Nutrients 15 02238

nutrients
Article
Regulation of Insulin Resistance, Lipid Profile and Glucose
Metabolism Associated with Polycystic Ovary Syndrome by
Tinospora cordifolia
Ritu Rani 1 , Havagiray R. Chitme 1, * , Neha Kukreti 1 , Pankaj Pant 1 , Basel A. Abdel-Wahab 2 ,
Masood Medleri Khateeb 2 , Mohammed Shafiuddin Habeeb 2 and Marwa B. Bakir 3
1 Faculty of Pharmacy, DIT University, Dehradun 248009, Uttarakhand, India; 1000013859@dit.edu.in (R.R.);
1000013858@dit.edu.in (N.K.); pankaj.pant@dituniversity.edu.in (P.P.)
2 Department of Pharmacology, College of Pharmacy, Najran University, Najran P.O. Box 1988, Saudi Arabia;
babdelnaem@nu.edu.sa (B.A.A.-W.); mmkhateeb@nu.edu.sa (M.M.K.); mshabeeb@nu.edu.sa (M.S.H.)
3 Department of Pharmacology, College of Medicine, Najran University, Najran P.O. Box 1988, Saudi Arabia;
mbbakir@nu.edu.sa
* Correspondence: hrchitme@gmail.com or havagiray.chitme@dituniversity.edu.in; Tel.: +91-135-710-034
Abstract: Background: The plant Tinospora cordifolia (TC), traditionally known as guduchi or giloy, is
used for a number of health conditions as a nutritional supplement and rejuvenation medicine. Its
nutritional supplementary products are traditionally recommended for a wide range of health issues,
including diabetes, menstruation discomfort, fever, obesity, inflammation, and more. Unfortunately,
there has not been extensive research into its effectiveness in treating or managing insulin resistance,
lipid and carbohydrate metabolism, hormonal imbalance, and metabolic syndrome-associated poly-
cystic ovary syndrome (PCOS). Methods: Consequently, the present study was designed to induce
insulin resistance, dyslipidemia, hormonal abnormality, hyperglycemia, and menstrual disturbance
of PCOS using dehydroepiandrosterone (DHEA) in mice and study the effect of oral TC extracts on
Citation: Rani, R.; Chitme, H.R.; these factors by using ancient and modern technologies. During the 21-day study, 6 mg/100 g/day
Kukreti, N.; Pant, P.; Abdel-Wahab, of DHEA was given to female mice. Levels of glucose, insulin, lipids, and hormones were esti-
B.A.; Khateeb, M.M.; Habeeb, M.S.; mated. In addition to being seen with the naked eye, the morphological and microscopic changes
Bakir, M.B. Regulation of Insulin were also observed on histology slides. Results: The study outcomes show that pretreatment with
Resistance, Lipid Profile and Glucose TC preparations significantly improved biochemical and histological abnormalities in female mice.
Metabolism Associated with
Diestrus phase was only observed in DHEA-treated animals, while cornified epithelial cells were
Polycystic Ovary Syndrome by
present in TC-treated mice. Pretreatment with TC satva showed significant (p < 0.001) reductions in
Tinospora cordifolia. Nutrients 2023, 15,
body weight compared to placebo. Fasting blood glucose, 1-h OGTT, and 2-h OGTT levels were all
2238. https://doi.org/10.3390/
significantly lower in TC satva- and oil-treated animals in comparison to the disease control group
nu15102238
(p < 0.001). Treatment with TC extracts resulted in a normalization of estradiol, progesterone, and
Academic Editors: Montserrat Esteve testosterone levels (p < 0.05). Treatment with TC extract improved lipid profiles (p < 0.001), LH/FSH
and Maria del Mar Romero
ratios (p < 0.01), fasting insulin levels (p < 0.001), HOMA-IR (p < 0.001), HOMA-Beta (p < 0.001), and
Received: 20 April 2023 QUICKI (p < 0.001). Both macroscopic and microscopic alterations were seen to be restored after TC
Revised: 3 May 2023 extract treatment. After being treated with TC satva, oil, and hydroalcoholic extract, the severity of
Accepted: 5 May 2023 PCOS decreased by 54.86%. Conclusions: These findings lead us to the conclusion that TC extracts
Published: 9 May 2023 and satva as nutritional supplements are useful for treating PCOS and associated symptoms. It is
recommended that additional research be conducted to determine the molecular mechanism of action
of TC nutritional supplements on PCOS-related changes in metabolic profiles. We also recommend
further clinical studies to explore the clinical efficacy and effectiveness of TC nutritional supplements
Copyright: © 2023 by the authors.
in treating and/or managing PCOS.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
Keywords: PCOS severity; insulin resistance; insulin sensitivity; guduchi satva; guduchi oil; guduchi
conditions of the Creative Commons hydroalcoholic extract
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Nutrients 2023, 15, 2238. https://doi.org/10.3390/nu15102238 https://www.mdpi.com/journal/nutrients

