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RESEARCH ARTICLE
Naringenin improves ovarian health by reducing the serum
androgen and eliminating follicular cysts in letrozole-induced
polycystic ovary syndrome in the Sprague Dawley rats
Rumaisa Rashid
1,2
| Rupal Tripathi
1,3
| Akanksha Singh
1,3
| Sudarsan Sarkar
1,3
|
Ajaykumar Kawale
1
| G. N. Bader
2
| Satish Gupta
1,3
| Rakesh Kumar Gupta
1
|
Rajesh Kumar Jha
1,3
1
Endocrinology Division, CSIR-Central Drug
Research Institute, Lucknow, India
2
Department of Pharmaceutical Sciences,
University of Kashmir, Jammu and Kashmir,
India
3
Academy of Scientific and Innovative
Research (AcSIR), Ghaziabad, India
Correspondence
Rumaisa Rashid and Rajesh Kumar Jha,
Endocrinology Division, Life Science North
101/111B, CSIR-Central Drug Research
Institute, B.S. 10/1, Sector-10, Jankipuram
Extension, Sitapur Road, Lucknow 226,031,
India.
Email: rumaisarashid54@gmail.com;
rajesh_jha@cdri.res.in
Funding information
Council of Scientific and Industrial Research
(CSIR), New Delhi, India, Grant/Award
Number: MLP2026; Department of Health and
Research, Human Resource Development,
New Delhi, India, Grant/Award Number:
12013/25/2020-HR/E; Department of
Biotechnology, New Delhi, India, Grant/Award
Number: BT-PR30749-MED-97-421-2018
Abstract
Polycystic ovary syndrome (PCOS) is most common in women of reproductive age,
giving rise to androgen excess and anovulation, leading to infertility and non-
reproductive complications. We explored the ameliorating effect of naringenin in
PCOS using the Sprague Dawley (SD) rat model and human granulosa cells.
Letrozole-induced PCOS rats were given either naringenin (50 mg/kg/day) alone or
in combination with metformin (300 mg/kg/day), followed by the estrous cycle, hor-
monal analysis, and glucose sensitivity test. To evaluate the effect of naringenin on
granulosa cell (hGC) steroidogenesis, we treated cells with naringenin (2.5 μM) alone
or in combination with metformin (1 mM) in the presence of forskolin (10 μM). To
determine the steroidogenesis of CYP-17A1, -19A1, and 3βHSD2, the protein
expression levels were examined. Treatment with naringenin in the PCOS animal
groups increased ovulation potential and decreased cystic follicles and levels of
androgens. The expression levels of CYP-17A1, -19A1, and 3βHSD2, were seen
restored in the ovary of PCOS SD rats' model and in the human ovarian cells in
response to the naringenin. We found an increased expression level of
phosphorylated-AKT in the ovary and hGCs by naringenin. Naringenin improves ovu-
lation and suppress androgens and cystic follicles, involving AKT activation.
KEYWORDS
AKT, CYP, DHEA, human granulosa cells, letrozole, metformin, naringenin, ovary, PCOS, SD
rats, SKOV3, steroidogenesis
1|INTRODUCTION
Polycystic ovary syndrome (PCOS) is a common endocrinological and
metabolic disorder in women of reproductive age, with a global preva-
lence of 6%–26% (Chen et al., 2008; Evanthia Diamanti-Kandarakis
et al., 1999; Knochenhauer et al., 1998; Lauritsen et al., 2014). PCOS
is one of the leading causes of infertility (Ghafarzadeh, 2020; Zehravi
et al., 2021). The prevalence of infertility among PCOS women is
about 75%, which is mainly due to its oligo or anovulation features
(Homburg, 2004). It is characterized by hyperandrogenism (evidence
of excess male hormone or clinically as hirsutism), anovulation
(absence of ovulation) or oligo-ovulation (irregular ovulation) (includ-
ing menstrual disturbances), and polycystic ovarian morphology
(depicted as excess preantral follicles in the ovaries of PCOS).
Abbreviations: 3βHSD2, 3-beta-hydroxysteroid dehydrogenase; AMH, anti-Müllerian
hormone; CYP17A1, cytochrome P450 17A1; CYP19A1, cytochrome P450 19A1; DHEAs,
dehydroepiandrosterone sulfate; hGC, human granulosa cell; PCOS, polycystic ovary
syndrome; StAR, steroidogenic acute regulatory protein.
Received: 8 June 2022 Revised: 3 April 2023 Accepted: 17 April 2023
DOI: 10.1002/ptr.7860
Phytotherapy Research. 2023;1–24. wileyonlinelibrary.com/journal/ptr © 2023 John Wiley & Sons Ltd. 1
Metabolic disorders, mainly insulin resistance (IR) and compensatory
hyperinsulinemia, are evident in the majority of women with PCOS
(Stepto et al., 2013; Vink et al., 2006), notably seen in women who
also have hyperandrogenism (Abinaya et al., 2019; Carmina &
Lobo, 1999; Kanbour & Dobs, 2022).
Due to the complexity of the pathogenesis of PCOS, the treat-
ment of PCOS is individualized depending on the signs and symptoms.
Apart from synthetic drugs used for the management of PCOS, a large
group of natural polyphenolic compounds, namely, quercetin, rutin,
apigenin, resveratrol, proanthocyanidins, and so forth, have shown
promising results in the management of PCOS (Banaszewska
et al., 2016; Darabi et al., 2020; Jahan et al., 2016; Wang et al., 2017).
Naringenin (polyphenol), a naturally occurring flavonoid, is pre-
dominantly found in citrus fruits (Mbaveng et al., 2014; Zobeiri
et al., 2018). It is endowed with broad biological activities. Several
preclinical and clinical studies have highlighted its beneficial role as an
antioxidant, anti-estrogenic, anti-inflammatory, anti-diabetic, anti-
hyperlipidemic, anti-cancer, and anti-Alzheimer (Ghofrani et al., 2015;
Kicinska et al., 2020; Roy et al., 2016; Sandeep & Nandini, 2017;
Zhang et al., 2016). Earlier studies have also shown favorable results
of naringenin in infertility, endometriosis, and pregnancy (Adana
et al., 2018; Lim & Song, 2016; Park et al., 2017). Naringenin
decreases IR, oxidative stress, body weight, and steroidogenic
enzymes, which are reported to be high in PCOS condition (Hong
et al., 2019; Wu, Yang, Hu, et al. 2022).
Metformin, the insulin sensitizing drug, is most commonly used as
a first-line therapy for the treatment of PCOS-related infertility as an
off-label drug. Studies have reported metformin-like effects of narin-
genin by upregulating AMP-activated protein kinase (AMPK) and non-
glycemic effects that mitigate inflammation and cell proliferation
events (Nyane et al., 2017). Furthermore, due to the structural similar-
ity of polyphenols with steroids, naringenin may act on altered ste-
roidogenic enzymes involved in PCOS. Being a bioactive natural
insulin sensitizer, naringenin may have a possible potential for the
management of PCOS. Therefore, we examined the effect of narin-
genin on ovarian health, follicles, corpora lutea, and serum androgen
levels at the preclinical level using the letrozole-induced PCOS model
in Sprague Dawley (SD) rats. Further, we explored the possible molec-
ular signaling associated with naringenin action in the ovary, including
the steroidogenic pathway rate-limiting enzymes CYP-17A1 and
-19A1. Collectively, our aim in the present study is to determine the
therapeutic potential of naringenin for the treatment of PCOS using
the SD rat model (preclinical model).
2|MATERIALS AND METHODS
2.1 |Experimental animal model
Rattus norvegicus, female SD strain (42 days old and 100–129 g), was
housed in polypropylene cages and maintained under standard condi-
tions (21 ± 2C, 50%–60% humidity and 12-h light/12-h dark cycle).
Free access to standard chow and water was available ad libitum. The
local animal ethical committee of Scientific and Industrial Research
(CSIR)-Central Drug Research Institute (CDRI), Lucknow, India,
reviewed the animal experimentation protocol and approved it via
IAEC/2021/48/Renew-0/ SI.No.16 /dated 15/03/2021. Animals
were inbred and received in-house experimental facilities at CSIR-
CDRI. Requirements considered to be relevant in recent guidelines for
the best practices in natural products pharmacological research have
been taken into account (Heinrich et al., 2020; Izzo et al., 2020).
2.2 |Ovarian/estrous cycle assessment
In female SD rats, 42 days old, the ovarian cycle (folliculogenesis) was
determined by confirmation of the estrous cycle by the presence of
various cell types in the vaginal smear, which was examined under the
microscope as described previously (Mettus & Rane, 2003; Ubba
et al., 2017). We examined the estrous cycle to check for changes that
occur during PCOS model induction and drug treatment periods.
