Journal of Traditional and Complementary Medicine 12 (2022) 575e583

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Journal of Traditional and Complementary Medicine

journal homepage: http://www.elsevier.com/locate/jtcme

Beneficial effect of polyherbal formulation in letrozole induced

Polycystic ovarian syndrome (PCOS)
Disha P. Prajapati a, *, Madhavi Patel b, Abhay Dharamsi b
a
Parul Institute of Pharmacy & Research, Parul University, Vadodara, Gujarat, India
b
Parul Institute of Pharmacy, Parul University, Vadodara, Gujarat, India

a r t i c l e i n f o a b s t r a c t

Article history: Aim of the study: PCOS is an endocrine condition that results in enlarged ovaries with tiny cysts on the
Received 13 December 2021 margins. The present study aims to investigate the beneficial effect of polyherbal formulation in Letrozole
Received in revised form induced PCOS in female Albino Wistar rats.
1 July 2022
Materials and methods: Acute toxicity study of the polyherbal formulation by administering a single dose
Accepted 6 August 2022
Available online 10 August 2022
(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
Keywords:
Polycystic ovarian syndrome
(1 mg/kg) was orally administered for 21 days in Group II to VI for induction of PCOS. After induction of
Letrozole PCOS, animals were treated with standard drug (Group III- Clomiphene citrate- 1 mg/kg) and polyherbal
Polyherbal formulation tablets (Group IV e 500 mg/kg, Group V- 750 mg/kg, and Group VI e 1000 mg/kg) up to 50 days. Vaginal
Clomiphene citrate smears were taken daily to check the estrous cycle. Body weight was measured weekly. Blood samples
Toxicity were withdrawn on 0,21 and 50 days for the determination of fasting blood glucose, lipid profile, LH, FSH,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.
© 2022 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier
Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

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
* Corresponding author.
E-mail addresses: disha.prajapati@paruluniversity.ac.in (D.P. Prajapati),
madhavi.patel@paruluniversity.ac.in (M. Patel), abhay.dharamsi@paruluniversity.
ac.in (A. Dharamsi).
Peer review under responsibility of The Center for Food and Biomolecules,
National Taiwan University.
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 hyper-
androgenism is associated with impaired hypothalamic-pituitary
feedback, luteinizing hormone (LH) hypersecretion, premature
granulosa cell luteinization, aberrant oocyte maturation, and pre-
mature 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

https://doi.org/10.1016/j.jtcme.2022.08.003
2225-4110/© 2022 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
D.P. Prajapati, M. Patel and A. Dharamsi
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)
challenges in defining individual components with the diagnostic
criteria, significant clinical heterogenicity generating a range of
phenotypes with or without obesity, ethnic differences, and vari-
ation 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 Ay-
urveda is applied.10,11 The selected herbs for the preparation of
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Journal of Traditional and Complementary Medicine 12 (2022) 575e583
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 (Rak-
tapitta).12,13 Kanchnara guggulu e Sarangadharasamhita, Madhya-
makhanda 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 medi-
cine 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.17e19 Thus, taking into consideration
the above traditional aspects, the drugs were selected for incor-
poration 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 dra-
vyaguna of the drug.12
TFG is an insulin sensitizer that is used to prevent diabetes and is
also used to treat PCOS.20e22 SA is used to manage menorrhagia and
other female reproductive abnormalities, which are also in-
dications of PCOS.23e27 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 de-
creases morphological abnormalities in ovarian follicles.29e31 BA
containing berberine as an active ingredient that treats PCOS
women with clinical, metabolic, and reproductive issues.32e34 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.35e37
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, hard-
ness, thickness, diameter, disintegration time, in-vitro dissolution,
and accelerated stability study.
2.2. Acute toxicity study
Acute toxicity study of the polyherbal formulation was per-
formed 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 200e250 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
D.P. Prajapati, M. Patel and A. Dharamsi
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 (200e250 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. Ani-
mals 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 3e4 months i.e., 3e4 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
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Journal of Traditional and Complementary Medicine 12 (2022) 575e583
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 accord-
ingly with 0.1% crystal violet and viewed under a light mi-
croscope using 10X and 45X lenses.41
c) Biochemical Parameters: Serum glucose, triglycerides, total
cholesterol, HDL, Triglycerides, Serum estradiol, progester-
one, 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 his-
topathological evaluation. The slides were observed in the
microscope. The change in ovary like corpus luteum, atretic
follicles and cystic follicles were evaluated43,44
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 pa-
rameters were evaluated in animals (Table 1). The histopathology
D.P. Prajapati, M. Patel and A. Dharamsi Journal of Traditional and Complementary Medicine 12 (2022) 575e583

study of the vital organs like the heart, liver, kidney, brain, lungs
and ovaries did not show any significant pathological changes
(Fig. 1).
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).
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).
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 pro-
gesterone 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 poly-
herbal 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).
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).
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).