Nutrients 2023, 15, 2238 2 of 23
1. Introduction
Polycystic ovary syndrome (PCOS) is a leading cause of infertility in women due
to hormonal imbalance. It manifests as polycystic ovaries, elevated testosterone levels,
and infertility [1]. It is estimated that between 5% and 10% of women aged 18–44 may be
affected by PCOS [2]. There are many prominent risk factors for this condition, includ-
ing hereditary and environmental components, according to epidemiological studies [3].
Menstrual problems, infertility, diabetes, elevated levels of masculinizing hormones, a
hyperinsulinemic metabolic syndrome, and endometrial cancer are all clinical symptoms
of PCOS [4]. Menstrual dysfunction, acne, hirsutism, and infrequent or absent ovulation
are all symptoms of hyperandrogenism associated with PCOS [5].
Increased luteinizing hormone (LH) or elevated blood insulin levels are considered
to be two prominent contributors to the cause of PCOS [6]. Patients with PCOS usually
undergo extensive clinical treatment, which may involve both lifestyle modification and
pharmaceutical interventions. Treatment options for polycystic ovarian syndrome are
severely limited. The symptoms of PCOS can be managed with symptomatic treatments [7].
The primary and realistic objectives of therapy in PCOS are fertility restoration, insulin
resistance (IR) improvement, acne and hirsutism management, menstrual cycle regular-
ization, and endometrial hyperplasia prevention [8]. Hot flashes, arthritis, joint or muscle
discomfort, irritability, mood swings, melancholy, and bloating are some of the psycholog-
ical and physiological side effects of contemporary PCOS treatment that can range from
mild to severe [9].
Medicines produced from natural sources may be an impressive substitute therapy
with a familiar mechanism of action for the creation of a powerful, safe, and cost-effective
treatment agent for this ailment [9]. However, so far, there is scant scientific basis for the
claims that complementary and alternative therapies are both safe and effective. This
necessitates testing some unconventional methods for preventing and treating PCOS, such
as medicines [7]. To this day, Tinospora cordifolia (TC) is one of Ayurveda’s most popular
medicinal plants [10]. In Ayurvedic and ethnic medicine, TC has several medical uses due
to its lack of side effects/minimal side effects and wide range of beneficial effects, including
for periods, allergies, arthritis, fever, leprosy, diabetes, stress, and malaria. The stem has
a number of medical purposes, including as a diuretic, a thirst quencher, a source of iron
and vitamins, a jaundice cure, and a bitter stomachic. There is evidence that using a plant
stem extract can assist with skin disorders. When combined with other drugs, TC serves
as an antidote for snakebite and scorpion stings [11]. Some of the other ailments that this
plant aids in treating include asthma, cough, wounds, and pneumonia. It is also found to
be effective against cancer and provides an improvement in the immune system, protection
of nerve cells, resistance to diabetes, a reduction in cholesterol, and protection of the liver.
Evidence suggests that TC can help diabetic foot ulcers heal faster and mitigate some of the
side effects of both chemotherapy and radiation [10].
As an anti-inflammatory herb, TC is believed to be extremely effective against PCOS
due to the fact that insulin dysregulation and ovarian cysts have a common root cause, i.e.,
chronic mild inflammation in the tissues. Reducing IR increases metabolism and stimulates
all tissues naturally [12]. The important constituents of TC are alkaloids (tinosporine, mag-
noflorine, berberine (BER), choline, jatrorrhizine, palmatine (PAL), tembeterine), flavonoids
(quercetin, luteolin, and kaempferol), steroids, terpenoids, glycosides, sesquiterpenoids,
essential oils, polysaccharides, and a mixture of fatty acids [13]. Syringin, berberine, and
rumphioside-I were the alkaloids studied, and in silico analyses showed that they signifi-
cantly inhibited insulin receptor substrate-1 (IRS-1) and IRS-2 receptors by the antagonistic
ligand. When docked against the human androgen receptor 1E3G, BER and PAL performed
better than other compounds. Isoquinoline alkaloids (BER and PAL) may have functions
against a variety of disorders; for example, virtual screening of an alkaloid from the stems
of TC against PCOS led to a scientific evaluation [14]. Furthermore, BER effectively re-
stored serum hormone levels and reduced insulin resistance. Apoptosis and morphological
damage to the ovaries were both repaired by BER therapy. BER’s effects on granulosa cell
Nutrients 2023, 15, 2238 3 of 23
proliferation and death were mitigated by blocking the PI3K/AKT pathway [15]. There
is hope that these medications will have promising outcomes in PCOS after they have
undergone comprehensive preclinical and clinical testing [16].
The Food Safety and Standards Authority of India (FSSAI) considered the root and
stem of Tinospora cardifolia as a nutritional supplement in the form of powder, decoction,
sattva (satva), and extract in a dose varying from 0.5 to 10 g [17]. The primary purpose of
conducting this study was to develop extracts, satva, and oil of TC as recommended by
FSSAI and examine their effects on DHEA-induced PCOS-related abnormalities such as
insulin resistance and sensitivity, glucose tolerance, menstrual disturbance, lipid profile,
and microscopic and morphological changes in the ovaries in a mice model, along with
elucidating the mechanism of action.
2. Materials and Methods

2.1. Plant Collection and Authentication
The plant specimens were obtained from the herbal garden of the institute and near
the campus and authenticated at HNBG University, Srinagar, Uttarakhand, India (ref.:
Plant Taxn/356/2822-A).
2.2. Reagents and Chemicals

Insulin testing kits were provided by Wuhan Fine Biotech Co., Ltd., Wuhan, China
(batch: M0260H030). Cholesterol and triglyceride testing kits were purchased from Transa-
sia Bio-medicals Ltd., Mumbai, India. Progesterone, testosterone, estradiol, LH, and FSH
levels were estimated using ELISA kits purchased from Abbott Laboratories, Abbott Park,
IL, USA.
2.3. Instruments
The following instruments were used to conduct this research: hemoglucometer (Dr.
Morepen Glucose Monitor-Gluco One BG03, Gurugram, India); rotary microtome; Kwality
fluorescent microscope with camera, Chandigarh, India; biochemical analyzer for lipid
profiles; microplate reader for ELISA tests (Micro lab, New Delhi, India).
2.4. Preparation of Hydroalcoholic Extract

TC hydroalcoholic (HA) extract was prepared from stems collected and sterilized. The
stems were trimmed into smaller bits and air-dried in the shade and then powdered in a
steel blender. For seven days, with occasional shaking and warming, 1 kg of powdered
TC stems was immersed in a hydroalcoholic solution (70:30) in a 5 L flat-bottomed flask
and cold-extracted. After seven days, the mixture was filtered through a Buchner funnel
to create a clear filtrate. Then, the filtrate was evaporated using a water bath and rotary
evaporator at 60–70 ◦ C. After evaporation, the hydroalcoholic extract was completely dry
and ready for use [18].
2.5. Formulation of Guduchi Satva

The fresh TC stems used to make guduchi satva (water extract by following FSSAI/
Ayurvedic-approved method of preparation) were washed well with clean water, then
chopped into pieces 1 to 2 inches in length, crushed into a coarse, slimy mass, and soaked
in water for an entire day. It was carefully macerated and filtered through four layers
of muslin the next day. After allowing the filtrate to settle for as long as five hours, the
supernatant liquid was skillfully drained, and the starchy sediment was scraped into a
tray. A white crystalline powder was made by lyophilizing the starchy material at −55 ◦ C;
this was then placed in an airtight container until pharmacological evaluation could be
undertaken [19].
Nutrients 2023, 15, 2238 4 of 23
2.6. Isolation of Essential Oil

The leaves of TC were harvested and then washed with running water to obtain
essential oil. The essential oil was obtained by subjecting 100 g of newly harvested TC
leaves to a Clevenger apparatus for hydrodistillation for a period of four hours. The
hydrodistillation process resulted in an average yield of oil, which was reported [20].
2.7. Animal Handling and Groups

Swiss albino female mice that were 6 weeks old with a body weight of 30–40 g were
purchased from NIB, Noida–India. After a week’s time for acclimatization, the animals
were used for experimentation. All 54 female mice were separated into 9 groups of 6 mice
each and treated every day for 20 days. DHEA of 6 mg/100 g/day BW diluted in 0.1 mL
sesame oil was given subcutaneously. The acute toxicity study was carried out as per the
OECD guidelines [21] and the recently published study [22]. The oral maximum dose of
2000 mg/kg did not cause any toxicity signs. Therefore, 10% of the dose was considered
optimal, i.e., 200 mg/kg/day, and its double, 400 mg/kg/day, was considered as the
maximum dose and used for this study. Group I: Normal group was administered 0.1 mL
sesame oil and 100 µL 0.5% methylcellulose s.c.; Group II: Disease control (DHEA + 100 µL
0.5% methylcellulose); Group III: Positive control (250 mg/kg/day metformin mixed in
0.5% methylcellulose oral + DHEA); Group IV: TC-satva-treated group, 200 mg/kg/day
oral + DHEA; Group V: TC-satva-treated group, 400 mg/kg/day oral + DHEA; Group
VI: TC-oil-treated group, 200 mg/kg/day oral + DHEA; Group VII: TC-oil-treated group,
400 mg/kg/day oral + DHEA; Group VIII: TC-HA-extract-treated group, 200 mg/kg/day
oral + DHEA; Group IX: TC-HA-extract-treated group, 400 mg/kg/day oral + DHEA.
2.8. Establishment of PCOS Model