2.3 |Letrozole-induced polycystic ovarian
syndrome (PCOS) in the SD rat model
This particular animal model represents the case of the adult human
PCOS stage. We used letrozole for PCOS model induction as it mimics
the human-like PCOS condition, which was also reported earlier
(Kafali et al., 2004; Maliqueo et al., 2013). Twenty-five (42-day-old)
female rats were randomly assigned into five groups of 5–6 rats each:
the control (sham), letrozole (PCOS) (PHR-1540-1G, Sigma-Aldrich
Chemicals Private Limited, Bangalore, India), metformin +letrozole
(M +L), naringenin +letrozole (N +L) (N5893, ≥95%, HPLC grade,
Sigma-Aldrich Chemicals Private Limited), and N +M+L groups. The
dose of naringenin has been used earlier as 20 mg/kg body weight (b.
wt.) for the determination of gut microbiota (Wu, Yang, Han, et al.
2022), steroidogenic enzymes and antioxidants (Hong et al., 2019),
and endometrial hyperplasia in the PCOS ovary (Yang et al., 2022).
Further, in our research based on 4-hydroxyisolucine (4-HIL) PCOS
management, we found 4-HIL MED 50 mg/kg body weight in a similar
PCOS model system (Indian patent reference no. 0229NF2019);
hence, we adhered to this dose for naringenin efficacy analysis. We
chose 50 mg/kg naringenin because it is the best possible pharmaco-
logical dose available based on the rats model. Further, an acute toxic-
ity study shows an LD50 value of naringenin with 0% mortality at
100 mg/kg b.wt. and also, the 100% mortality was found in the dose
of 600 mg/kg b.wt. Selecting 1/10th of the LD50 values of naringenin
as a pharmacological dose, 50 mg/kg naringenin was selected as the
effective and best possible pharmacological dose available for rats
(Mathi & Murugaiyah, 2015).
The PCOS, M +L, and N +L groups were administered a gavage
(p.o.) of 1.0 mg/kg body weight of letrozole solution once daily for
21 consecutive days as therapeutic protocol. The control group was
administered a gavage of corn oil. After 21 days of letrozole treat-
ment, that is, Day 22, the M +L and N +L groups were administered
metformin 300 mg/kg b.wt. oral and naringenin 50 mg/kg b.wt. oral,
along with letrozole for 20 consecutive days. The N +M+L group
2RASHID ET AL.
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received the combination of naringenin (50 mg/kg b.wt.) and metfor-
min (300 mg/kg b.wt.) with letrozole. Changes in the estrous cycle
were observed during the 42 days of the experiment. At the end of
the experiment, ovaries were collected after the animals were sacri-
ficed by cervical dislocation under the anesthesia of xylazine and keta-
mine, followed by excision. The left ovaries were kept in 4%
paraformaldehyde for histological analysis, the right ovaries for immu-
noblotting (IB), and other tissue and serum were stored at 80C until
further use.
2.4 |Ovarian tissue histology
We used hematoxylin (51275, Chemicals Private Limited) and eosin
(318960, Chemicals Private Limited) staining to check the histopathol-
ogy of the ovaries in the developed PCOS model as described earlier
(Ubba et al., 2017). The ovarian tissues were fixed in 4% paraformal-
dehyde (PFA) at 4C and dehydrated using a gradient of isopropanol,
followed by incubation with xylene. Thereafter, the mold of tissue in
paraffin wax was prepared. Ovarian tissue sections of 5 μm were cut
serially using a microtome (Leica Biosystem, Germany) and mounted
on glass slides coated with Vectabond reagent (SP-1800, Vector Labo-
ratories, Inc., California, USA). Ovarian tissue sections were cleared
from paraffin in xylene (overnight incubation) and allowed to rehy-
drate with subsequent changes in an ethanol gradient. Then the ovar-
ian tissue sections were washed gently, stained with hematoxylin,
followed by blue color development in 0.5% ammonium hydroxide,
and counterstained with 0.5% eosin. Dehydration was performed with
incubation in a gradient of alcohol and xylene, which were finally
mounted with DPX mountant. Finally, the tissue sections were imaged
under the light microscope (CKX41 Trinocular with Cooled CCD Cam-
era Model Q Imaging MP5.0-RTV-CLR-10-C from Olympus, Tokyo,
Japan).
2.5 |Protein extraction, SDS-PAGE, Western
blotting, and immunoblotting
Estimation of protein concentration in the extracts of ovarian tissues
and ovarian cell lysates (hGC and SKOV3) was done using the bicinch-
oninic acid (BCA) protein assay kit (23225, ThermoFisher Scientific,
Bangalore, India). Proteins were denatured with Laemmli buffer, fol-
lowed by heating at 95C for 5 min. A total of 20 μg of protein was
loaded into each well and electrophoresed on 10% and 12% sodium
dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for
the CYP17A1, CYP19A1, follicle stimulating hormone receptor
(FSHR), 3βHSD2, pAKT, phosphatase and tensin homolog (PTEN),
AMPK, and steroidogenic acute regulatory protein (StAR) proteins
expression analysis. SDS-PAGE were run on a tetra protean cell verti-
cal electrophoretic system (BioRad Pvt. India Ltd.) in a running buffer
containing 0.10% SDS, 247 mMTris, pH 8.3, 192 mM glycine at
100 V, and then electro-blotted onto PVDF membrane using transfer
buffer containing 20% v/v methanol, 247 mM Tris, pH 8.2, 187 mM
glycine at 50 mA for 12 h (Kumari et al., 2022; Towbin et al., 1979).
The blotted membrane was incubated in a 5% non-fat milk pow-
der blocker for 1 h and washed with PBST buffer. Further, the mem-
brane was incubated with the antibodies against CYP17A1 (DF6398),
CYP19A1 (DF3564), 3βHSD2 (DF6639), StAR (DF6192), and FSHR
(DF7229), which were purchased from Affinity Bioscience, Australia.
pAKT (4051) and PTEN (9188) were purchased from Cell Signaling
Technology, CA, USA, and AMPK (07–181) from Sigma-Aldrich Che-
micals Private Limited . Antibodies were used in a 1:1000 dilution
overnight at 4C. Anti-β-actin was used in 1:40,000 dilutions as gel
loading internal control. After washing with PBST, the membrane was
incubated with diluted HRP-conjugated secondary antibodies
(1:50,000, goat anti-rabbit IgG linked with HRP) (A0545, Sigma-
Aldrich Chemicals Private Limited) for 1 h. Membranes were washed
and subjected to the enhanced chemiluminescence reaction
(WBKLS0500, Merck-Millipore, Bangalore, India), and the image was
developed with the help of the ImageQuant LAS 4000 gel documenta-
tion system (GE Life Sciences, Chicago, Illinois), and the band intensity
was analyzed using Total Lab Quant software (Nonlinear Dynamics,
Newcastle-upon-Tyne, United Kingdom).
2.6 |Human ovarian cells (SKOV3) culture in the
presence of naringenin
Since we wanted to study the steroidogenesis process in the presence
of naringenin; therefore, we used the human ovarian cell line
(SKOV3). The SKOV3 cell lines (HTB-77) were purchased from (ATCC,
USA, by Dr. Dipak Datta, Biochemistry Division, CSIR-CDRI, Lucknow,
India), and we used the cell passage number 9. SKOV3 ovarian cells
were maintained in RIPA-1640 medium (R4130-1 L, Sigma- Aldrich,
Bangalore, India, Chemicals Private Limited) supplemented with 10%
FBS (10437–028, Thermo Fisher Scientific) and 1% penicillin/
streptomycin (15240–062, Sigma- Aldrich, Bangalore, India, Chemicals
Private Limited). MTT assay was done to check the percent survival of
SKOV3 cells with letrozole, metformin, and naringenin for
24 and 48 h.
2.7 |Steroidogenesis induction in the human
granulosa cells (hGC)
A human granulosa cell line (HGL5, Applied Biological Materials,
Canada) was used to correlate with humans (Cell Passage No.15). The
cells were maintained in DMEM high glucose medium supplemented
with 2.5% cosmic calf serum (SH30087.03, HyClone, Logan, USA),
10% FBS, and 1% penicillin–streptomycin having the temperature
37C and 5% CO
2
under humid conditions.
To check the effect of naringenin on steroidogenesis, we first
checked the cytotoxic effects of naringenin, letrozole, and metformin
using MTT assays (24 and 48 h) in hGCs (Figure S1). Letrozole 10 μM,
naringenin 2.5 μM and metformin 1 mM were used for final treat-
ments in hGC cells. Steroidogenesis induction was done using 10 μM
forskolin (FSK) as described previously (Attia et al., 2001). Upon
reaching 70% confluency, cells were starved for 5 h in Opti-MEM
RASHID ET AL.3
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reduced media (31985–070, ThermoFisher Scientific, Bangalore,
India) and treated for 24 h. Both cell lysate and supernatant were col-
lected for steroidogenesis.
2.8 |Organ weight measurements
The weight of the ovary (left and right) and uterus was measured
using a weighing balance (ME204, Mettler Toledo, Mumbai, India).
The weight was measured in grams. The ovary size was calculated
using a centimeter scale.
