Effect of herbo-mineral formulation (Shilajatu Rasayana) in letrozole-induced polycystic ovarian syndrome

Vanitha Hosur Kumari; Revanasiddappa S Sarashetti; Kashinath S Hadimur; Kasturi A Patil

doi:10.4103/2468-838X.196093

ORIGINAL ARTICLE

Year : 2016 | Volume : 1 | Issue : 2 | Page : 108-112

Effect of herbo-mineral formulation (Shilajatu Rasayana) in letrozole-induced polycystic ovarian syndrome

Vanitha Hosur Kumari, Revanasiddappa S Sarashetti, Kashinath S Hadimur, Kasturi A Patil
Department of Rasashastra and Bhaishajya Kalpana, BLDEA's AVS PGCRC Ayurveda Mahavidyalaya, Bijapur, Karnataka, India

Date of Submission	18-Aug-2016
Date of Acceptance	10-Sep-2016
Date of Web Publication	19-Dec-2016

Correspondence Address:
Dr. Vanitha Hosur Kumari
Department of Rasashastra and Bhaishajya Kalpana, BLDEA's AVS PGCRC Ayurveda Mahavidyalaya, Bijapur, Karnataka
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/2468-838X.196093

Abstract

The major cause of female infertility in recent years is polycystic ovarian syndrome commonly called as PCOS, compared to yonivyapath (disorders of female reproductive system) described in Ayurveda. Shilajatu Rasayana herbo-mineral preparation mentioned in "Rasendra Chudamani" indicated in yonyaamaya (disorders of female reproductive system), gulma (tumors), meha (hyperinsulinemia), pandu (anemia), etc. is expected to contribute positive and better result in letrozole-induced PCOS, considering the previous analytical and experimental studies. Letrozole, Shilajatu Rasayana, clomiphene citrate, ghee, and female albino rats formed the materials for the study. Totally thirty female rats were initially induced to develop PCOS by injecting letrozole. The induction of PCOS in rats was checked through vaginal smear analysis and further confirmed by hormonal assay. PCOS-induced rats were treated with clomiphene citrate (standard), Shilajatu Rasayana (test drug), and ghee (control). Ovarian and uterine weight, hormonal assay, and histomorphometric changes were observed and recorded. Shilajatu Rasayana has shown a significant result in letrozole-induced PCOS by regulating hormones, reduction of cystic follicles, maturation of ovarian follicles, and decreasing the increased ovarian and uterine weight.

Keywords: Clomiphene citrate, ghee, letrozole, polycystic ovarian syndrome, Shilajatu Rasayana

How to cite this article:
Kumari VH, Sarashetti RS, Hadimur KS, Patil KA. Effect of herbo-mineral formulation (Shilajatu Rasayana) in letrozole-induced polycystic ovarian syndrome. BLDE Univ J Health Sci 2016;1:108-12

How to cite this URL:
Kumari VH, Sarashetti RS, Hadimur KS, Patil KA. Effect of herbo-mineral formulation (Shilajatu Rasayana) in letrozole-induced polycystic ovarian syndrome. BLDE Univ J Health Sci [serial online] 2016 [cited 2023 Mar 24];1:108-12. Available from: https://www.bldeujournalhs.in/text.asp?2016/1/2/108/196093

Infertility is the common global problem, failure to conceive over 1-year period of continuous exposure to normal unprotected coitus regularly, during the appropriate period of menstrual cycle.^[1] Female infertility accounts to 35%-40% of the overall infertility.^[2] Polycystic ovarian syndrome (PCOS) is one of the most common female endocrine disorders affecting approximately 5%-10% of women of reproductive age, and it is thought to be one of the leading causes of female subfertility.^[3],[4] It is characterized by hyperandrogenism, oligo-anovulation, obesity, and polycystic ovaries. It is not only recognized as an endocrine disorder but also as the one highly associated with metabolic risk factors such as insulin resistance often associated with type 2 diabetes and high cholesterol level.^[5] In Ayurveda, many conditions such as yonivyapath, arthavadushti, rasapradoshaja vyadhi, santharpanottha vyadhi, kaphameda vikara, or granthi\gulma have clinical features, which simulate PCOS.^[6]

