and FSH were measured on days 0, 21 and 50. On the last day of the experiment, hormonal levels were measured and the ovary was collected for histopathology study.

2.3.5. Parameters evaluated

• a)Physical parameters: The body weight of all animals was recorded at the beginning and weekly intervals throughout the study.
• b)Vaginal smear test: A vaginal swab technique was used for the collection of vaginal smears. The vaginal smears were obtained using cotton-tipped swab wetted with ambient temperature physiological saline and introduced into the vagina of the rat. The swab was gently turned and rolled against the vaginal wall and then removed. Cells collected were then transferred to a dry glass slide by moving a swab across the slide. The slide was air-dried and stained accordingly with 0.1% crystal violet and viewed under a light microscope using 10X and 45X lenses.⁴¹
• c)Biochemical Parameters: Serum glucose, triglycerides, total cholesterol, HDL, Triglycerides, Serum estradiol, progesterone, testosterone and FSH:LH ratio were evaluated.⁴²
• d)Histopathology of the ovary: On the 50th day of the study, both ovaries were collected from each animal. It was removed, cleaned up and weighed. The ovaries were fixed in 10% formalin solution and embedded in paraffin blocks. Tissue sections were cut and stained and subjected to histopathological evaluation. The slides were observed in the microscope. The change in ovary like corpus luteum, atretic follicles and cystic follicles were evaluated^43,44
• e)Statistical analysis: All the values are expressed as the mean ± standard deviation. The results were statistically analyzed by two-way analysis of variance (ANOVA) followed by the Bonferroni comparison test using graph pad prism software 9.1.2.

3. Results

3.1. Preparation and evaluation of polyherbal tablet

The polyherbal tablet was evaluated using post-compression parameters. The optimized polyherbal tablet exhibited a hardness of 1.5 ± 0.06 kg/m³, disintegration time of 30 ± 1 min, and friability of 0.58 ± 0.02%. In vitro dissolution study showed that there was 90% drug release at the end of 2 h. The accelerated stability study revealed that the content of markers in the tablet does not deviate from more than 10% of the initial content indicating the stability of the tablet.⁴⁵

3.2. Acute toxicity study

In an acute toxicity study after oral administration of a single dose of polyherbal formulation (2000 mg/kg) the rats were observed for 30 min, at the hourly intervals for the next 24 h and thereafter for 14 days twice daily (morning and evening). The body weight was recorded daily for 14 days. No mortality and clinical signs of ataxia, convulsion, lacrimation, nasal/oral discharge and polyuria were observed. No remarkable changes or differences were observed in body weight. All the animals had normal behavior throughout the study. The blood, biochemical and hormone parameters were evaluated in animals (Table 1). The histopathology study of the vital organs like the heart, liver, kidney, brain, lungs and ovaries did not show any significant pathological changes (Fig. 1).

Table 1.

Acute toxicity study: Blood, biochemical and hormone parameters.

Parameters	Control Group	Test Group (2000 mg/kg)
Body weight (gm)	249 ± 1	252.66 ± 1.52
Haemoglobin (gm%)	14.3 ± 0.1	13.39 ± 0.15
Total RBC (millions/ml)	7.14 ± 0.01	7.11 ± 0.01
Total WBC (/cmm)	3670 ± 78.58	3779.66 ± 75.43
Platelet (/cmm)	813573.3 ± 825.91	814,592 ± 1450.65
Glucose (mg/dl)	156 ± 1	156.33 ± 2.08
Total cholesterol (mg/dl)	122 ± 1	124.33 ± 2.08
HDL (mg/dl)	55.14 ± 0.60	54.82 ± 0.71
Triglycerides (mg/dl)	159.33 ± 5.50	166 ± 17.57
Testosterone (ng/ml)	32.8 ± 0.50	33.25 ± 0.37
Progesterone (ng/ml)	29.50 ± 0.69	29.35 ± 0.49
Estradiol (ng/ml)	45.39 ± 0.89	44.81 ± 0.72
LH: FSH	0.24 ± 0.01	0.24 ± 0.01

∗Values are represented as Mean ± SD, n = 6.

Fig. 1.

Acute toxicity study (Histopathophysiology of vital organs).

(Nu-Neurons; Mf-Myofibre; MR-Medullary rays; RC-Renal corpuscle; Bc-Bronchiole; AS-Alveolar sac; Hc-Hepatocytes; CV-Central vein; CL-Corpus luteum; AF- Atretic follicles).