2.9 |Body composition analysis
The EchoMRI body composition analyzer E26-226-RM (EchoMRI
tLLC, Houston, TX, USA) was used to measure the body composition
of experimental live rodents, namely, whole body fat, lean, free water,
and total water masses. Live rats were weighed and loaded into a long
cylindrical tube one by one, guided toward the bottom of the tube,
and locked into inserts. The tube was loaded horizontally in the Echo-
MRI system, and the total body composition—lean mass, fat mass, free
water, and total water content—was analyzed with the help of the
Echo-MRI analyzer.
2.10 |Biochemical analysis
The oral glucose tolerance test (OGTT) was performed on fasting
experimental rats. Glucose (2.0 g/kg body weight) was administered
orally to 6 h fasted rats. Blood samples were collected with a single
cut at the tip of the tail at different time points 0, 30, 60, 90, and
120 min. Blood glucose levels were measured with a portable glucose
meter (ACCU-CHEK active, Roche Diabetes Care, India) (Brooks
et al., 1986).
2.11 |Blood circulatory steroids analysis
Serum levels of testosterone (TT), dehydroepiandrosterone sulfate
(DHEAs), and Anti-Müllerian hormone (AMH) levels were determined
using a competitive enzyme-linked immunosorbent assay using com-
mercially available ELISA kits (E-EL-0155, Universal ELISA Testoster-
one kit; and E-EL-R0325, Rat DHEA-S ELISA Kit, Elabscience,
Houston, TX, USA) (Universal AMH ELISA kit; ITEU00033, G-Biosci-
ence, MO, USA) and analyzed using ELISA reader (iMark™Microplate
Absorbance Reader, BioRad, USA).
2.12 |Statistical analysis
All the experiments were conducted on 3–5 replicates (both in vitro
and in vivo). The band intensity on the blots of the specific protein
was determined by Totallab Quant software (Newcastle upon Tyne,
England); values were normalized with β-actin values and the mean
deviation and error calculated using MS Office 2016 (Version 2112,
USA). The histograms were plotted using Graph Pad Prism (version
5.01, California Corporation, USA), and one-way analysis of variance
(ANOVA) was performed, followed by Tukey's multiple comparison
test. p< 0.05 was considered statistically significant.
3|RESULTS
3.1 |Naringenin ameliorates PCOS by the
reduction in ovarian follicular cysts in the letrozole-
induced PCOS in the SD rat model
We have checked the therapeutic effect of naringenin on ovarian folli-
culogenesis in a letrozole-induced-PCOS rat (SD) model as described
earlier (Hong et al., 2019), and we have used a therapeutic approach
with naringenin treatment. As per the earlier studies, the dose of nar-
ingenin was used as 50 mg/kg body weight with mild modification
(Wu, Yang, Han, et al. 2022; Yang et al., 2022). First, we recapitulated
the PCOS model in SD rats. To achieve our goals, we mimicked the
human PCOS condition in rodents by administering letrozole (1 mg/kg
body weight p.o.) for 21 days, as described earlier (Kafali et al., 2004).
We confirmed the PCOS condition histologically by employing hema-
toxylin and eosin staining. In the PCOS rats' model (N=5 in each
group), the ovary shows multiple cystic follicles and fewer corpus
luteum, confirming the model's development (Figure 1a,e,f). However,
in healthy ovarian tissue, less number of cystic follicles were observed,
which represented the normal development of follicles.
We performed the ovarian histological analysis, and the ovarian
follicles were categorized based on the basis of diverse cells surround-
ing the oocyte. Primordial follicles are identified by oocytes sur-
rounded by squamous granulosa cells (GCs), and primary and
secondary follicles are recognized by oocytes encased with monolayer
and multilayers of cuboidal GCs, respectively. The antral follicle is
identified by the presence of an antrum or cavity, 8–10 layers of GCs,
and theca cells. Likewise, a Graafian follicle is identified by a fully
grown antrum or cavity with 8–10 layers of GCs and theca cells sur-
rounding the oocyte with cumulus cells. A follicular cyst is recognized
by a fluid-filled antrum surrounded by a thin GCs layer with degener-
ate/atretic oocytes, and multilayers of theca cells. Corpus luteum is
identified by luteinized granulosa cells and invaginated septi contain-
ing luteinized theca cells, and both types of cells present a convoluted
appearance with trabeculae of vessels and fibrous tissues surrounded
by luteinized theca cells (Kauffman et al., 2015; Oktem &
Urman, 2010; Stubbs et al., 2007; Young & McNeilly, 2010).
Ovarian histological analysis revealed a high number of corpus
luteum and a few cystic follicles in a sham group compared to letro-
zole, with significant changes between the two groups (***p< 0.001,
*p< 0.05) (Figure 1a,e,f).
We found an increased number of follicular cysts and less number
of corpus luteum in the letrozole-induced PCOS model, but naringenin
4RASHID ET AL.
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oral route administration of (50 mg/kg) for 2 weeks decreased the fol-
licular cysts and increased primordial and primary follicles in compari-
son to PCOS (letrozole alone) (***p< 0.001) (Figure 1a,b,c). Ovarian
follicular cysts were decreased (**p< 0.01) in the naringenin
treatment in the letrozole-induced PCOS animal group (Figure 1e). In
addition, we also found that the naringenin sequel on pre-ovulatory
follicles and corpus luteal number was better than the metformin (pos-
itive control/standard drug/comparator) treatment (Figure 1a,f).
FIGURE 1 Histological analysis of ovarian follicles in response to naringenin treatment in the letrozole-induced polycystic ovary syndrome
(PCOS) model in the (Sprague Dawley) SD rat. (a) Hematoxylin and eosin staining was performed in the ovarian tissue from the control (sham/
vehicle), PCOS, metformin, and naringenin groups from the letrozole-induced SD rat PCOS model. A treatment (Oral) of PCOS with naringenin
(50 mg/kg body weight) for consecutive 21 days, after the 21 days of letrozole dosing, promotes (b) primordial (c) primary follicles development
(e) reduced ovarian follicular cysts and effects are seen parallel to that of Metformin (Positive control group). (f ) In parallel, the increase in the
corpus luteal number was also seen with naringenin treatment, ***p< 0.001 compared to letrozole. Image magnification is 4(scale bar 500 μm)
and 40(scale bar 50 μm) objectives lens. PrF indicates primordial follicle; PF, primary follicle; SF, secondary follicle; AF indicates antral follicle;
AtF, atretic follicle; CL, corpus luteum; GF, Graafian follicle; PCOS, polycystic ovary syndrome. Each value represents Mean ± SEM. The study was
conducted on five different animals as replicates (N=5). Ns (non-significant) p> 0.05, *p< 0 0.05, **p< 0.01, ***p< 0.001.
RASHID ET AL.5
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Naringenin ameliorated PCOS by increasing the ovulation potential
indicator, corpus luteal numbers (**p< 0.01) (Figure 1f). A non-
significant difference in Graafian follicles was observed between the
letrozole and naringenin groups (ns p> 0.05) (Figure 1d). Each value
represents Mean ± SEM, N=5.
3.2 |In a PCOS rat model, naringenin with
metformin combination does not show an additive
effect on ovarian follicular cyst reduction compared to
naringenin stand-alone treatment
We also examined whether combination therapy of naringenin with
metformin offers a better therapeutic option for PCOS, rather than
naringenin alone as a treatment. The ovarian histological analysis
revealed increased primordial (**p< 0.01) and primary follicles
(*p< 0.05) with naringenin and metformin combinations in PCOS con-
ditions, when compared with letrozole (Figure 2a–c). With comparison
to letrozole-PCOS, naringenin alone has shown a similar significant
effect on preovulatory follicles in PCOS condition (Primordial
*p< 0.05, and Primary*p< 0.05). However, no changes were seen in
Graafian follicles in PCOS conditions both in combination and with
naringenin alone when compared with letrozole (ns p> 0.05)
(Figure 2a,d). Ovarian follicular cysts were decreased in both the com-
bination and naringenin alone treatments in contrast to the letrozole-
induced PCOS group (***p< 0.001) (Figure 2a,e). In addition, combina-
tion treatment also ameliorated PCOS by increasing corpus luteal
numbers (*p< 0.05) (Figure 2a,f). Naringenin alone and in combination
has shown increased corpus luteum number when compared to letro-
zole (**p< 0.01). The combination of naringenin with metformin does
not result in better outcomes. Each value represents Mean
± SEM, N=5.
3.3 |Naringenin decreased the serum androgen
levels in PCOS condition
We also investigated whether naringenin has any effect on serum
androgen levels in PCOS. The DHEAs and TT were at the basal level
in the non-PCOS Sham group of SD rats (Figure 3a,b). In the
letrozole-induced PCOS model, we found an increase in serum andro-
gen, DHEAs, and TT levels compared with the control non-PCOS
group (***p< 0.001, *p< 0.05). The treatment with naringenin
decreased the levels of serum steroids, DHEAS, and TT ( p*** < 0.001,
p* < 0.05), which were otherwise elevated in PCOS conditions
(Figure 3a,b). Moreover, naringenin treatment showed an equally effi-
cacious pattern in reducing serum androgen levels in PCOS when
compared with metformin (ns p> 0.05).