It is established that conventional therapy has an untoward effect on immuno-metabolic functions, which interfere with the total health of an individual, with few adverse reactions, namely, headache, gastrointestinal effects (nausea, diarrhea, and flatulence) at initiation of therapy, weight loss, abdominal discomfort, constipation, vaginal bleeding or irritation, and psychological side effects such as mood swings, depression, and bloating.^[4] Ayurveda has a wide range of herbs and minerals to strengthen ovarian functions. The goal is to reduce circulating androgens, optimize ovarian functions, and support optimal endocrine function. "Shilajatu Rasayana" mentioned in "Rasendra Chudamani" indicated in yonyaamaya, gulma, meha, pandu, etc.^[7],[8],[9] Research study conducted at BLDEA'S AVS PGCRC, Bijapur,^{[10],[11],[12]} shown its efficacy as antioxidant, immunomodulatory, hypoglycemic, and hypolipidemic, and Shilajatu Rasayana is expected to contribute positive and better result in PCOS. Hence, this study was undertaken to assess its effect on ovarian volume, reduction of cysts, hormones, and induction of ovulation in letrozole-induced PCOS.

Materials and Methods

Materials

Drugs

Letrozole (1 mg/kg in 1 ml carboxymethylcellulose [CMC]) to induce PCOS (oral)
Clomiphene citrate (oral) (standard drug)
Shilajatu Rasayana (oral) (test drug)
Ghee (oral) (control drug).

Animals

Female albino rats; thirty female albino rats were taken, ten in each group and maintained under standard laboratory conditions.

Methods

Materials required and method of preparation of Shilajatu Rasayana^[7]

Shilajatu Rasayana contains shilajatu bhasma,^[13] loha bhasma, vaikranta bhasma, triphala choorna, and trikatu choorna^[14] which were prepared as per the reference in Rasendra Chudamani.^[7]

Method of selection of animals

Inclusion criteria

Healthy female albino rats, aged 6 weeks (42 days) and weight between 150 and 200 g were included in the study.

Exclusion criteria

Unhealthy female and male albino rats, aged below 6 weeks and weight below 150 g and above 200 g were excluded from the study.

Method of preparation of drug and administration

Group I, Group II, and Group III: 1 mg/kg of letrozole was taken with 1 ml of CMC and fed to thirty female albino rats orally to induce PCOS.

Group I: 25 μg of clomiphene citrate/200 g body weight was taken with 1 ml of CMC and fed to female albino rats.

Group II: 25.31 mg of Shilajatu Rasayana/200 g body weight was taken with 1 ml of ghritha (ghee) and fed to female albino rats.

Group III: 1 ml of ghrita/200 g body weight was taken and fed to female albino rats.

Sample size

The sample size included thirty female albino rats, ten in each group for PCOS induction. After inducing PCOS, five female albino rats were included in each group for drug (standard, test, and control) administration.

Method of experimental study^[4]

Determination of ovarian cycle

Vaginal smears were collected daily using normal saline and evaluated microscopically for estrus cycle determination. Only those animals displaying two consecutive normal 4-5-day cycles were selected for the study.

Induction of polycystic ovaries

As a first step, PCOS was induced by the administration of letrozole suspension at the concentration of 1 mg/kg dissolved in 1 ml CMC once daily for 28 days.

After 28 days, after 24 h of last dose of letrozole and 18 h of fasting period, half of the animals, i.e., 5 from each group were sacrificed. Ovaries and uterus were excised, weighed, and sent for histomorphometry and blood was sent for hormonal assay
Then, the remaining animals, i.e., 5 in each group were subjected for the drug administration, i.e., standard drug (clomiphene citrate), test drug (Shilajatu Rasayana), and control drug (ghrita) for a period of 40 days (8 estrus cycle). Later they were sacrificed, ovaries and uterus were weighed, and sent for histomorphometry and blood was sent for hormonal assay.

Tissue sampling

After 24 h of the last dose of letrozole and after 40 days of drug administration (standard drug - clomiphene citrate, test drug - Shilajatu Rasayana, and control drug - ghritha) and after 18 h of fasting period, animals were sacrificed, ovaries and uterus were excised, cleaned of fat, and weighed. Ovaries were sent for histomorphometry.