3.3. Letrozole induced PCOS study

3.3.1. Body weight

During the administration of letrozole for 21 days there was a significant increase in the body weight of all groups as compared to the normal control group. After the treatment with the standard drug and polyherbal formulation, remarkable decrease in weight was observed. Treatment groups with polyherbal formulation at a dose of 500, 750 and 1000 mg/kg showed a significant decrease (P < 0.0001) in body weight as compared to the disease control group on Day 50 (Fig. 2).

Fig. 2.

Effect of polyherbal formulation on body weight in letrozole induced PCOS rat. (NC: Normal control, DC: Disease control, SC: Standard control, TG1-500: Polyherbal formulation 500 mg/kg, TG2-750: Polyherbal formulation 750 mg/kg, TG3-1000: Polyherbal formulation 1000 mg/kg).

Values are expressed as Mean ± SEM (n = 6).

∗Indicates P < 0.0001 vs NC on day 21.

#Indicates P < 0.0001 vs DC on day 50.

As evaluated by two-way ANOVA followed by the Bonferroni comparison test.

3.3.2. Vaginal smear test

A vaginal smear test showed the proestrus, estrous, metestrus, and diestrus phases of the reproductive cycle of the rats. After 21 days of administration of letrozole, the reproductive cycle becomes irregular. During that time the control group had a regular estrous cycle. The irregularity in the estrous cycle indicates the induction of the PCOS in rats. From 21 days to 50 days, the disease control group showed an irregular estrous cycle and exhibited a constant diestrus phase. The treatment group with polyherbal formulation and the standard control group showed improvement in the irregularity of the estrous cycle and a decrease in the length of the diestrus phase as compared to the disease group (Fig. 3).

Fig. 3.

Estrous cycle phase in rats.

(A: Estrous phase shows a large number of anucleated keratinized epithelial cells, few small and large nucleated epithelial cells; B: Diestrous phase shows a greater number of neutrophils; C: Metestrus phase shows a large number of neutrophils and anucleated keratinized epithelial cells; D: Proestrus phase shows small nucleated epithelial cells.).

3.3.3. Biochemical Parameters

Glucose: On Day 0 there was no significant difference in blood glucose between all the groups. On day 21 after administration of letrozole, there was a significant increase (P < 0.0001) in blood glucose observed in all the groups as compared to the normal control group. After treatment with polyherbal formulation, significant decrease (P < 0.0001) in blood glucose level was observed as compared to the disease control group. The decrease in the blood glucose level indicates a positive effect on hyperglycemia which is the major pathophysiological condition in PCOS (Table 2).

Table 2.

Effect of polyherbal formulation on biochemical parameters in letrozole induced PCOS rat.

	NC	DC	SC	TG1-500	TG2-750	TG3-1000
Glucose (mg/dl)
0 day	61.19 ± 0.42	60.39 ± 0.23	61.06 ± 0.69	61.73 ± 1.16	62.06 ± 2.54	60.73 ± 0.56
21 days	61.43 ± 0.26	70.60 ± 7.54a, ∗∗∗∗	71.43 ± 0.26	71.69 ± 6.90	71.43 ± 0.26	71.43 ± 0.26
50 days	61.54 ± 0.35	73.93 ± 2.82a, ∗∗∗∗	54.78 ± 9.91b, ∗∗∗∗	58.65 ± 3.73c, ∗∗∗∗	58.21 ± 4.34d, ∗∗∗∗	55.15 ± 8.67e, ∗∗∗∗
Total Cholesterol
0 day	37.82 ± 2.43	38.84 ± 1.79	37.48 ± 1.29	38.42 ± 0.62	38.75 ± 1.09	39.16 ± 0.82
21 days	37.58 ± 1.35	38.75 ± 1.86	40.11 ± 2.20	40.80 ± 1.75	39.75 ± 0.51	40.47 ± 1.19
50 days	38.75 ± 1.86	39.44 ± 1.73	38.51 ± 1.62	37.56 ± 1.26	38.75 ± 0.48	38.18 ± 1.32
Triglycerides
0 day	26.37 ± 045	24.56 ± 0.86	27.39 ± 0.90	25.39 ± 0.74	27.09 ± 1.27	26.61 ± 0.23
21 days	25.95 ± 0.30	38.02 ± 0.40a, ns	36.21 ± 0.28	35.69 ± 1.02	35.41 ± 0.87	35.48 ± 0.98
50 days	26.26 ± 0.49	38.54 ± 0.61a, ∗∗	33.60 ± 0.67b, ∗∗∗∗	32.68 ± 0.15c, ∗∗∗∗	32.08 ± 1.32d, ∗∗∗∗	32.31 ± 1.31e, ∗∗∗∗
HDL
0 day	54.49 ± 0.93	54.71 ± 0.12	55.11 ± 1.12	53.04 ± 0.83	55.83 ± 0.83	53.89 ± 0.45
21 days	54.78 ± 1.24	32.57 ± 0.49a, ∗∗∗∗	32.15 ± 0.71	31.13 ± 0.67	30.92 ± 0.47	31.24 ± 0.80
50 days	54.44 ± 0.94	30.91 ± 0.48a, ∗∗∗∗	45.05 ± 0.57b, ns	39.50 ± 1.05c, ns	40.26 ± 0.40d, ∗∗∗	42.13 ± 0.64e, ∗

Values are expressed as Mean ± SEM (n = 6).

∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns-non significant.

As evaluated by two-way ANOVA followed by Bonferroni comparison. (NC: Normal control, DC: Disease control, SC: Standard control, TG1-500: Polyherbal formulation 500 mg/kg, TG2-750: Polyherbal formulation 750 mg/kg, TG3-1000: Polyherbal formulation 1000 mg/kg).

^aNC vs DC.

^bDC vs SC.

^cDC vs TG1-500.

^dDC vs TG2-750 and.

^eDC vs TG3-1000.

3.3.4. Lipid profile

The lipid profile was evaluated by measuring total cholesterol, triglycerides and HDL on days 0, 21 and 50. There was no significant difference found in the levels of total cholesterol while triglycerides levels were elevated and HDL was decreased in the animals after administration of letrozole on day 21 as compared to the normal control group. Post-treatment with polyherbal formulation and standard drug from 21 to 50 days normalized the elevated levels of triglycerides and HDL (Table 2).

3.3.5. Serum hormonal assay

Serum testosterone was significantly increased whereas progesterone and estradiol were decreased markedly in the animals after administration of the letrozole for 21 days as compared to the normal control group. Treatment with standard drug and polyherbal formulation cause a significant decrease in testosterone and the level of estradiol and progesterone was improved as compared to the disease control group (Table 3).

Table 3.

Effect of polyherbal formulation on hormones in letrozole induced PCOS rat.

	NC	DC	SC	TG1-500	TG2-750	TG3-1000
Estradiol
0 day	46.15 ± 0.33	45.02 ± 0.40	45.54 ± 0.73	45.47 ± 0.90	45.48 ± 0.86	43.42 ± 0.90
21 days	44.72 ± 0.83	25.07 ± 0.62a, ∗∗∗∗	24.50 ± 0.10	24.36 ± 0.82	24.85 ± 0.36	24.43 ± 0.82
50 days	44.90 ± 0.47	20.78 ± 0.42a, ∗∗∗∗	40.03 ± 0.47b, ∗∗∗∗	37.18 ± 1.36c, ∗∗∗∗	40.71 ± 0.41d, ∗∗∗∗	40.66 ± 0.42e, ∗∗∗∗
Progesterone
0 day	30.96 ± 0.49	31.25 ± 0.80	31.24 ± 0.80	31.25 ± 0.82	31.16 ± 0.84	30.85 ± 0.43
21 days	31.91 ± 0.48	20.42 ± 0.71a, ∗∗∗∗	21.56 ± 1.24	21.32 ± 0.82	21.31 ± 0.81	21.27 ± 0.50
50 days	31.59 ± 0.51	20.69 ± 0.47a, ∗∗∗∗	30.23 ± 0.00b, ∗∗∗∗	29.75 ± 1.19c, ∗∗∗∗	29.54 ± 0.13d, ∗∗∗∗	30.37 ± 0.13e, ∗∗∗∗
Testosterone
0 day	33.24 ± 2.77	30.71 ± 0.41	31.96 ± 0.50	30.84 ± 0.42	31.25 ± 0.83	31.27 ± 0.87
21 days	33.26 ± 1.41	45.45 ± 0.13a, ∗∗∗∗	45.28 ± 0.87	43.95 ± 0.51	43.06 ± 1.87	44.67 ± 0.42
50 days	33.07 ± 2.22	44.52 ± 0.93a, ∗∗∗∗	35.29 ± 1.59b, ∗∗∗∗	34.12 ± 0.63c, ∗∗∗∗	32.23 ± 0.80d, ∗∗∗∗	29.96 ± 0.48e, ∗∗∗∗

Values are expressed as Mean ± SEM (n = 6).

∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns-non significant.

As evaluated by two-way ANOVA followed by Bonferroni comparison. (NC: Normal control, DC: Disease control, SC: Standard control, TG1-500: Polyherbal formulation 500 mg/kg, TG2-750: Polyherbal formulation 750 mg/kg, TG3-1000: Polyherbal formulation 1000 mg/kg).