Further, we also studied the effect of naringenin treatment on
the ovarian reserve indicator, AMH, under PCOS conditions and
found that the levels of AMH were unaltered in all groups
(ns p> 0.05) (Figure 3c) of the letrozole-induced PCOS rat model.
Each value represents Mean ± SEM, N=3.
3.4 |The expression level of steroidogenesis
associated proteins in PCOS are modulated with
naringenin treatment
We determined whether naringenin could regulate the expression level
of steroidogenic enzymes in the ovary. Ovarian dysregulation led to an
alteration in the steroidogenic enzymes functions in PCOS. The ste-
roidogenic pathway-associated molecules StAR, CYP17A1, 3βHSD2,
and CYP19A1 play an important role in steroidogenesis in the ovary
and are altered in women with PCOS. For this, we studied the expres-
sion levels of enzymes involved in the steroidogenesis process, namely,
StAR, CYP17A1, 3βHSD2, and CYP19A1 (aromatase), in the ovary
from the letrozole-induced model of PCOS in the SD rat (Figure 3d-g).
During biosynthesis in the ovary, aromatase encoded by the cyto-
chrome P450 family 19 subfamily A member 1 (CYP19A1) gene prod-
uct catalyze the androgens to estrogen (Stocco, 2008). Aromatase is
expressed in the granulosa cells of preovulatory follicles and the cor-
pus luteum. The cholesterol side-chain cleavage cytochrome P-450
(P450scc) and 17 alpha-hydroxylase cytochrome P450 (P450[17 alpha])
are mainly expressed in the ovarian theca cells, and low levels of
CYP17A1 are expressed in granulosa cells and is involved in the cataly-
sis of androgen production (Doody et al., 1990;Lietal.,2023;Xu
et al., 2011). β-hydroxysteroid dehydrogenase (3Beta HSD-2) converts
estrone to estradiol (Emami et al., 2021). Steroidogenic acute regula-
tory protein (StAR) is a transport protein that regulates cholesterol
transfer within the mitochondria and is primarily present in steroid-
producing cells, including theca cells and luteal cells in the ovary. StAR
is involved in the cholesterol into pregnenolone conversion (Lin
et al., 1995). Thus, we studied these notable enzymes involved in ste-
roid biosynthesis in the ovary during naringenin treatment.
Using IB and densitometric analysis, we analyzed the expression
level of StAR to know whether naringenin can regulate the early stage
of steroidogenesis production in the ovary. A significantly low expres-
sion level of StAR was seen in the sham group, and the expression
level of StAR was increased in the PCOS group (*p< 0.05) and found
no change in response to naringenin treatment (ns p> 0.05)
(Figure 3d). The rate-limiting enzyme in steroid synthesis, CYP17A1,
was increased in PCOS (***p< 0.001) and restored to normal levels by
naringenin treatment in the SD rat model (*p< 0.05) (Figure 3e). The
expression level of 3βHSD2 remained unaffected in all the studied
groups (ns p> 0.05) (Figure 3f). We also found that PCOS decreased
the expression level of CYP19A1, which was reversed by naringenin
treatment (*p< 0.05) (Figure 3g). Values are Mean ± SEM, N=3.
3.5 |Naringenin modulated the expression of
steroidogenic proteins StAR, 3βHSD2, CYP19A1 in
human granulosa cells (hGC)
First, using an MTT assay, we checked the cytotoxic effects of narin-
genin, letrozole, and metformin on the hGC for 24 and 48 h, as shown
in Figure S1a–c. The concentrations of letrozole (10 μM), naringenin
(2.5 μM), and metformin (1 mM) were determined by MTT assay using
6RASHID ET AL.
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FIGURE 2 Histological analysis of ovarian follicles in response to naringenin and metformin combination therapy in the letrozole-induced SD
rat PCOS model. (a) Hematoxylin and eosin staining was performed in the ovarian tissue from the sham (vehicle), PCOS, metformin, naringenin
and naringenin +metformin combination groups from the letrozole-induced SD rat PCOS model. Treatment of PCOS with naringenin and
metformin combination (50 and 300 mg/kg body weight) for consecutive 21 days, after the 21 days of letrozole dosing, promotes (b) primordial
(c) primary and (d) Graafian follicles development and (e) reduced ovarian follicular cysts (**p< 0.001), and effects are seen parallel to that of
naringenin alone, but (f) increases in the corpus luteal number was also seen with combination therapy, *p< 0.05 compared to letrozole. Image
magnification is at 4(scale bar 600 μm) and 40(scale bar 15 μm) objectives lens. PF indicates primary follicle; Pr, primordial follicle; SF,
secondary follicle; AF indicates antral follicle; AtF, atretic follicle; CL, corpus luteum; GF, Graafian follicle. Each value represents Mean ± SEM,
N=5. ns (non-significant) p> 0.05, *p< 0 0.05, **p< 0.01, ***p< 0.001.
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FIGURE 3 Legend on next page.
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hGC cells. FSK's dose was selected as described previously (Attia
et al., 2001; Kumar et al., 2008). FSK stimulates granulosa cells to differ-
entiate and also serves to accumulate cAMP, which helps in progester-
one production. We have used letrozole to mimic an in vitro model of
PCOS-related conditions as it suppresses estradiol production in granu-
losa cells as well as breast cancer cells and increases androgen levels in
theca cells (Lu et al., 2012; Bonelli et al., 2010;Ortegaetal.,2013).
We checked the expression levels of the steroidogenic enzymes
StAR, 3βHSD2, and CYP19A1 in FSK-induced steroidogenesis in
human granulosa cells (hGC) in the presence or absence of naringenin
treatment by IB and densitometric analysis (Figure 4).
The expression level of StAR, which was downregulated with FSK
treatment and was increased in response to naringenin, but changes
were non-significant (ns p> 0.05). Further, letrozole treatment also
increased the expression level of StAR in the background of FSK when
compared with the FSK alone treatment; however, the changes were
not significant. The treatment of naringenin with FSK showed the level
of sham but without statistical significance (ns p> 0.05) (Figure 4a).
The basal level of expression of 3βHSD2 was seen without FSK
treatment and was upregulated with FSK treatment, which remained
unaltered with the addition of letrozole in the hGC (Figure 4b). Narin-
genin downregulated the expression of 3βHSD2 to normal (*p< 0.05
compared to FSK) (Figure 4b). Similar, results were also seen with
metformin (*p< 0.05 compared to FSK). No changes in the expression
levels were seen between the study groups with respect to CYP19A1
(ns p> 0.05) (Figure 4c).
Follicle stimulating hormone (FSH) mediates activation of AKT
signaling in granulosa cells of the ovaries, which promotes estrogen
production (Baumgarten et al., 2014). We studied the expression
levels of FSHR with respect to naringenin treatment in FSK-induced
steroidogenesis (Figure 4d). We found increased expression levels of
FSHR in the presence of FSK as to sham (*p< 0.05), which was a little
decreased by letrozole (ns p> 0.05), and naringenin treatment signifi-
cantly decreased the FSK-mediated increased level of FSHR to normal
in the hGC (*p< 0.05) (Figure 4d). Similar findings were seen with
metformin (*p< 0.05). Values are Mean ± SEM, N=3.
3.6 |Combination therapy of naringenin and
metformin modulated the expression level of
steroidogenic proteins in human granulosa cells (hGC)
In order to assess the efficacy of naringenin and metformin combina-
tion therapy over naringenin stand-alone therapy, expression level
analysis of the steroidogenic enzymes StAR, 3βHSD2, CYP19A1, and
FSHR was studied in FSK-induced steroidogenesis in hGC (Figure 5).
The expression level of StAR showed a basal level in the FSK treat-
ment, with mildly increased expression levels in both combination and
stand-alone naringenin therapy, but changes were non-significant
(ns p> 0.05) (Figure 5a).
FSK has shown a stimulatory effect on 3βHSD2 expression com-
pared with sham in the hGC (*p< 0.05) (Figure 5b), which was not
altered by letrozole and letrozole+metformin; however, naringenin
treatment has significantly decreased the expression level compared
with the sham group (*p< 0.05). Combination therapy of naringenin
and metformin showed downregulation of 3βHSD2 levels, which was
high in the FSK group, though the changes were not significant
(ns p> 0.05) (Figure 5b). Naringenin stand-alone therapy proves to be
more effective in reducing high levels of 3βHSD2 than combination
therapy in PCOS (Figure 5b).
The expression level of CYP19A1 was seen at the basal level
in the FSK treatment, and a statistically non-significant decrease in
the presence of letrozole and naringenin stand-alone, and in
combination with metformin with FSK in the background showed
decreased expression of aromatase CYP19A1 without a statistical
significance (Figure 5c). Combination therapy has shown decreased
expression levels of aromatase in comparison to FSK; however,
changes were non-significant (ns p> 0.05).