Ovarian histomorphology

The radius of the largest cystic follicle after PCOS induction was measured. The radius of the largest ovarian follicle and granulosa thickness after 40 days of drug administration in letrozole-induced PCOS were measured. The ovarian follicles and corpus luteum at different stages of development were observed and analyzed at Department of Pathology, BLDEAU's Shri. B. M. Patil Medical College.

Blood sampling

After sacrificing the rats, blood was collected into different Eppendorf tubes. The blood samples with 11% trisodium citrate dehydrate anticoagulant were centrifuged at 3000 rpm for 15 min, and the plasma was separated for hormonal assay. The plasma was kept in a freezer at −20°C. Plasma testosterone, progesterone, and estradiol (estrogen) were assayed by EIA Kit.

Observations and Results

[Figure 1] shows cystic follicles in letrozole-induced PCOS before treatment (BT) in rat ovaries and [Figure 2] shows ovarian follicles and corpus leuteum in letrozole-induced PCOS after treatment (AT) in rat ovaries.

Figure 1: Rat ovaries showing cystic follicles in letrozole-induced polycystic ovarian syndrome efore treatment. (a) Group I (standard). (b) Group II (test). (c) Group III (control)

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Figure 2: Rat ovaries showing ovarian follicles and corpus leuteum in letrozole-induced polycystic ovarian syndrome fter treatment. (a) Group I (standard). (b) Group II (test). (c) Group III (control)

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Weight changes in ovary and uterus due to drug administration (shown in [Table 1])

Table 1: The effect of drugs (standard, test, and control) on the weight of ovaries and uterus in letrozole-induced polycystic ovarian syndrome, mean±standard deviation (n=5)

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Weight of ovaries which was increased after PCOS induction in all the three groups was reduced after treatment. The increase in weight is shown as follows: in clomiphene citrate-treated group (standard group - Group I) from 0.618 ± 0.133 g to 0.116 ± 0.018 g, in Shilajatu Rasayana-treated group (test group - Group II) from 0.372 ± 0.118 g to 0.189 ± 0.025 g, and in ghritha-treated group (control group - Group III) from 0.304 ± 0.037 g to 0.182 ± 0.024 g. Similarly, the weight of uterus which was increased after PCOS induction was reduced after treatment in standard group (Group I) and test group (Group II), i.e., from 0.687 ± 0.165 g to 0.199 ± 0.053 g and from 0.497 ± 0.278 g to 0.269 ± 0.030 g, respectively. However, it increased in control group (Group III) from 0.292 ± 0.072 g to 0.303 ± 0.099 g.

Changes in the hormonal assay due to drug administration (shown in [Table 2])

Table 2: The effect of drugs (standard, test, and control) on the hormonal assay in letrozole - induced polycystic ovarian syndrome, mean±standard deviation (n=5)

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In standard group (Group I), the testosterone level decreased after treatment from 915 ± 511.8 pg/ml to 48.25 ± 0.975 pg/ml, estrogen level increased from 16.15 ± 3.19 pg/ml to 18.53 ± 1.62 p/ml, and the progesterone decreased from 3.57 ± 1.17 ng/ml to 2.01 ± 0.36 ng/ml. In test group (Group II), the testosterone level decreased from 995 ± 927.5 pg/ml to 207.5 ± 157.8 pg/ml, estrogen level increased from 16.96 ± 4.63 pg/ml to 27.58 ± 9.38 pg/ml, and the progesterone level increased from 2.185 ± 0.50 ng/ml to 4.31 ± 1.133 ng/ml. In control group (Group III), the testosterone level decreased from 1020 ± 690.74 pg/ml to 325.5 ± 112.40 pg/ml, estrogen level increased from 14.19 ± 2.54 pg/ml to 22.395 ± 3.76 pg/ml, and the progesterone level increased from 2.56 ± 1.35 ng/ml to 3.185 ± 0.451 ng/ml.