^aNC vs DC.

^bDC vs SC.

^cDC vs TG1-500.

^dDC vs TG2-750 and.

^eDC vs TG3-1000.

3.3.6. Ovary weight

There were a significant increase in ovary weight in letrozole induced PCOS rats (82.67 ± 2.05) as compared to the normal group (41 ± 0.82). After treatment with polyherbal formulation and standard drug, this condition was normalized with SC (54 ± 0.82), TG1-500 (55 ± 0.82), TG2-750 (51 ± 0.82) and TG3-1000 (51 ± 0.82) (Fig. 4).

Fig. 4.

Effect of polyherbal formulation on ovary weight in letrozole induced PCOS rats. (NC: Normal control, DC: Disease control, SC: Standard control, TG1-500: Polyherbal formulation 500 mg/kg, TG2-750: Polyherbal formulation 750 mg/kg, TG3-1000: Polyherbal formulation 1000 mg/kg).

Values are expressed as Mean ± SEM (n = 6).

∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns-non significant.

As evaluated by two-way ANOVA followed by Bonferroni comparison. (NC: Normal control, DC: Disease control, SC: Standard control, TG1-500: Polyherbal formulation 500 mg/kg, TG2-750: Polyherbal formulation 750 mg/kg, TG3-1000: Polyherbal formulation 1000 mg/kg).

^a NC vs DC, ^b DC vs SC, ^c DC vs TG1-500, ^d DC vs TG2-750 and ^e DC vs TG3-1000.

As evaluated by two-way ANOVA.

3.3.7. Ovarian morphology

Letrozole administered group showed many small and multiple ovarian follicles and cysts with a smaller number of corpus luteum. No histological abnormalities were observed in the control group. The treatment group showed normal follicular development as compared to the disease control group, which showed a decrease in the number of cyst formation (Fig. 6).

Fig. 6.

Effect of polyherbal formulation on ovarian morphology.

(NC: Normal control shows the cortex with primary follicles with aggregation of granulosa cells showing intact oocyte and corpus luteum showing intact cells; DC: Disease control shows follicular cysts with degrading granulosa cells in the thin layer of corpus luteum; SC: Standard control, TG1-500: Polyherbal formulation 500 mg/kg, TG2-750: Polyherbal formulation 750 mg/kg, TG3-1000: Polyherbal formulation 1000 mg/kg shows normal development of primary follicles and aggregation of the granulosa cells and corpus luteum was found which was distorted in the disease control group. It also showed the development of oocytes in the corpus luteum).

(GC-Granulosa cells; CL-Corpus luteum; Oo-Oocyte).

4. Discussion

In the present study, letrozole was used for the induction of PCOS. Letrozole is a non-steroidal aromatase inhibitor. Oral administration of letrozole (1 mg/kg once daily for 21–28 days) in prepubertal or post-pubertal female rats can induce PCOS-like features. Letrozole increases free testosterone, FSH, and LH while decreasing progesterone and estrogen hormones in rats. There is also an increase in insulin resistance and weight gain observed in rats induced with letrozole.⁴⁶ Due to the increased levels of androgens, there is follicular atresia and abnormal follicular development in the ovary.⁴²

In PCOS, hormone activity becomes irregular as ovulation is not occurring expectedly. Therefore, the body gives mixed signals and the menstrual cycle gets disturbed. It can change from irregular, infrequent periods (oligomenorrhea) or heavy to absent periods (amenorrhea). In our study, letrozole produces estrous irregularity due to an imbalance of hormones, circulating hyperandrogenism and excess intraovarian androgen. Administration of letrozole for 21 days caused the diestrus phase to continue for a longer period time in the disease control group and other groups. The treatment group and standard group after administration of polyherbal formulation and clomiphene citrate respectively showed improvement by regularizing estrous cycle and decreasing the length of the diestrus phase as compared to the disease control group. Moreover, the polyherbal formulation contains SA which is used to manage menorrhagia as it possesses estrogenicity activity.²⁶ Further methanolic extract of SA reduces the thickening of the endometrium proliferation in the uterus by lowering the levels of lipopolysaccharides induced COX-2 enzymes in the rat uterus. It possesses antiproliferative and anti-keratinizing effects in the uterus through its anti-estrogenic and anti-inflammatory properties.²³