We also studied the expression levels of FSHR with respect to com-
bination therapy with naringenin and metformin in FSK-induced steroido-
genesis (Figure 5d). High expression levels of FSHR were seen both in
FSK and combination-treated groups (ns p>0.05)(Figure5d). Naringenin
alone treatment showed downregulation of FSHR expression in compari-
son to FSK; however, the changes were not significant (ns p> 0.05). Simi-
lar findings were seen with metformin treatment as well (ns p> 0.05). A
significant increase in the expression level of FSHR in combination ther-
apy was seen in contrast to naringenin alone therapy (*p< 0.05), metfor-
min (*p< 0.05), and letrozole +FSK groups (**p< 0.01).
Naringenin alone therapy has been found to be more effective
than its combination with metformin to promote CYP19 expression
levels. Each value represents Mean ± SEM, N=3.
3.7 |Naringenin regulated the expression of
steroidogenic proteins in human ovarian cells, SKOV3
We first examined the cytotoxic effects of naringenin, letrozole, and
metformin by MTT assay using SKOV3 ovarian cells for 24 and 48 h.
FIGURE 3 Analysis of serum hormonal androgen levels and expression level of steroidogenesis associated proteins; StAR, CYP17A1,
3βHSD2, and CYP19A1 in the ovary in response to naringenin treatment to the letrozole-induced SD rat model. In the letrozole-induced PCOS
SD rats, serum levels of (a) DHEAs (b) Testosterone and (c) Anti-Mullerian hormone (AMH) were determined. The expression level of (d) StAR,
(e) CYP17A1, (f) 3βHSD2 and (g) CYP19A1 was analyzed in response to naringenin treatment in the letrozole-induced SD rat model. DHEAs
stands for dehydroepiandrosterone sulfate; AMH, Anti-Mullerian hormone; CYP19A1, Cytochrome P450 Family 19 Subfamily A Member 1;
CYP17A1, cytochrome P450 family 17 subfamilies A member 1; 3βHSD2, 3-beta (β)-hydroxysteroid dehydrogenase; StAR, steroidogenic acute
regulatory protein; SD, Sprague Dawley. The study was conducted on three different animals as replicates (N=3). The values are represented as
Mean ± SEM. ns (non-significant) p> 0.05, *p< 0.05, ***p< 0.001.
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As shown in Figure S1d,e,f, the percent survival of cells was highest at
10 μM letrozole (an in vitro inducer of PCOS-like traits), 5 μM narin-
genin, and 1 mM metformin concentrations. These concentrations
were used for final treatments in SKOV3 cells.
We assessed the expression levels of the steroidogenic enzymes
StAR, CYP17A1, and CYP19A1 in the ovarian cells, SKOV3, in
response to naringenin treatment by IB and densitometric analysis
(Figure 6). The expression level of StAR was examined and found to
FIGURE 4 Analysis of expression level of StAR, 3βHSD2, FSHR, and CYP19A1 in hGCs in response to steroidogenesis. The expression levels
of (a) StAR, (b) 3βHSD2, (c) CYP19A1 and (d) FSHR was analyzed in the ovary in response to naringenin and letrozole treatment to human
granulosa cells (hGC) in the presence of forskolin. hGC denotes human granulosa cells; CYP19A1, Cytochrome P450 Family 19 Subfamily A
Member 1; 3βHSD2, 3-beta (β)-hydroxysteroid dehydrogenase; StAR, steroidogenic acute regulatory protein; FSHR, Follicle Stimulating Hormone
Receptor. The study was conducted on three different replicates (N=3). The values are represented as Mean ± SEM. ns (non-significant)
p> 0.05, *p< 0.05.
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be significantly increased in the SKOV3 cells in the presence of letro-
zole compared to sham (*p< 0.05). Metformin treatment showed a
reduction in expression levels of StAR, which are high in the letrozole
alone group (*p< 0.05), while naringenin treatment also decreased
the expression level of StAR in SKOV3 cells, but non-significantly
(ns p> 0.05) (Figure 6a).
FIGURE 5 Expression level analysis of StAR, 3βHSD2, FSHR, and CYP19A1 in hGCs in response to steroidogenesis in combination therapy.
The expression level of (a) StAR, (b) 3βHSD2, (c) CYP19A1 and (d) FSHR was analyzed in the hGCs in response to naringenin stand-alone therapy
and its combination with metformin in presence of forskolin. hGC denotes human granulosa cells; CYP19A1, Cytochrome P450 Family
19 Subfamily A Member 1; 3βHSD2, 3-beta (β)-hydroxysteroid dehydrogenase; StAR, steroidogenic acute regulatory protein; FSHR, Follicle
Stimulating Hormone Receptor. The values are represented as Mean ± SEM (N=3). ns (non-significant) p> 0.05, *p< 0.05.
RASHID ET AL.11
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The expression level of CYP17A1 was increased in the letrozole
group that was less in the sham group (**p< 0.01). Treatment with
naringenin significantly restored the expression level of CYP17A1
to the normal level (**p< 0.01) (Figure 6b). Metformin treatment
slightly decreased the CYP17A1 levels without any statistical signif-
icance (ns p> 0.05). Letrozole treatment showed down-regulation
of the expression level of CYP19A1, whereas naringenin and met-
formin upregulated its expression level to the normal level
(**p<0.01; *p<0.05) (Figure 6c). Each value represents Mean
±SEM,N=3.
3.8 |AKT is the target of naringenin action in the
ovary
IR and compensatory hyperinsulinemia affect most of the women with
PCOS (E. Diamanti-Kandarakis & Dunaif, 2012), and it was seen that
the ameliorating effect in PCOS is exerted via the PI3/AKT pathway
(Xie et al., 2021). AKT signaling plays a central role in insulin-
stimulated glucose uptake in both muscle and adipose tissue and
decreases IR. Hence, we also assessed ovarian tissue molecular sen-
sors and pathways involved in insulin signaling in the pathophysiology
FIGURE 6 Expression level analysis of StAR, CYP17A1, and CYP19A1 in SKOV3 ovarian cells. We analyzed the expression level of (a) StAR
(b) CYP17A1 and (c) CYP19A1 in the ovary in response to naringenin and letrozole treatment to SKOV3 cells in presence of forskolin. CYP19A1
indicates Cytochrome P450 Family 19 Subfamily A Member 1; CYP17A1, cytochrome P450 family 17 subfamilies A member 1; StAR,
steroidogenic acute regulatory protein; SKOV3, ovarian serous cystadenocarcinoma. The study was conducted on three replicates (N=3). The
values are represented as Mean ± SEM. ns (non-significant) p> 0.05, *p< 0.05, **p< 0.01.
12 RASHID ET AL.
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of PCOS in response to naringenin. Further, we also evaluated the
potential of naringenin as an insulin sensitizer, as described earlier
(Kannappan & Anuradha, 2010). We found that the treatment with
naringenin significantly up-regulated (restored) the expression level of
pAKT, which is otherwise decreased in PCOS conditions (*p< 0.05
compared to letrozole) (Figure 7a). In a positive (standard drug)
control, metformin treatment also showed increased expression levels
of pAKT compared to letrozole (**p< 0.01).
Phosphatase and tensin homolog deleted on chromosome 10, a
kind of tumor suppressor gene and phosphatase, play a role in granu-
losa cell proliferation and regulate the differentiation process in PCOS
(Iwase et al., 2009; Onal et al., 2023), where PTEN downregulation
FIGURE 7 Analysis of expression level of pAKT, PTEN, and AMPK in the ovary of letrozole-induced SD rat model in response to naringenin
treatment. The expression level of (a) pAKT, (b) PTEN and (B) AMPK were determined in the letrozole-induced SD rat model after the naringenin
treatment. pAKT stands for Phosphorylated protein kinase, strain AK, Thymoma (Phosphorylated protein kinase B); PTEN, Phosphatase and
tensin homolog; AMPK, AMP-activated protein kinase; SD, Sprague Dawley; PCOS, polycystic ovary syndrome. The values are represented as
Mean ± SEM. The study was conducted on three different animals as replicates (N=3). ns (non-significant) p> 0.05, *p< 0.05, **p< 0.01.
RASHID ET AL.13
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prevents PCOS development (Ouyang et al., 2013); however, theca
cell-specific deletion leads to hyperandrogenism and ovary dysfunc-
tion (Lan et al., 2017). The expression levels of PTEN were downregu-
lated in the PCOS SD rat model without statistically significant
changes, and treatment with naringenin could not restore them
(ns p> 0.05) (Figure 7b).
In addition, we also examined AMPK, a molecular sensor involved
in insulin sensitivity. AMP-activated protein kinase (AMPK) is a key
regulator of cellular metabolism and energy balance. It inhibits IR and
promotes glucose metabolism and uptake (Cokorinos et al., 2017).