Histomorphometric changes due to drug administration (shown in [Table 3])

Table 3: The effect of drugs (standard, test, and control) on the histomorphometry in letrozole - induced polycystic ovarian syndrome, mean±standard deviation (n=5)

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After PCOS induction, cystic follicles were seen in all the three groups. In standard group (Group I), the radius of the largest cystic follicle was 189.931 ± 48.569 μm, in test group (Group II), it was 255.261 ± 52.523 μm, and in control group (Group III), it was 213.405 ± 31.683 μm. After 40 days of drug administration, the cystic follicles were not seen in standard group (Group I) and test group (Group II), but in control group (Group III), the radius of the largest cystic follicle was 178.652 ± 120.030 μm.

Ovarian follicles which were not seen after PCOS induction were present after 40 days of drug administration. The radius of the largest ovarian follicle was measured; it was 167.470 ± 11.135 μm in standard group (Group I), 185.440 ± 56.640 μm in test group (Group II), and 104.681 ± 85.110 μm in control group (Group III). Similarly, granulosa of ovarian follicle was seen after 40 days of drug administration; its thickness was measured, it was 60.430 ± 18.896 μm in standard group (Group I), 84.289 ± 22.778 μm in test group (Group II), and 55.751 ± 54.194 μm in control group (Group III).

Significant results in weight changes, hormonal assay, and histomorphometry were observed in test group than standard and control groups.

Discussion

The test drug Shilajatu Rasayana (Group II) has shown a significant effect on all the parameters in letrozole-induced PCOS. The main line of treatment in PCOS is hormonal replacement therapy, since PCOS has a direct link with obesity and hyperinsulinemia, it is also treated with metformin-like drugs, diet mainly of nutritional supplements, and exercise to reduce weight. Obesity causes hormonal imbalance, which further leads to increased testosterone resulting in PCOS and the increased insulin in blood which stimulates androgen secretion by the ovarian stroma causing increased level of free testosterone resulting in PCOS. The main activity required in treating PCOS is antioxidant, hypolipidemic, hypoglycemic, and gonadotrophic effects.^[16],[17]

Shilajatu Rasayana is a herbo-mineral preparation which is a combination of multimineral drugs such as shilajatu bhasma, loha bhasma,^[15] vaikranta bhasma, triphala choorna, and trikatu choorna having antioxidant, immunomodulatory, hypoglycemic, and hypolipidemic effects, and various elements such as zinc, magnesium, chromium, Vitamins B, A, omega fatty acids, enzymes,^[18] and pytosterols^[19],[20] having hormonal balancing and estrogenic effects, which have shown a significant result in letrozole-induced PCOS by their synergistic activity. It might have acted by regulating gonadotrophins (hormones) by its multiminerals, multivitamins, and phytosterols; decreasing blood insulin and causing decrease in testosterone levels by its hypoglycemic effect leading to maturation of follicles, reduction in cysts, and ovulation; and decreasing serum lipids by its hypolipidemic effect leading once again to hormonal balance. It nourished the affected follicle by its antioxidant and immunomodulatory properties.

Because of multidimensional pharmacological activities (antioxidant, immunomodulatory, hypoglycemic, hypolipidemic, and gonadotrophic effects), Shilajatu Rasayana might have contributed to a significant result in letrozole-induced PCOS.

Conclusion

Shilajatu Rasayana has shown a significant result in letrozole-induced PCOS by regulating hormones, reduction of cystic follicles, maturation of ovarian follicles, and decreasing the increased weight of ovaries and uterus.

Scope

Shilajatu Rasayana has shown a significant effect in letrozole-induced PCOS by experimental studies. These scientific data will give further scope for clinical research.

Acknowledgment

I am highly grateful to the authors of all those books, articles, and dissertations which have helped in the project referred. My sincere gratitude to my respected and esteemed guide, Dr. Revanasiddappa S. Sarashetti M.D. (Ayu), PhD. Professor and HOD, the teachers, and other staff of the Department of Rasashastra and Bhaishajya Kalpana. I also express my sincere thanks to Dr. Mahesh Karigoudar, Pathologist, Dr. Surekha Hippargi, Pathologist, Department of Pathology, Shri. B. M. Patil Medical College, Vijayapura, and Dr. N. M. Kalyani, Principal, BLDEA's College of Pharmacy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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