Oral administration of TC preparations (satva, oil, and HA extract) began 2 h before
the first DHEA dose and continued for 20 days to assess the preventative benefits of TC
extracts in PCOS. The animals were weighed on days 1 and 21. According to a group of
researchers [7], blood glucose levels were checked on days 1 and 21. On day 20 of the
study, an Oral Glucose Tolerance Test (OGTT) was carried out. Measurements of insulin,
testosterone, progesterone, estradiol, cholesterol, LH, FSH, and triglycerides were taken
from the animals’ blood at the conclusion of the study. The ovaries were removed for
histological examination.
2.9. Estrous Cycle Monitoring and Vaginal Smear Test

To measure the variations in the estrous cycle of the test mice, vaginal lining smears
were collected. There are four stages to the estrous cycle: estrus, diestrus, metestrus, and
proestrus. After flushing the mice’s vaginas with 0.2 to 0.3 mL of normal saline using a
micropipette, we aspirated vaginal fluid to analyze the estrous cycle. Droplets of this cell
solution were placed on slides under coverslips and observed under a microscope with
10× and 50× lenses to identify methylene blue-stained cells. The presence of cornified
epithelial cells indicates the estrous phase; the presence of mucus and leukocytes confirms
the diestrus stage; the presence of epithelial cells, leukocytes, and cornified cells with nuclei
indicates the metestrus phase; and epithelial cells with nuclei with few leukocytes indicate
the proestrus stage [23].
2.10. Change in Body Weight

Regular changes in the body weight of female mice were monitored on day 1, day 14,
and day 21 [24].
2.11. Measurement of Fasting Blood Glucose (FBG) and Oral Glucose Tolerance Test (OGTT)
On day 20, for the OGTT measurement, the mice were given glucose orally at a rate of
2 g/kg of body weight after they had been deprived of food for a period of six hours. A
hemoglucometer (Dr. Morepen Glucose Monitor-Gluco One BG03, Gurugram, India) was
Nutrients 2023, 15, 2238 5 of 23
used to measure the amount of glucose in the blood at 60 and 120 min. The fasting blood
glucose was measured on days 1 and 21 [25].
2.12. Blood Collection, Collection of Plasma, and Detection of Biochemical Indexes

On day 21 of the study, retro-orbital and cardiac blood samples were collected and well
preserved in BD Vacutainers with potassium EDTA to avoid clotting until use. Hemolysis,
clotting, or clot-forming samples were discarded. Following the collection of blood on the
21st day, serum samples were taken following the centrifugation of blood at 3000 rpm and
4 ◦ C for 10 min. These samples were then stored in a deep refrigerator at a temperature of
−80 ◦ C [26]. We assessed the levels of cholesterol and triglycerides by using a biochemical
analyzer, in addition to fasting insulin (FINS) levels, followed by the calculation of HOMA-
IR, HOMA-Beta, and QUICKI. Serum estradiol, progesterone, and testosterone levels were
also estimated according to the manufacturer’s instructions using a microplate reader.
There were limited serum samples available for LH and FSH estimation. Therefore, the
serum samples of two animals, 150 µL each, were pooled into one, and LH and FSH were
estimated using the ELISA test kit [24].
2.13. Measurement of Weight, Size, and Morphological Changes in the Ovaries

After blood collection, animals were euthanized with an overdose of ketamine and
their ovarian weight, size, and morphology were assessed [27].
2.14. Histological Examination

After 21 days, the mice had their ovaries removed and preserved in 10% formalin.
The previously reported method was followed for the histological examination. In brief,
blocks were serially sectioned at 5 µm thickness using a microtome after undergoing xylene
deparaffinization, rehydration, hematoxylin and eosin staining, dehydration, clearing, and
mounting on DPX (dibutylphthalate polystyrene xylene) beneath glass coverslips. After
that, we observed the slides under a microscope [26].
2.15. Severity of PCOS

Based on previous literature [28] and our own understanding, we propose the follow-
ing method of calculating the severity score of PCOS in mice and categorize it as nil, mild,
moderate, severe, and very severe.
PCOS Severity Score = ( Presence or Absence o f Oocyte + Number o f Cystic f ollicles)×

Ovary diameter (mm)+Ovarian weight (mg)/10
IU + Testosterone nmol )
10×( Insulin ( mL ) ( L )
where the presence of oocytes = 0 and the absence of oocytes = 1.

Then, we categorize the severity of PCOS based on the numerical score:
Severity score: ≥10 (very severe), 7–10 (severe), 4–7 (moderate), 1–4 (mild), <1 (nil).
2.16. Statistical Analysis