Further, DHEA negatively affects AMPK signaling in the granulosa
cells (Tao et al., 2017), and the expression level of it was seen lower in
the primary GCs from lean women with PCOS than in the control
group (Froment et al., 2022). The dysregulation of AMPK is correlated
to both IR and PCOS; therefore, we also accessed its activity in
response to naringenin. The expression level of AMPK was decreased
in the ovary during the PCOS condition and was not restored by met-
formin or naringenin treatment (ns p> 0.05) (Figure 7c). The expres-
sion level of AMPK was seen to be downregulated in the letrozole-
induced PCOS SD rats (*p< 0.05 compared with sham), and neither
naringenin nor metformin treatment could restore it to the sham level
(ns p> 0.05) (Figure 7c). Each value represents Mean ± SEM, N=3.
3.9 |Naringenin treatment causes a significant
increase in the expression level of pAKT in human
granulosa cells (hGC)
The molecular sensors and pathways involved in insulin signaling were
also studied with respect to naringenin in human granulosa cells
(Figure 8). The expression level of pAKT was higher in the non-
steroidogenesis control group, where FSK-induced steroidogenesis
and letrozole treatment significantly decreased the expression level of
pAKT (***p< 0.001) (Figure 8a). As soon as we treated hGC with nar-
ingenin, the increased expression level of pAKT was seen with statisti-
cal significance for FSK (*p< 0.05) (Figure 8a). However, metformin
treatment has significantly improved the pAKT levels to normal com-
pared to the FSK group (**p< 0.01).
The expression level of PTEN was high in non-steroidogenesis
and FSK-induced steroidogenesis, which was reduced by letrozole in
the FSK background, and naringenin significantly increased the
expression levels of PTEN (*p< 0.05) (Figure 8b).
We also assessed the expression level of AMPK, and found
letrozole-dependent downregulation and restoration by metformin
and naringenin (Figure 8c), but without the statistical significance
(ns p> 0.05). Values are Mean ± SEM, N=3.
3.10 |Combination therapy of naringenin with
metformin treatment modulated the expression level
of pAKT in human granulosa cells (hGC)
The expression level of pAKT was higher under normal non-
steroidogenic conditions, which was reduced in response to
steroidogenesis by FSK without any statistical significance
(ns p> 0.05). Upon treatment with naringenin alone, the expression
level of pAKT was significantly upregulated compared with FSK
(*p< 0.05). Treatment with both metformin and combination therapy
has significantly increased the expression levels of pAKT in the hGC
(**p< 0.01) (Figure 9a). A significant increase in pAKT level was also
seen with metformin treatment compared to letrozole in the FSK
background group (*p< 0.05).
The expression level of PTEN did not show any significant change
between the sham and FSK groups (ns p> 0.05). A significant increase
in PTEN levels was seen in letrozole, metformin, and combination
therapy with the FSK groups (***p< 0.001) (***p< 0.001) (*p< 0.05).
Nonetheless, PTEN expression levels were unaltered between narin-
genin alone therapy and FSK (ns p> 0.05). However, a significant
increase in the expression level of PTEN was seen in the FSK+letro-
zole group with sham (***p< 0.001) (Figure 9b), which was reduced a
little by metformin without any statistical significance (ns p> 0.05),
but higher than FSK alone group. However, combination with narin-
genin therapy restored the expression leel of PTEN levels to near the
normal, sham group (**p< 0.01) (***p< 0.001).
The expression level of AMPK was seen high in the hGC from the
non-steroidogenic than FSK-induced steroidogenic group (*p<0.05),
whichwasdecreasedbyletrozole(***p<0.001),metformin(**p<0.01),
naringenin (***p< 0.001) and combination groups (**p< 0.01) compared
to FSK alone group. Values are Mean ± SEM, N=3.
3.11 |Naringenin causes no significant change in
estrous/ovarian cycles in PCOS rats
PCOS is characterized by an irregular menstrual cycle, and likewise in
letrozole-induced PCOS rats. The estrous cycle in rats, like the men-
strual cycle in humans, lasts for 4 days and is categorized by proestrus,
estrus, metestrus, and diestrus (Figure 10a). A significant change in
the number of days in the diestrus was seen between the sham and
letrozole groups (PCOS) (***p< 0.001) (Figure 10b). The vaginal smear
examination revealed that the disturbed estrous cycle in PCOS condi-
tion (increased number of days in diestrus) was not reversed with the
naringenin treatment in the given duration of the treatment
(ns p> 0.05). Similar results were seen with metformin when com-
pared to letrozole (ns p> 0.05). Further, the number of days in the
estrus stage (ovulatory stage) of the estrous cycle was increased in
the sham group compared to letrozole (***p< 0.001). However, narin-
genin treatment did not increase the number of days in estrus, which
are less in letrozole (ns p> 0.05). Similar results were seen with met-
formin treatment when compared to letrozole (ns p> 0.05). Each
value represents Mean ± SEM, N=5.
3.12 |Naringenin treatment slightly restored
organ weights
We conducted an Echo-MRI body composition analysis to determine
if naringenin treatment affects body weight in PCOS condition. An
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analysis of the data showed a non-significant difference between the
naringenin treatment group and the PCOS group in terms of percent
fat and lean mass (ns p> 0.05) (Figure 10g,h). The body weight and
body mass remained unaltered in all the groups: Sham, PCOS, PCOS
+metformin, and PCOS +Naringenin (ns p> 0.05) (Figure 10c,f). Fat
mass was reduced in the PCOS group and could not be restored to
the normal by naringenin alone or the positive control, metformin
group (Figure 10h).
The ovarian weight remained unaffected by the naringenin treat-
ment in the letrozole-PCOS animal groups (Figure 10f). Nevertheless,
a slight decrease in uterine weight was seen in the PCOS group and
was not affected by naringenin (ns p> 0.05) (Figure 10e,g). Values are
Mean ± SEM, N=5.
3.13 |Naringenin treatment does not affect
glucose metabolism in PCOS
An OGTT was done prior to the ovarian tissue collection. Our result
showed that basal glucose levels were comparable in all the study
groups (ns p> 0.05 between sham and letrozole, naringenin, and
letrozole). Naringenin administration maintains plasma basal as well
FIGURE 8 Analysis of pAKT, PTEN and AMPK in the hGC in response to naringenin treatment during in vitro steroidogenesis. The
expression level of (a) pAKT, (b) PTEN and (c) AMPK was examined in response to naringenin and forskolin treatment. PTEN indicates
Phosphatase and tensin homolog; AMPK, AMP-activated protein kinase; pAKT, Phosphorylated protein kinase, strain AK, Thymoma
(Phosphorylated protein kinase B. The study was conducted using the three replicates (N=3). The values are represented as Mean ± SEM. ns
(non-significant) p> 0.05, *p< 0.05.
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as postprandial glucose levels in parallel to those of metformin. At
90 min time interval, the serum glucose level in the naringenin group
was significantly decreased (***p< 0.001) compared to the control;
however, non-significant changes were seen in comparison to letro-
zole (ns p>0.05) (Figure 10i). At a 120 min time interval, the glu-
cose level of the naringenin treatment group was comparable to
that of letrozole (ns p> 0.05). Each value represents Mean
±SEM,N=5.
3.14 |Naringenin with metformin combination
causes a significant change in estrous/ ovarian cycles
in PCOS rats
We also checked whether combination therapy rather than naringenin
alone treatment is useful in maintaining the ovarian cycle in rats. The
vaginal smear's examination revealed that the disturbed estrous cycle
in the PCOS condition (increased in the number of days in diestrus,
FIGURE 9 Expression analysis of pAKT, PTEN, and AMPK in the hGC in response to combination therapy of naringenin and metformin
during in vitro steroidogenesis. The relative expression level of (a) pAKT, (b) PTEN and (c) AMPK was examined in response to naringenin and
forskolin treatment. hGC indicates human granulosa cell; pAKT, Phosphorylated protein kinase, strain AK, Thymoma (Phosphorylated protein
kinase B); PTEN, Phosphatase and tensin homolog; AMPK, AMP-activated protein kinase. Each value represents Mean ± SEM (N=3). ns (non-
significant) p> 0.05, *p< 0.05.
16 RASHID ET AL.
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FIGURE 10 Legend on next page.
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***p< 0.001 between letrozole and sham) was reversed with narin-
genin (***p < 0.001 with letrozole) and additive naringenin
(***p< 0.001 with letrozole) in the given duration of the treatment
(Figure 11a,b). Further, a significant increase in the estrus stage (ovu-
lation) was seen both in combination and with naringenin alone when
compared with the PCOS condition (letrozole group) (***p< 0.001).
Values are Mean ± SEM, N=5.
3.15 |Add on therapy of naringenin with
metformin treatment slightly restored organ weights
We conducted an Echo-MRI body composition analysis to determine
if naringenin treatment or the combination therapy of naringenin and
metformin affects body weight in PCOS condition. An analysis of the
data showed a non-significant difference between the combination
group and the naringenin treatment group with the PCOS group in
terms of body weight and ovarian weights (ns p> 0.05) (Figure 11c,d).