Insulin resistance accompanied by compensating hyperinsulinemia is an important biochemical feature of PCOS which puts women at increased risk for diabetes. The results from our study showed that the glucose level was elevated in the rats after administration of letrozole for 21 days. We found that the treatment with polyherbal formulation significantly decreased (P < 0.0001) the glucose level in rats as compared to the disease control group. The activity of the polyherbal formulation may be attributed to the presence of TFG in the polyherbal formulation which is an insulin sensitizer and known to prevents diabetes.^22,44 Moreover, TFG seed extract showed significant results in 94% of patients regulating the menstrual cycle. It also significantly reduces ovary volume and size of cyst.⁴⁷

The ovarian and adrenal glands of women with PCOS are usually the sites of the production of elevated androgens. It is also proposed that women with PCOS have a hyperproduction of CYP17 enzymes, which are found to be responsible for forming androgens in the ovaries and adrenals. Ovaries make several androgens of which testosterone is the most prominent, others include androstenedione and dehydroepiandrosterone (DHEAS). The most typical feature of a polycystic ovary is the stroma and theca cells make excessive testosterone.⁴⁸ CM which is one of the major ingredients of the polyherbal formulation helps to regulate the normal hormonal levels as well as decreases morphological abnormalities in the ovarian follicles (Fig. 5).^22,30,43 The mechanism of action of CM can be explained by antihyperglycemic, insulinogenic activity due to the steroidal lipids guggulsterone-E and guggulsterone-Z which possess antioxidant properties due to the presence of hydroxyl group at α-position of double bonds similar to antioxidant vitamins.²⁹

Fig. 5.

Effect of polyherbal formulation on LH: FSH ratio in letrozole induced PCOS rats. (NC: Normal control, DC: Disease control, SC: Standard control, TG1-500: Polyherbal formulation 500 mg/kg, TG2-750: Polyherbal formulation 750 mg/kg, TG3-1000: Polyherbal formulation 1000 mg/kg).

Values are expressed as Mean ± SEM (n = 6).

∗Indicates P < 0.0001 vs NC on day 21.

#Indicates P < 0.0001 vs DC on day 50.

PCOS is found to be associated with various patterns of dyslipidemia including higher levels of triglycerides, total cholesterol and low-density lipoprotein (LDL), and low levels of high-density lipoproteins (HDL). In our study, there was not any significant difference found in the total cholesterol level in disease control and treatment groups. The triglyceride levels were significantly increased (P < 0.05) in disease induced group as compared to the normal control group after administration of letrozole for 21 days. Post-treatment with polyherbal formulation at all the dose levels significantly decreased (P < 0.0001) triglycerides levels in treatment group as compared to the disease control group. Administration of letrozole significantly decreased (P < 0.0001) the level of HDL in disease induced group as compared to the normal control group. The treatment with polyherbal formulation significantly increased (P < 0.001) the levels of HDL in the treatment group at 750 mg/kg and 1000 mg/kg dose levels.

Polycystic ovaries are six-fold larger than normal ovaries in size. There is a large number of immature follicles which causes the change in the shape of the ovary. The ovary becomes whitish and there are multiple cystic follicles covered by a dense fibrous capsule. There is luteinization of the theca cells and thickening of tunica albuginea (connective tissue covering the ovaries). In our study, the treatment group showed a decrease in the ovary weight as compared to the disease control group. BV present in the polyherbal formulation corrects the pathophysiology of PCOS by diminishing cysts and preventing them from becoming larger in the ovaries.^14,43

PCOS is treated by BA with berberine as an active component by addressing clinical, metabolic, and reproductive concerns.^32,34,49 Curcumin an active ingredient of CL rectifies the abnormalities in the serum lipid, glucose and glycosylated hemoglobin levels. It also possesses anti-inflammatory activity on the granulosa layer of the corpus luteum.^{35, 36, 37} From the results, it can be identified that the polyherbal formulation in all the doses i.e., 500, 750 and 1000 mg/kg does not show any significant changes in the various parameters associated with the PCOS. Thus, it can be stated that the polyherbal formulation at the dose of 500 mg/kg can be effective in the treatment and prevention of PCOS.

5. Conclusion

Polyherbal formulation demonstrated a beneficial effect similar to Clomiphene citrate in treating PCOS conditions and inducing ovulation. It restored the hormone and lipid profile, glucose levels as well as ovarian morphology in letrozole induced PCOS animals. This activity may be attributed to the multiple pharmacological activities like estrogenic, antihyperlipidemic, hypoglycemic and antioxidant activity of various phytoconstituents present in the polyherbal formulation which could be useful in the effective management of PCOS and thereby preventing ovarian cell dysfunction and improving fertility. Together broad spectrum of the biological effects of polyherbal formulation makes it a promising alternative for treating clinical and pathological abnormalities in PCOS conditions.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.