Statistical analysis was performed, and the data are presented as mean ± SEM. The
unpaired T test was used to analyze the categorical data, while the paired T test, one-way
analysis of variance (ANOVA), and Dennett’s multiple comparisons test were used to
analyze the continuous data. Statistical significance was assumed when the p-value was
less than 0.05. The results are indicated in figures as * p < 0.05, ** p < 0.01, and *** p < 0.001
compared to disease control mice.
3. Results
3.1. Estrous Cycle Determination
In Figure 1, it is observed that Group I had all four phases of a normal estrous cycle
throughout the study, showing a healthy mouse’s estrous cycle. Group II animals with
PCOS had delayed estrous cycles that lingered in diestrus/PCOS. As most vaginal smear
3. Results
3.1. Estrous Cycle Determination
Nutrients 2023, 15, 2238 6 of 23
In Figure 1, it is observed that Group I had all four phases of a normal estrous cycle
throughout the study, showing a healthy mouse’s estrous cycle. Group II animals with
PCOS had delayed estrous cycles that lingered in diestrus/PCOS. As most vaginal smear
cells
cells were
were leukocytes,
leukocytes, Group
Group III
III (Metformin)
(Metformin) remained
remained inin diestrus.
diestrus. Groups
Groups IV,
IV, V,
V, VI,
VI, VII,
VII,
VIII,
VIII, and
and IX
IX exhibited
exhibitedthethepresence
presenceofofcornified
cornifiedepithelial
epithelialcells.
cells.
Figure 1.
Figure 1. The
The different
different stages
stages of
of the
the estrous
estrous cycle
cycle were
were estimated
estimated inin normal
normal healthy
healthymice.
mice. (A)
(A) In
In the
the
estrus stage, predominantly cornified epithelial cells (brown arrow) are present. (B) The
estrus stage, predominantly cornified epithelial cells (brown arrow) are present. (B) The pro-estrus pro-estrus
stageisischaracterized
stage characterizedby bynucleated
nucleated cornified
cornified epithelial
epithelial cells
cells (black
(black arrow)
arrow) withwith leukocytes
leukocytes (red(red ar-
arrow).
row). (C) The metestrus stage contains cornified epithelial cells (brown arrow), leukocytes (red ar-
(C) The metestrus stage contains cornified epithelial cells (brown arrow), leukocytes (red arrow),
row), and nucleated epithelial cells (black arrow). (D) In the diestrus stage or PCOS stage, predom-
and nucleated epithelial cells (black arrow). (D) In the diestrus stage or PCOS stage, predominantly
inantly leukocytes (red arrow) are present.
leukocytes (red arrow) are present.
3.2. Body
3.2. Body Weight
Weight
Figure2A
Figure 2Ashows
showsthatthatDHEA-treated
DHEA-treatedmice micegained
gained considerably
considerably more
more weight
weight by by
dayday
21
21 than their normal control counterparts did (p < 0.05). Mice that were
than their normal control counterparts did (p < 0.05). Mice that were given TC satva hadgiven TC satva
a
had a significant
significant (p < 0.001)
(p < 0.001) decrease
decrease in BWincompared
BW compared to those
to those givengiven a placebo.
a placebo. The treat-
The treatment
ment group
group receiving
receiving 400 mg/kg400 mg/kg
of TC oilof TC
andoil
HA and HA extract
extract also lostalso lost significantly
significantly more
more weight
weight
than thethan
DHEA thecontrol
DHEA groupcontrol(pgroup (p < 0.01).
< 0.01).
Nutrients 2023, 15, x FOR PEER REVIEW 7
Nutrients 2023, 15, 2238 7 of 23
Figure
Figure 2. Effects 2. Eﬀectscordifolia
of Tinospora of Tinospora cordifolia
extract treatmentextract treatment in DHEA-induced
in DHEA-induced PCOS mice onPCOS body mice on
weight
weight (A), fasting (A),glucose
blood fasting blood glucose
(B), and OGTT(B),(C) and OGTT (C) measurement.
measurement. Every value isEvery valueasisapresented
presented
mean along withmean along with
standard error.standard
Data wereerror. Data were
analyzed analyzed
by ANOVA by ANOVA
followed followed
by DMCT. bygroups,
In all DMCT. In all gr
0 h of OGTT was compared with 1 and 2 h of OGTT. * p < 0.05, ** p < 0.01, and *** p < 0.001. *** p < 0.001.
0 h of OGTT was compared with 1 and 2 h of OGTT. * p < 0.05, ** p < 0.01, and
3.3. Fasting Blood Glucose Level

Nutrients 2023, 15, x FOR PEER REVIEW 8 of 23
Nutrients 2023, 15, 2238 8 of 23
3.3. Fasting Blood Glucose Level

As presented in Figure 2B, on day 1, the blood glucose level was normal in all groups.
3.3. Fasting
On day Blood
21, the Glucose
DHEA Levelhad highly significantly (p < 0.001) increased BG levels when
group
As presented
compared in Figure
to normal control2B,mice.
on day 1, thesignificant
Highly blood glucose
(p < level
0.001)was normal ininallBG
reductions groups.
were
On day 21,inthe
observed DHEA
groups of group had highly
mice treated withsignificantly
TC satva and(poil
< 0.001)
on dayincreased BG levels
21 compared to thewhen
neg-
compared
ative controlto group.
normalTreatment
control mice.
withHighly
metforminsignificant (p < 0.001)
significantly reductions
(p < 0.05) reducedintheBG were
DHEA-
observed in groupsblood
induced increased of mice treated
glucose levelwith
but TC
not satva
at the and
leveloil on treatment
of TC day 21 compared
groups. to the
negative control group. Treatment with metformin significantly (p < 0.05) reduced the
DHEA-induced
3.4. Oral Glucoseincreased blood glucose level but not at the level of TC treatment groups.
Tolerance Test
As shown
3.4. Oral Glucosein Figure 2C,
Tolerance Test on day 20, 1 h OGTT of TC satva and oil treatment signifi-
cantly (p < 0.01) raised glucose levels compared to 0 h OGTT of TC satva and oil, but it
As shown in Figure 2C, on day 20, 1 h OGTT of TC satva and oil treatment significantly
was normal after 2 h OGTT of TC satva and oil. When compared to the 0 h OGTT of the
(p < 0.01) raised glucose levels compared to 0 h OGTT of TC satva and oil, but it was normal
DHEA group, the 1 h OGTT and 2 h OGTT of Group II demonstrated a significant rise in
after 2 h OGTT of TC satva and oil. When compared to the 0 h OGTT of the DHEA group,
BG levels. There was no alteration in OGTT for the normal control group. The 2-hour
the 1 h OGTT and 2 h OGTT of Group II demonstrated a significant rise in BG levels. There
OGTT results indicate that TC treatment for 20 days significantly improves glucose toler-
was no alteration in OGTT for the normal control group. The 2-h OGTT results indicate
ance in mice.
that TC treatment for 20 days significantly improves glucose tolerance in mice.
3.5. Hormonal
3.5. Hormonal Profile
Profile
As presented
As presented in in Figure
Figure 3A,B,
3A,B, the
the estradiol
estradiol and
and progesterone
progesterone levels
levels were
were observed
observed toto
be considerably (p < 0.05 and p < 0.01) lower in DHEA-treated mice compared to Group I.
be considerably (p < 0.05 and p < 0.01) lower in DHEA-treated mice compared to Group I.
However, testosterone
However, testosterone levels
levels (Figure
(Figure 3C)
3C) were
were found
found to to be
be statistically
statistically (p
(p << 0.001)
0.001) greater.
greater.
In all
In all TC
TC treatment
treatment groups,
groups, there
there was
was aa decrease
decrease in in serum
serum testosterone,
testosterone, but
but the
the highest
highest
doses of satva and oil brought it down to nearly normal levels. Metformin
doses of satva and oil brought it down to nearly normal levels. Metformin counteracted counteracted
the effect
the effect that
that DHEA
DHEA had hadon
onestradiol
estradiol(p (p<< 0.05).
0.05). When
When compared
comparedto toGroup
GroupIIII(20.5
(20.5± ± 2.59),
2.59),
the maximum dose (400 mg/kg) of TC satva, oil, and HA extracts significantly
the maximum dose (400 mg/kg) of TC satva, oil, and HA extracts significantly increased increased
levels (TC
estradiol levels (TC satva
satva (29.33
(29.33 ± ± 1.406), oil (29
(29 ± ± 2.017), and HA extracts
extracts (27
(27 ± ± 1.75)).
Treatment with
Treatment with TC
TC extract
extract prominently
prominently raised
raised (p
(p << 0.01)
0.01) serum
serum progesterone
progesterone levels
levels when
when
compared to
compared to the
the placebo
placebo group.
group.
40 A
30
** *
Estradiol (pg/ml)
* * *
*
20
10
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ol
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on
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Figure 3. Cont.
Nutrients2023,
Nutrients 2023,15,
15,2238
x FOR PEER REVIEW 9 of
9 of2323
20
B
Progesterone (ng/ml)
15
10 ** * ** ** **
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l l ) ) ) ) ) ) )
ro ro kg kg kg kg kg kg kg
nt nt g/ g/ g/ g/ g/ g/ g/
Co Co m m m m m m m
al A 25
0 00 00 00 00 00 00
rm E
t( (2 (4 (2 (4 (2 (4
No DH e tv
a
tv
a
O O HA
il
HA
il
A +M Sa Sa A+ A+ A+ A+
E + + E E E E
DH EA EA DH DH DH DH
DH DH
15
C
***
Testosterone (nmol/L)
10
*
** ** **
5 **
*** ***
0
l l ) ) ) ) ) ) )
Co Co m m m m m m m
al A 50 00 00 20
0
40
0 00 00
rm E (2 (2 (4 l( l( (2 (4
No DH et tv
a
tv
a i i A A
+M Sa Sa +O +O +H +H
EA A+ A+ H EA H EA EA EA
H E E D D DH D H
D
DH DH
7
6.42 D
6
LH/FSH Level (mIU/ml)
4
2.87 2.88
3 2.49 2.43 2.59
2.42 2.43 2.33 2.44 2.32
2.1 2.03 2.23 2.23
1.92 2.16 1.98 1.88
2 1.52
1.061 0.87 0.93 0.9 0.99 0.94
1 0.79
0
LH FSH LH:FSH Ratio
Normal control DHEA Control DHEA +Met (250 mg/kg)