Letrozole-induced PCOS groups also showed a significant decrease in
uterus weight compared with the sham group (**p< 0.01) (Figure 11e),
which was not changed by naringenin alone or in combination with
metformin treatment (Figure 11e). The body mass was analyzed with
respect to percentile fat and lean mass, and we did not find any signifi-
cant change. However, % fat mass was decreased by the PCOS condi-
tion, and naringenin with metformin combination treatment showed
some restoration effect but without statistical significance
(ns p> 0.05) (Figure 11h). Each value represents Mean ± SEM, N=5.
3.16 |Adjuvant therapy causes a significant
change in glucose metabolism in PCOS
Our result showed that at 0 min, the glucose level in letrozole was sig-
nificantly higher among the rest groups, including the sham and com-
bination groups (***p< 0.01). At 30 min, the sham group showed a
significantly decreased compared with the letrozole serum glucose
level. Naringenin administration maintains plasma basal as well as
postprandial glucose levels in parallel to those of the metformin as
well as with combination therapy. At 90 min time interval, the serum
glucose level in the naringenin group was significantly increased
(**p< 0.01) and maintained at 120 min when compared to letrozole-
induced PCOS and stabilized along with the sham group. Adjuvant
therapy of naringenin with metformin maintained normal glucose
levels significantly (*p< 0.05) at 120 min when compared to letrozole
(Figure 11i). Values are Mean ± SEM, N=5.
4|DISCUSSION
The multifaceted ovarian process of folliculogenesis begins with the
activation of the primordial follicle and culmination at ovulation, which
is followed by luteogenesis. There is emerging evidence for a basic
abnormality of folliculogenesis in PCOS that affects the very earliest
stages of follicle development. PCOS, the commonest cause of anovu-
latory infertility, is characterized by the arrested growth of antral folli-
cles. The management of PCOS is demanding and depends on the
signs and symptoms observed in the patients. New pharmacological
interventions are being attempted to assess their potential in the
management of PCOS with fewer side effects and enhanced efficacy.
Naringenin exhibits biological and pharmacological properties like
hypoglycemia, increased insulin sensitivity (via an increase in AMPK
and tyrosine phosphorylation), anti-inflammatory, lipid-lowering, and
antioxidant activities (Kannappan & Anuradha, 2010; Li et al., 2019;
Mulvihill et al., 2009). Therefore, in the present study, we have used
naringenin, trihydroxyflavanone, the predominant flavanone in citrus
fruits, as a new pharmacological entity to check its efficacy in the
management of PCOS in the pre-clinical setup in comparison to met-
formin as an off-label, standard drug.
We recapitulated PCOS-like conditions in the SD rat model using
letrozole, an effective aromatase inhibitor, where we observed an
increased number of ovarian cystic follicles and serum androgen. At
the same time, we found a decreased number of corpus luteum and
different stages of follicles as reported in the model of PCOS
(Homburg, 2009; Kafali et al., 2004; Maliqueo et al., 2013), confirming
the traits of human like PCOS in the SD rat model. Our study has
shown the mitigated effect of naringenin on ovarian pathology by
decreasing the number of ovarian follicular cysts, but increasing the
numbers of corpus luteum along with improved folliculogenesis
(increased number of primordial and primary follicles) in the SD rat
model of PCOS. The number of preantral follicles, primordial, and pri-
mary were also increased, suggesting the restoration of normal
folliculogenesis.
PCOS is attributed to the arrest of growth of the antral follicle at
5–8 mm size, which further leads to the development of a fluid-filled
cystic follicle. Naringenin possibly releases the antral follicle from the
arrest state by decreasing the androgen (DHEAs and TT) levels and
thereby reducing the number of follicular cysts, although we need a
confirmatory study. Further, the increased corpus luteal number with
naringenin treatment depicts naringenin may take part in mature folli-
cle development, ovulation potential, and luteinization.
We also checked whether combination therapy of naringenin and
metformin exerts stronger efficacy than naringenin stand-alone
FIGURE 10 Estrous cycle/ovarian cycle analysis, investigation of weights in body and organ and response to oral glucose tolerance test in
response to naringenin treatment. (a,b) The estrous cycle was examined in response to naringenin treatment. The effect of naringenin on (c) body
weight (d) ovary weights (e) uterus weight (f) Echo-MRI body composition analysis measuring fat, lean, free and total water mass (g) Percent Lean
mass and (h) Percent fat mass shows using letrozole-induced PCOS model in the SD. However, naringenin treatment shows a decrease in % fat
mass to control. (i) Glucose concentration in plasma at 0, 30, 60, 90, and 120 min was assessed in response to naringenin using the letrozole-
induced PCOS model in the SD rats. The values are represented as Mean ± SEM. The study was conducted on five different animals as replicates
(N=5). ns (non-significant) p> 0.05, *p< 0.05, **p< 0.01, ***p< 0.001.
18 RASHID ET AL.
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FIGURE 11 Legend on next page.
RASHID ET AL.19
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treatment in PCOS. In our study, the combination therapy has shown
to decline ovarian pathology by reducing ovarian follicular cysts,
increasing corpus luteal number, and preantral follicles. However,
combination therapy does not seem to be superior to naringenin alone
and shows neither additive nor synergistic effects.
Women with PCOS represent hyperandrogenism (androgen
excess), a hallmark of PCOS, in their biochemistry profile. The treat-
ment of hyperandrogenic manifestations is an important consideration
in PCOS. We determined the hyperandrogenism by determining the
androgen species DHEAs and TT in the blood circulation of the
letrozole-induced SD rat model in response to treatment with narin-
genin. We found decreased levels of TT and DHEAs in the blood cir-
culation, suggesting a beneficial role of naringenin in PCOS
management by normalizing ovarian folliculogenesis and, at the same
time, restoring normal serum levels of TT and DHEAs (dehydroepian-
drosterone sulfate) in PCOS state, to regularize the ovarian function
at the preclinical level. These results were consistent with the earlier
study done by Hong et al. (2019). It has been reported that the resto-
ration of antioxidant and steroidogenic enzyme activity occurs
through naringenin (Hong et al., 2019). In our study, the letrozole-
induced PCOS group of female SD rats showed an increased level of
TT, which was brought to normal levels by naringenin treatment. Simi-
larly, another study showed ameliorated hormone levels and improved
IR in the letrozole-induced PCOS in rats model (Wu, Yang, Hu, et al.
2022). The above studies support our findings that hyperandrogenism
features are improved by naringenin administration.
Finding a naringenin treatment-mediated reduction in androgen
species, TT, and DHEA in the SD rat model, we examined the expres-
sion level of the notable enzymes: CYP17 A1 and 19 A1, StAR, and
3βHSD2 of steroidogenesis. CYP17A1 is a key component of the
androgen synthesis pathway in the ovary, a pathway that is dysregu-
lated in PCOS. CYP17A1 helps convert pregnenolone to DHEA and
progesterone to androstenedione in the ovary. An increased expres-
sion of the CYP17A1 enzyme is reported in women with PCOS (Li
et al., 2013). Naringenin treatment restored the expression levels of
CYP17A1, which otherwise were dysregulated in the PCOS rat model.
Another cytochrome family enzyme, CYP19A1 (aromatase), plays an
important role in estrogen biosynthesis in granulosa cells, and an over-
all decrease in aromatase activity is reported in women with PCOS
(Aghaie et al., 2018). CYP19A1 converts androstenediol and TT to
estrone and estradiol in granulosa cells. We found an increased
expression level of CYP19A1/aromatase cytochrome P450
(P450arom; product of the CYP19 gene) by naringenin treatment,
which possibly favors C19 steroids/androgens conversion to estro-
gens to help in restoring normal steroidogenesis.
StAR, a transport protein, governs the rate-limiting step in ste-
roidogenesis and is considered a major candidate for the high level of
steroid hormones in PCOS (Aghaie et al., 2018). It regulates choles-
terol transfer from the outer to the inner mitochondrial membrane.
We observed that none of the treatment, metformin or naringenin
could restore the normal level of the StAR expression in the ovary.
3βHSD2 catalysis the biosynthesis of progesterone from pregneno-
lone, and its levels are reported to be high in women with PCOS
(Belani et al., 2018). However, in our experimental setup, the expres-
sion level of 3βHSD2 was not restored to the non-PCOS level for
unknown reasons.
Steroidogenesis in the granulosa cell is very important and
undergoes luteinization, and we assessed this phenomenon in
response to naringenin in the hGCs. Experimentally, FSKstimulates
granulosa cells to differentiate and also serves to accumulate the
cAMP that helps in progesterone/steroid production. FSK increases
cAMP and progesterone production with the help of FSH stimulation
but acts as an inhibitor of LH receptor formation (Ranta et al., 1984).