4. Discussion
3.3.3. Biochemical Parameters
Glucose: On Day 0 there was no significant difference in blood In the present study, letrozole was used for the induction of
glucose between all the groups. On day 21 after administration of PCOS. Letrozole is a non-steroidal aromatase inhibitor. Oral
letrozole, there was a significant increase (P < 0.0001) in blood administration of letrozole (1 mg/kg once daily for 21e28 days) in
glucose observed in all the groups as compared to the normal prepubertal or post-pubertal female rats can induce PCOS-like
control group. After treatment with polyherbal formulation, sig- features. Letrozole increases free testosterone, FSH, and LH while
nificant decrease (P < 0.0001) in blood glucose level was observed decreasing progesterone and estrogen hormones in rats. There is
as compared to the disease control group. The decrease in the blood also an increase in insulin resistance and weight gain observed in
glucose level indicates a positive effect on hyperglycemia which is rats induced with letrozole.46 Due to the increased levels of an-
the major pathophysiological condition in PCOS (Table 2). drogens, there is follicular atresia and abnormal follicular devel-
opment in the ovary.42
3.3.4. Lipid profile In PCOS, hormone activity becomes irregular as ovulation is not
The lipid profile was evaluated by measuring total cholesterol, occurring expectedly. Therefore, the body gives mixed signals and
triglycerides and HDL on days 0, 21 and 50. There was no significant the menstrual cycle gets disturbed. It can change from irregular,
difference found in the levels of total cholesterol while triglycerides infrequent periods (oligomenorrhea) or heavy to absent periods
levels were elevated and HDL was decreased in the animals after (amenorrhea). In our study, letrozole produces estrous irregularity
administration of letrozole on day 21 as compared to the normal due to an imbalance of hormones, circulating hyperandrogenism
control group. Post-treatment with polyherbal formulation and 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
Table 1
Acute toxicity study: Blood, biochemical and hormone parameters. group and standard group after administration of polyherbal
formulation and clomiphene citrate respectively showed
Parameters Control Group Test Group (2000 mg/kg)
improvement by regularizing estrous cycle and decreasing the
Body weight (gm) 249 ± 1 252.66 ± 1.52 length of the diestrus phase as compared to the disease control
Haemoglobin (gm%) 14.3 ± 0.1 13.39 ± 0.15 group. Moreover, the polyherbal formulation contains SA which is
Total RBC (millions/ml) 7.14 ± 0.01 7.11 ± 0.01
Total WBC (/cmm) 3670 ± 78.58 3779.66 ± 75.43
used to manage menorrhagia as it possesses estrogenicity activ-
Platelet (/cmm) 813573.3 ± 825.91 814,592 ± 1450.65 ity.26 Further methanolic extract of SA reduces the thickening of the
Glucose (mg/dl) 156 ± 1 156.33 ± 2.08 endometrium proliferation in the uterus by lowering the levels of
Total cholesterol (mg/dl) 122 ± 1 124.33 ± 2.08 lipopolysaccharides induced COX-2 enzymes in the rat uterus. It
HDL (mg/dl) 55.14 ± 0.60 54.82 ± 0.71
possesses antiproliferative and anti-keratinizing effects in the
Triglycerides (mg/dl) 159.33 ± 5.50 166 ± 17.57
Testosterone (ng/ml) 32.8 ± 0.50 33.25 ± 0.37 uterus through its anti-estrogenic and anti-inflammatory
Progesterone (ng/ml) 29.50 ± 0.69 29.35 ± 0.49 properties.23
Estradiol (ng/ml) 45.39 ± 0.89 44.81 ± 0.72 Insulin resistance accompanied by compensating hyper-
LH: FSH 0.24 ± 0.01 0.24 ± 0.01 insulinemia is an important biochemical feature of PCOS which
*Values are represented as Mean ± SD, n ¼ 6. puts women at increased risk for diabetes. The results from our
578
D.P. Prajapati, M. Patel and A. Dharamsi
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).
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 ¼ 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.
study showed that the glucose level was elevated in the rats after
administration of letrozole for 21 days. We found that the treat-
ment 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
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Journal of Traditional and Complementary Medicine 12 (2022) 575e583
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 in-
gredients of the polyherbal formulation helps to regulate the
normal hormonal levels as well as decreases morphological ab-
normalities 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 a-position of double bonds similar to
antioxidant vitamins.29
PCOS is found to be associated with various patterns of dysli-
pidemia 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 dif-
ference 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 treat-
ment group as compared to the disease control group. Adminis-
tration 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 poly-
herbal formulation corrects the pathophysiology of PCOS by
diminishing cysts and preventing them from becoming larger in the
ovaries.14,43
D.P. Prajapati, M. Patel and A. Dharamsi Journal of Traditional and Complementary Medicine 12 (2022) 575e583

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.).

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 ¼ 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.

PCOS is treated by BA with berberine as an active component by the serum lipid, glucose and glycosylated hemoglobin levels. It also
addressing clinical, metabolic, and reproductive concerns.32,34,49 possesses anti-inflammatory activity on the granulosa layer of the
Curcumin an active ingredient of CL rectifies the abnormalities in corpus luteum.35e37 From the results, it can be identified that the

580
D.P. Prajapati, M. Patel and A. Dharamsi Journal of Traditional and Complementary Medicine 12 (2022) 575e583

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 ¼ 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.

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 ¼ 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.
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.
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 ¼ 6).
*Indicates P < 0.0001 vs NC on day 21.
#Indicates P < 0.0001 vs DC on day 50.
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 prom-
ising alternative for treating clinical and pathological abnormalities
in PCOS conditions.

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.
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.

581
D.P. Prajapati, M. Patel and A. Dharamsi
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).
Acknowledgments
The authors are thankful to the Student Startup Innovation
Policy, Education Department, Government of Gujarat for providing
grant and Parul University, Vadodara for providing facilities for
carrying out the research work.
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