DHEA +Satva (200 mg/kg) DHEA +Satva (400 mg/kg) DHEA +Oil (200 mg/kg)
DHEA +Oil (400 mg/kg) DHEA +HA (200 mg/kg) DHEA +HA (400 mg/kg)
Figure 3. Treatment with Tinospora cordifolia extracts was given to mice that had DHEA-induced
Figure
PCOS, 3.
andTreatment
the levelswith Tinospora
of estradiol (A),cordifolia extracts
progesterone (B),was given to (C),
testosterone miceand
that had DHEA-induced
LH/FSH (D) were
PCOS, and the levels of estradiol (A), progesterone (B), testosterone (C),
measured. Every value is presented as a mean along with standard error. Data were and LH/FSH (D)by
analyzed were
measured. Every value
ANOVA followed is presented
by DMCT. as **
* p < 0.05, a mean along
p < 0.01, and with
*** p <standard
0.001. error. Data were analyzed by
ANOVA followed by DMCT. * p < 0.05, ** p < 0.01, and *** p < 0.001.
Nutrients 2023, 15, 2238 10 of 23
3.6. LH and FSH Level

3.6. LH and in
As shown FSH Level 3D, DHEA treatment resulted in greater increases in levels of
Figure
FSH (2.23AsmIU/mL)
shown in and
FigureLH3D, DHEA
(6.42 treatment
mIU/mL) in resulted
mice thanin greater
in the increases
controls in levels
(FSH of FSH
(1.98
(2.23 mIU/mL)
mIU/mL) and LH (2.1 and LH (6.42 LH
mIU/mL)). mIU/mL)
and FSH in levels
mice than
wereinfound
the controls (FSHin(1.98
to be lower micemIU/mL)
that
wereand LHTC
given (2.1 mIU/mL)).
extracts comparedLH toand FSH
those levels
given were found
a placebo to be
for their lower inThe
condition. mice that were
LH/FSH
ratiogiven
of DHEATC extracts
groups compared
was increasedto those
(2.87)given a placebo
as compared to for theirmice
normal condition.
(1.06) .The LH/FSH
In all TC
ratio of DHEA groups was increased (2.87) as compared to normal
extract treatment groups, the FSH was higher than LH. However, metformin treatment mice (1.06). In all TC
extract treatment groups, the FSH was higher
was slightly significant in reversing the effects of DHEA. than LH. However, metformin treatment was
slightly significant in reversing the effects of DHEA.
3.7. Lipid Profiles
3.7. Lipid Profiles
Figure 4A,B shows that the DHEA group had significantly higher levels of cholesterol
Figure 4A,B
and triglycerides thanshows that the
the control DHEA
group (p group
< 0.01).had significantly
Metformin higher
reduced levels
blood of cholesterol
cholesterol
and triglycerides than the control group (p < 0.01). Metformin reduced blood cholesterol
levels significantly (p < 0.01) in mice when administered at a dose of 250 mg/kg. All groups
levels significantly (p < 0.01) in mice when administered at a dose of 250 mg/kg. All groups
treated with TC extracts had significantly decreased serum cholesterol and triglyceride
treated with TC extracts had significantly decreased serum cholesterol and triglyceride
levels compared to the DHEA group (p < 0.01 to p < 0.001).
levels compared to the DHEA group (p < 0.01 to p < 0.001).
250
A
200 **
Cholesterol (g/L)
** ** ** **
** **
***
150
100
50
0
l l ) ) ) ) ) ) )
Co Co m m m m m m m
al A 50 00 00 00 00 00 00
rm E (2 (2 (4 (2 (4 (2 (4
No DH et tv
a
tv
a
O O HA
il
HA
il
M
A+ + Sa + Sa EA+ EA+ A+ A+
E E E
DH DH
250
B
200 ***
Triglycerides (g/L)
** * ** ** **
**
150 ***
100
50
0
)
)
)
)
ol
o l
kg
kg
kg
kg
kg
kg
kg
tr
tr
g/
g/
g/
g/
g/
g/
g/
on
on
m
m
m
C
00
00
50
00
al
EA
20
20
20
(4
(4
(2
(4
rm
(
(
(
DH
et
il
il
va
va
A
No
+O
+O
+H
+H
+M
at
at
EA
EA
+S
+S
EA
EA
EA
EA
EA
DH
DH
DH
DH
DH
DH
DH
FigureFigure 4. Effects
4. Effects of Tinospora
of Tinospora cordifolia
cordifolia extract
extract treatment
treatment in DHEA-induced
in DHEA-induced PCOSPCOS
mice mice on cholesterol
on choles-
terol (A)
(A) and
and triglyceride
triglyceride (B)
(B) levels.
levels. Every value is presented as a mean alongalong with
with standard
standard error.
error. Data
Datawere
wereanalyzed
analyzedby byANOVA
ANOVAfollowed
followedby DMCT.**pp<<0.05,
byDMCT. **pp<< 0.01,
0.05,** 0.01, and *** pp << 0.001.
and *** 0.001.
Nutrients 2023, 15, 2238 11 of 23
3.8. Insulin Profile