FSHR is found exclusively on granulosa cells of the ovary and its func-
tion is mediated by FSH, which plays a role in follicle development. It
has been shown that granulosa cells from women with PCOS have
higher levels of FSHR expression compared with those from normal
ovaries (Catteau-Jonard et al., 2008). Our study was consistent with
the above literature, in which increased expression levels of FSHR
were seen with FSK in hGCs. The expression level of FSHR was
brought down to the normal level in the hGC by naringenin, suggeting
crucial role of the naringenin in the granulosa cell. In hGC cells, the
expression level of 3β-HSD2 was restored by naringenin, which sug-
gests a mitigatory effect of naringenin on the steroidogenic protein
that was otherwise dysregulated by FSK-induced steroidogenesis.
Interestingly, SKOV3 cells are sensitive to naringenin along with letro-
zole to upregulate the expression levels of CYP17A and StAR whereas
the expression level of CYP19A is positively affected by naringenin
and acts to normalize the expression level in SKOV3.
The majority of the studies found that letrozole-induced PCOS
causes a change in the corresponding estrous cycle (ovarian cycle) of
the rats, with the estrous cycle being arrested in the diestrus phase
(Reddy et al., 2016; Vani et al., 2018). In the present research, we
observed a continuous diestrus in the letrozole (PCOS) group. Similar
results were seen, possibly due to the low duration of naringenin
treatment and without any other combination, Daine-35, as used ear-
lier (Wu, Yang, Han, et al. 2022). However, naringenin treatment did
not show normalization of the estrous cycle within the given duration
of treatment as reported in the earlier studies (Hong et al., 2019; Yang
et al., 2022). Add-on therapy of naringenin with metformin has been
FIGURE 11 Analysis of Estrous cycle/ovarian cycle analysis, body and organ weights, and oral glucose tolerance test in response to
combined therapy of naringenin and metformin. (a,b) The estrous cycle was examined in response to naringenin treatment. The effect of
naringenin on (c) body weight (d) ovary weights (e) uterus weight (f) Echo-MRI body composition analysis measuring fat, lean, free, and total
water mass (g) Percent Lean mass and (h) Percent fat mass using letrozole-induced PCOS model in the SD rat. However, combination treatment
shows an increase in % fat mass than control. (i) Glucose concentration in plasma at 0, 30, 60, 90 and 120 min was assessed in response to
adjuvant therapy using the letrozole-induced PCOS model in the SD rats. (***p< 0.001at 0 h with letrozole), (*p< 0.05 at 120 h with letrozole).
The values are represented as Mean ± SEM (N=5). ns (non-significant) p> 0.05, *p< 0.05, **p< 0.01, ***p< 0.001.
20 RASHID ET AL.
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shown to normalize the estrous cycle with a decrease in diestrus and
an increase in estrus phases of the estrous cycle.
IR and compensatory hyperinsulinemia play a major role in the
pathogenesis of hyperandrogenism in PCOS. Naringenin has been
shown to ameliorate IR and also inhibit steroidogenic enzymes, which
play an important role in androgen synthesis (Hong et al., 2019;
Murugesan et al., 2020; Papiez, 2004). Naringenin administration in
the PCOS model led to basal and postprandial glucose levels to normal
levels. A combination therapy of naringenin with metformin regulates
basal and postprandial glucose levels to normal and has proven to be
more effective in reducing postprandial glucose levels than naringenin
alone. Since metformin is a well-known insulin sensitizer, an additive
effect was seen when combined with naringenin.
AKT is the downstream signal transduction mediator of the
Ras/MAPK and PI3K pathways (Aksamitiene et al., 2012). FSH-
mediated activation of AKT signaling in granulosa cells of the ovaries
promotes estrogen production (Baumgarten et al., 2014). AKT signal-
ing plays a central role in insulin-stimulated glucose uptake in both
muscle and adipose tissue. The effect of insulin on glucose uptake in
peripheral tissue via AKT is through its ability to translocate GLUTs to
the cell membrane, thereby facilitating glucose uptake. PTEN is a lipid
phosphatase that converts phosphatidylinositol 3,4,5-trisphosphate
(PIP3) to phosphatidylinositol 4,5-bisphosphate (PIP2), thus inhibiting
the activation of AKT (Nguyen Huu et al., 2021). AMPK is a key regu-
lator of cellular metabolism and energy balance. It inhibits IR and pro-
motes glucose metabolism and uptake (Cokorinos et al., 2017). Our
study was consistent with the literature, showing enhanced insulin
response with naringenin treatment by upregulating pAKT in the
ovary of the SD rat model of PCOS and in the hGC.
Insulin signaling impairment also affects the overall body metabo-
lism and mass; hence, we assessed the same. Comparable body
weights were seen in the PCOS and naringenin groups. However, per-
cent lean and total water mass were higher in all the groups and nar-
ingenin did not affect those. Naringenin treatment groups reversed
the condition of lerozole-PCOS by incareasing the uterine weight. In
the letrozole group, the increase in ovary weight appears to be due to
cystic follicles, which was unaffected by the naringenin treatment.
Further, a decrease in uterus weight in the letrozole group is a result
of the decrease in corpus luteal number, which is responsible for pro-
gesterone synthesis; however, treatment with naringenin reversed the
condition. A combination of naringenin with metformin could not
result in better outcomes than naringenin alone in terms of body and
organ weight.
The detailed mechanism of naringenin on its ameliorating effect
on PCOS is not clear. However, there are several studies showing
AKT as one of the targets to ameliorate PCOS (Xie et al., 2021; Zhang
et al., 2020; Zheng et al., 2021), and we studied the same in our inves-
tigation. We observed that AKT seems to be one of the targets of nar-
ingenin in the ovary, as the phosphorylation of AKT was increased in
response to it and metformin as well.
Further, the human equivalent dose of naringenin is well within
the accepted 8.10 mg/kg as per the earlier study, and the dose for an
adult weighing 60 kg in a single dose would be 486.22 mg (Nair &
Jacob, 2016).
Collectively, naringenin, a natural citrus flavonoid, has revealed
encouraging results in our preclinical studies in the letrozole-induced
SD rat model by improving PCOS conditions and may be promising as
emerging therapeutics in the management of PCOS in clinical setup
prospectively. Naringenin promotes primordial and primary follicles
and the corpus luteum, restores the normal circulatory levels of andro-
gens, and, at the same time, eliminates the ovarian follicular cysts pos-
sibly involving the AKT pathway.
5|CONCLUSION
Naringenin promotes primordial and primary follicles and the corpus
luteum, restores the normal circulatory levels of androgen, and at the
same time, eliminates the ovarian follicular cysts possibly involving
the AKT pathway in the letrozole-induced SD rat model.
5.1 |Limitations
The limitation of this study includes the duration of naringenin treat-
ment, which was only 20 days. The dose-ranging studies of the drug
might provide us with more insight about the minimum effective dose.
The disturbed estrous cycle was reversed only during the later days of
the treatment. Further, naringenin being the natural insulin sensitizer,
the insulin tolerance test (ITT), and HOMA-IR were not confirmed in
PCOS conditions.
AUTHOR CONTRIBUTIONS
Rumaisa Rashid: Conceptualization; data curation; formal analysis;
funding acquisition; investigation; project administration; resources;
supervision; visualization; writing –original draft; writing –review and
editing. Rupal Tripathi: Data curation; software. Akanksha Singh:
Data curation; formal analysis; software. Sudarsan Sarkar: Data cura-
tion; formal analysis. Ajaykumar Kawale: Data curation; formal analy-
sis; software. Ghulam Nabi Bader: Conceptualization; funding
acquisition; methodology. Satish Gupta: Data curation. Rakesh Kumar
Gupta: Data curation. RAJESH KUMAR Kumar JHA: Conceptualiza-
tion; formal analysis; funding acquisition; investigation; project admin-
istration; resources; supervision; validation; visualization;
writing –original draft; writing –review and editing.
ACKNOWLEDGEMENTS
The authors thank Mr. Hasham Shafi for his timely help during cell cul-
ture work. The author thanks the laboratory animal house facility. The
manuscript number 136/2022/RKJ.
FUNDING INFORMATION
We would like to thank Department of Health and Research, Human
Resource development, New Delhi, India for their funding under
women scientist scheme (No. 12013/25/2020-HR/E).
CONFLICT OF INTEREST STATEMENT
Authors declare no conflict of interest.
RASHID ET AL.21
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DATA AVAILABILITY STATEMENT
All the data supporting the findings of this study are available on
request from the corresponding author.
ORCID
Rajesh Kumar Jha https://orcid.org/0000-0002-0418-7699
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How to cite this article: Rashid, R., Tripathi, R., Singh, A.,
Sarkar, S., Kawale, A., Bader, G. N., Gupta, S., Gupta, R. K., &
Jha, R. K. (2023). Naringenin improves ovarian health by
reducing the serum androgen and eliminating follicular cysts in
letrozole-induced polycystic ovary syndrome in the Sprague
Dawley rats. Phytotherapy Research,1–24. https://doi.org/10.
1002/ptr.7860
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