DHEA-treated mice had significantly increased insulin levels (Figure 5A) compared
3.8. Insulin Profile
to controls (p < 0.01). Serum insulin was significantly decreased (p < 0.001) in the TC-
DHEA-treated mice had significantly increased insulin levels (Figure 5A) compared to
treated group of DHEA-pretreated animals. Similarly, the HOMA-IR index (Figure 5B)
controls (p < 0.01). Serum insulin was significantly decreased (p < 0.001) in the TC-treated
wasgroup
highly significantly (p <animals.
of DHEA-pretreated 0.001) reduced
Similarly,by
theTC treatment
HOMA-IR in (Figure
index mice. Treatment with TC
5B) was highly
oil significantly
and HA extract (p < 0.001) reduced by TC treatment in mice. Treatment with TC oil and HAin the
failed to stimulate beta cells in DHEA-treated mice as indicated
HOMA-Beta
extract failedindex (Figure
to stimulate 5C).
beta cellsAindose-independent
DHEA-treated mice as effect of TC
indicated on HOMA-Beta
in the DHEA-induced
QUICKI (Figure5C).
index (Figure 5D)Awas observed andeffect
dose-independent was of highly
TC onsignificant
DHEA-induced(p < 0.01
QUICKIand(Figure
p < 0.001)
5D) with
was observed and was highly significant (p < 0.01 and p < 0.001) with HA
HA extract, oil, and satva compared to the negative control. However, the lower doses ofextract, oil, and
TCsatva compared
satva, oil, and toHAtheextracts
negativesignificantly
control. However, the lower
improved doses of TC satva, oil, and HA
QUICKI.
extracts significantly improved QUICKI.
2.0
A
Fasting Insulin (IU/mL)
1.5 **
1.0 **
**
*** ***
***
*** ***
0.5
0.0
l l g) g) g) g) g) g) g)
ro ro k k k k k k k
ont nt g/ g/ g/ g/ g/ g/ g/
lC Co m m m m m m m
a EA 50 00 00 00 00 00 00
rm (2 (2 (4 (2 (4 (2 (4
No DH e t
tv
a
tv
a
O
i l
O
i l
HA HA
M
A+ +Sa +Sa E A+ E A+ A+ A+
E E E
DH DH
0.8
B
0.6 ***
HOMA-IR
0.4
**
***
*** ***
0.2 *** ***
***
0.0
l l g) g) g) g) g) g) g)
ntro n tro g /k g /k g/k g/k g/k g/k g/k
Co Co m m m m m m m
al E A 2 50 2 00 4 00 2 00 4 00 2 00 4 00
r m ( ( ( ( ( ( (
No DH et va va il il A A
+M S at S at A+O A+O +H +H
A + + E E E A E A
E
DH DH
Figure 5. Cont.
Nutrients 2023, 2023,
Nutrients 15, x 15,
FOR PEER REVIEW
2238 12 of 23
12 of 23
8
C
HOMA-Beta 4 *
g)
g)
g)
g)
g)
g)
g)
ol
ol
tr
tr
k
k
k
g/
g/
g/
g/
g/
g/
g/
on
on
m
m
m
C
0
0
0
al
0
5
0
E
(2
(4
m
(2
(4
(2
(2
(4
H
or
va
va
et
il
il
A
D
+O
+O
N
+H
+H
+M
at
at
A
+S
+S
A
A
E
E
H
H
D
D
H
D
4
D
3
**
QUICK-I
2 **
* ***
*** ***
1 *
0
)
)
)
)
ol
ol
kg
kg
kg
kg
kg
kg
kg
tr
tr
g/
g/
g/
g/
g/
g/
g/
on
on
m
m
m
C
00
00
00
00
0
00
00
al
EA
(2
(4
m
(2
(4
(2
(2
(4
H
or
va
va
et
il
il
A
D
+O
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N
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+H
+M
at
at
EA
EA
+S
+S
EA
EA
EA
EA
EA
H
H
D
D
H
D
Figure
Figure 5. 5.
Effects Tinosporacordifolia
EffectsofofTinospora cordifolia extract
extract treatment
treatmentininDHEA-induced
DHEA-induced PCOS
PCOSmice
miceon fasting
on fasting
insulin levels (A), HOMA-IR (B), HOMA-Beta (C), and QUICKI levels (D).
insulin levels (A), HOMA-IR (B), HOMA-Beta (C), and QUICKI levels (D). Every value Every value is presented
is pre-
as a mean
sented along with
as a mean alongstandard error. Data
with standard wereData
error. analyzed
wereby ANOVAby
analyzed followed
ANOVA by DMCT.
followed * p by
< 0.05,
DMCT. *
p < 0.01,
p <**0.05, ** p and *** pand
< 0.01, < 0.001.
*** p < 0.001.
3.9. Ovarian Size and Weight
3.9. Ovarian
In thisSize andwe
study, Weight
used the right ovary of each mouse for morphological examina-
Inand
tion thisthe
study, we used
left ovary for the right ovary ofstudy.
histopathological each mouse for diameter
The ovary morphological
(Figureexamination
6B) of
DHEA-treated mice was substantially (p < 0.01) larger than that of the
and the left ovary for histopathological study. The ovary diameter (Figure 6B) ofcontrol group. This
DHEA-
demonstrates
treated mice was that ovarian hypertrophy
substantially (p < 0.01)islarger
brought about
than thatby
ofDHEA therapy.
the control The ovarian
group. This demon-
diameter
strates that was found
ovarian to be significantly
hypertrophy (p < 0.01)
is brought aboutreduced
by DHEA aftertherapy.
TC preparation therapy.
The ovarian diame-
Similar results were seen with metformin treatment. The weight of the ovaries, as shown
ter was found to be significantly (p < 0.01) reduced after TC preparation therapy. Similar
in Figure 6A, was also computed in this study. DHEA treatment significantly (p < 0.05)
results werethe
increased seen withofmetformin
weight the ovaries treatment.
compared toThe weight
control of Treatment
mice. the ovaries, as shown
of TC in Figure
for 21 days
6A, was also computed in this study. DHEA treatment significantly (p <
did not allow the weight of the ovaries to increase, and they maintained almost a normal0.05) increased
the weight of the ovaries compared to control mice. Treatment of TC for 21 days did not
allow the weight of the ovaries to increase, and they maintained almost a normal weight.
TC treatment reduced the DHEA-induced ovarian hypertrophy and ovarian weight very
significantly (p < 0.01).
Nutrients 2023, 15, 2238 13 of 23

weight. TC treatment reduced the DHEA-induced ovarian hypertrophy and ovarian weight
very significantly (p < 0.01).
80
A
*
Ovary weight (mg) 60
* *
** **
40 **
20
)
)
)
ol
o l
kg
kg
kg
kg
kg
kg
kg
tr
tr
g/
g/
g/
g/
g/
g/
g/
on
on
m
m
m
C
00
00
50
00
00
al
0
E
(2
(4
(2
(4
(2
4
m
(
H
or
et
il
il
va
va
A
D
+O
+O
+H
+H
N
+M
at
at
EA
EA
+S
+S
A
EA
E
A
H
H
D
D
H
D
4
** B
3 *
Ovary size (mm)
* *
* **
**
**
2
0
)
)
)
g)
)
ol
ol
kg
kg
kg
kg
kg
kg
tr
tr
/k
g/
g/
g/
g/
g/
g/
on
on
g
m
m
m
m
C
00
00
00
00
50
00
00
al
EA
(2
(4
m
(2
(4
(2
(2
(4
H
or
va
et
il
il
A
D
tv
+O
+O
N
+H
+H
M
at
a
+
EA
EA
+S
+S
EA
EA
EA
EA
EA
H
H
D
D
H
D
Figure
Figure The effect
6. effect
6. The that treatment
that treatment with with Tinospora
Tinospora cordifolia
cordifolia extracts
extracts had onhadovary
on ovary weight
weight (A) and
(A) and
size (B).
size Every valuevalue
(B). Every is presented as a as
is presented mean alongalong
a mean withwith
standard error.error.
standard Data Data
werewere
analyzed by by
analyzed
ANOVA followed by DMCT. * p < 0.05, ** p <
ANOVA followed by DMCT. * p < 0.05, ** p < 0.01.0.01.
3.10.3.10. Morphological
Morphological Observation
Observation of Ovaries
of Ovaries
Observation
Observation of theofovaries
the ovaries with
with the the naked
naked eye showed
eye showed that thethat
DHEAthe DHEA treatment
treatment in-
creased the ovary volume and weight and led to the appearance of black spots and gran- and
increased the ovary volume and weight and led to the appearance of black spots
ular granular
changes, changes, as demonstrated
as demonstrated in Figure in Figure 7. Treatment
7. Treatment with metformin
with metformin reduced thereduced
black the
spots and granulation, but not as good as in the TC-treated groups. Treatment of mice of
black spots and granulation, but not as good as in the TC-treated groups. Treatment
withmice with and
TC satva TC satva
oil in and oil in DHEA-induced
DHEA-induced PCOSinresulted
PCOS resulted normalin normalTreatment
ovaries. ovaries. Treatment
with
with TC HA extract also significantly reduced the alterations in the appearance
TC HA extract also significantly reduced the alterations in the appearance of ovaries. of ovaries.
Nutrients 2023, 15, 2238 14 of 23
(A) Normal control (B) DHEA control (C) DHEA+Met (250 mg/kg)
(D) DHEA+Satva (200 mg/kg) (E) DHEA+Satva (400 mg/kg) (F) DHEA+Oil (200 mg/kg)
(G) DHEA+Oil (400 mg/kg) (H) DHEA+HA (200 mg/kg) (I) DHEA+HA (400 mg/kg)
Figure 7. Effects of Tinospora cordifolia extract treatment in DHEA-induced PCOS mice on morpho-
7. Effects of Tinospora
Figure examination
logical cordifolia
of the ovaries. extract treatment
Histological analysisinofDHEA-induced PCOS micewith
normal ovaries compared on morpholog-
PCOS and
ical examination of the ovaries. Histological analysis of normal ovaries compared
ovaries treated with TC preparations. (A) Normal control, (B) DHEA treated, (C) positive with PCOS and
control
ovaries by
treated treated with TC
metformin, preparations.
(D) TC satva 200(A) Normal
mg/kg control,
treated, (B) DHEA
(E) TC treated,
satva 400 mg/kg(C) positive
treated, (F)control
TC oil
treated
200 by treated,
mg/kg metformin,
(G) TC (D)oilTC
400satva
mg/kg 200treated,
mg/kg(H) treated,
TC HA(E) 200TC satvatreated,
mg/kg 400 mg/kg
(I) TCtreated, (F) TC
HA 400 mg/kg
treated.
oil 200 mg/kg treated, (G) TC oil 400 mg/kg treated, (H) TC HA 200 mg/kg treated, (I) TC HA
400 mg/kg treated.
3.11. Histopathological Examination of the Ovaries
3.11. Histopathological Examination of the Ovaries
As presented in Figure 8, oocytes were found in the normal control group’s ovary
As presented
follicles. These oocytesin Figure 8, oocytes were
were surrounded found
by theca inand
cells the granulosa
normal control group’s
cells. Theca ovary
cells are
follicles. These oocytes were surrounded by theca cells and granulosa
polarized mesenchymal cells having a spindle-like form and typically have two to threecells. Theca cells
are polarized
layers mesenchymal
of thickness. These cellscells
werehaving
presenta outside
spindle-like
of theform and typicallybasal
follicle-associated havelamina.
two to
An immature interstitial compartment was found outside of the theca layer. The cellsbasal
three layers of thickness. These cells were present outside of the follicle-associated that
lamina.
made upAnthisimmature interstitial
compartment compartment
were formed was found outside
from steroidogenic of thethat
theca cells theca
hadlayer.
been The
left
cells from
over that made
atreticupfollicles.
this compartment were formed
After TC therapy, from steroidogenic
PCOS-induced mice ovaries theca cells that
showed had
a larger
number of cystic follicles and fewer preantral follicles, corpus luteum, stomal nodules,
Nutrients 2023, 15, 2238 15 of 23
been left over from atretic follicles. After TC therapy, PCOS-induced mice ovaries showed
astromal
larger hyperplasia,
number of cystic follicles
and antral and fewer
follicles preantral
compared follicles, In
to controls. corpus luteum,
addition, stomal
during the
nodules, stromal hyperplasia, and antral follicles compared to controls.
ovarian histopathology study after TC treatment, there were no corpora lutea present, In addition, during
the ovarian
which histopathology
is proof that ovulation study
had after TC treatment,
not taken place. Ovarythere were no
sections corpora
from lutea present,
a TC-satva-treated
which is proof that ovulation had not taken place. Ovary sections from a
mouse demonstrated the presence of a medulla, a follicular cyst, and a Graafian follicle. TC-satva-treated
mouse
Medulla, demonstrated the presence
cortex, and follicular cystsofwere
a medulla, a follicular
seen in the cyst, and a group.
metformin-treated Graafian follicle.
When the
Medulla, cortex, and follicular cysts were seen in the metformin-treated
organ was cut transversely, the components of the normal control group’s ovary could be group. When the
organ was cut
seen, each withtransversely, the components
a clearly defined structure.ofThethe cortex
normalofcontrol group’s
the ovary ovary different
revealed could be
seen, each with a clearly defined structure. The cortex of the ovary revealed different stages
stages of follicle development. There were unruptured primordial follicles, as well as pri-
of follicle development. There were unruptured primordial follicles, as well as primary
mary follicles, secondary follicles, and Graafian follicles. The blood arteries were normal,
follicles, secondary follicles, and Graafian follicles. The blood arteries were normal, and the
and the surface epithelium was bordered with low cuboidal to flattened cells. The stroma
surface epithelium was bordered with low cuboidal to flattened cells. The stroma is made
is made up of theca cells that range in shape from round to spindle. The mice that were
up of theca cells that range in shape from round to spindle. The mice that were given DHEA
given DHEA showed stromal nodules and follicular cysts coupled with infrequent Graaf-
showed stromal nodules and follicular cysts coupled with infrequent Graafian follicles.
ian follicles. Additionally, these mice had multiple follicular cysts linked with hyperplasia
Additionally, these mice had multiple follicular cysts linked with hyperplasia of theca cells.
of theca cells. The follicular cysts, medulla, and Graafian follicles were all visible in the
The follicular cysts, medulla, and Graafian follicles were all visible in the ovaries of mice
ovaries of mice that had been treated with TC oil. The ovary of a mouse that had been
that had been treated with TC oil. The ovary of a mouse that had been treated with TC
treated with TC hydroalcoholic extract exhibited a medulla, cortex, and follicular cyst.
hydroalcoholic extract exhibited a medulla, cortex, and follicular cyst. Because of this, the
Because suggest
findings of this, the
thatfindings
there wassuggest that there was
an improvement an improvementsymptoms
in histopathological in histopathological
of the mice
symptoms of the mice that were given TC satva and oil as a treatment
that were given TC satva and oil as a treatment and that these mice ovulated. and that these mice
ovulated.
A B C
D E F
Figure 8. Cont.
Nutrients 2023, 15, 2238 16 of 23
G H I
G H I
Figure 8. Effects of TC extracts on histopathology of DHEA-induced PCOS ovaries in mice. Com-

Figure 8. Effects of TC extracts on histopathology of DHEA-induced PCOS ovaries in mice. Compar-
parative histological examination of normal and PCOS ovarian tissue, as well as ovarian tissue that
Figure
ative 8. Effects of
histological TC extractsofonnormal
histopathology ofovarian
DHEA-induced PCOS ovaries intissue
mice.that
Com-
was treated with examination
TC formulations. and PCOS
Hematoxylin and tissue, staining,
eosin (H&E) as well as10×
ovarian
magnification, was
100
parative
treated histological
with TC examination
formulations. of normal
Hematoxylin and
and PCOS
eosin ovarian
(H&E) tissue,
staining,as well
10 × as ovarian
magnification, tissue
100 that
μM scale, as detailed in Section 2. (A) The untreated normal control, (B) the treated positive con-µM
was treated with TC formulations. Hematoxylin and eosin (H&E) staining, 10× magnification, 100
scale, as detailed
trol with DHEA, in (C)Section 2. (A)positive
the treated The untreated
controlnormal control, (B)
with metformin, the(D–I)
and treated positive
mice treatedcontrol with
with TC
μM scale, as detailed in Section 2. (A) The untreated normal control, (B) the treated positive con-
satva 200,
DHEA, (C)TCthesatva 400,positive
treated TC oil 200, TC oil
control 400,
with TC HA 200,
metformin, andand TC HA
(D–I) mice400 mg/kg,
treated respectively.
with AF
TC satva 200,
trol with DHEA, (C) the treated positive control with metformin, and (D–I) mice treated with TC
is short
TC satva for
400,antral
TC follicle,
oil 200, FCoil
TC stands
400, for HA
TC cystic follicle,
200, and TC CLHAstands
400 for corpus
mg/kg, luteum, GC
respectively. AF refers
is to for
short
satva 200, TC satva 400, TC oil 200, TC oil 400, TC HA 200, and TC HA 400 mg/kg, respectively. AF
granulosa
antral cell,FC
PFstands
stands forcystic
primary follicle,
CL and SF for
stands for secondary follicle.
is shortfollicle,
for antral follicle,for
FC standsfollicle,
for cystic stands
follicle, CLcorpus
standsluteum,
for corpusGC refers toGC
luteum, granulosa
refers tocell,
PF stands for primary follicle, and SF stands for secondary follicle.
granulosa cell, PF stands for primary follicle, and SF stands for secondary follicle.
3.12. Severity Score Measurement
3.12. In
Severity Score study,
this 21-day Measurement
we estimated the severity score for PCOS (Figure 9). The severity
3.12. Severity Score Measurement
In this 21-day
in DHEA-treated study, we estimated thethan
severity
in thescore for PCOS (Figure 9). Thedecrease
severity
In this 21-daymice was
study, wehigher (54.86)
estimated the severity control.
score A highly
for PCOS significant
(Figure 9). The severity
in DHEA-treated
in DHEA-treated mice
severity was observed was higher
with (54.86) than
TC preparation in the control. A highly significant decrease
in mice was higher (54.86) thantreatment. The A
in the control. results
highlywith metformin
significant treat-
decrease
in severity
ment were was
not observed
more with TC
significant. preparation
Treatment of treatment.
mice with TC The results
satva and with
oil in metformin
DHEA-in-
in severity was observed with TC preparation treatment. The results with metformin treat-
treatment
ducedwerePCOSwere not more
resulted significant.
in highly Treatment
reduced ofTreatment
severity. mice with with
TC satvamg/kg
and oil in HA
DHEA-
ment not more significant. Treatment of mice with TC satva400and oil in TC
DHEA-in-ex-
induced
tract also PCOS resulted
significantly in highly
reduced the reduced
severity severity. Treatment with 400 mg/kg TC HA
score.
duced PCOS resulted in highly reduced severity. Treatment with 400 mg/kg TC HA ex-
extract also significantly reduced the severity score.
tract also significantly reduced the severity score.
60 54.86
60 54.86
50
50
scorescore
40
40
Severity
30
Severity
30
20 14.37 15.81
11.57
20 14.37 6.99 15.81 7.51
10 3.11 2.69 11.57 3.15
6.99 7.51
100 3.11 2.69 3.15
Normal Let Let +Met Let Let Let +Oil Let +Oil Let +HA Let +HA
0
control Control (250 +Satva +Satva (200 (400 (200 (400
Normal Let Let +Met Let Let Let +Oil Let +Oil Let +HA Let +HA
mg/kg) (200 (400 mg/kg) mg/kg) mg/kg) mg/kg)
control Control (250 +Satva +Satva (200 (400 (200 (400
mg/kg) mg/kg)
mg/kg) (200 (400 mg/kg) mg/kg) mg/kg) mg/kg)
mg/kg) mg/kg)
Figure 9. The effect that treatment with Tinospora cordifolia extracts had on severity score for PCOS.
4. Discussion
4. Metabolic instability is commonly connected with polycystic ovarian syndrome, a
4. Discussion
Discussion
common reproductive condition.
Metabolic Insulinconnected
resistance,with
diabetes mellitus, and dyslipidemia
Metabolic instability
instability is commonly
is commonly connected with polycystic
polycystic ovarian
ovarian syndrome, aa
syndrome,
are all conditions that are more likely to occur in women with
common reproductive condition. Insulin resistance, diabetes mellitus, and PCOS. Obesity, insulin
dyslipidemia
resistance,
are genetic that
all conditions predisposition, inflammation,
are more likely to occur in and oxidative
women withstress are Obesity,
PCOS. just someinsulin
of the
resistance, genetic predisposition, inflammation, and oxidative stress are just some ofwith
theories proposed to explain PCOS [29]. PCOS is a complicated illness, and women the
theories proposed to explain PCOS [29]. PCOS is a complicated illness, and women with
Nutrients 2023, 15, 2238 17 of 23
resistance, genetic predisposition, inflammation, and oxidative stress are just some of the
theories proposed to explain PCOS [29]. PCOS is a complicated illness, and women with it
often experience ovarian and neuroendocrine dysfunction [30,31]. Insulin has been shown
to increase androgen release from ovarian stromal and thecal cells in in vitro studies [24].
Hyperandrogenism is caused by an increase in ovarian androgen production due to insulin
resistance and hyperinsulinemia in women with polycystic ovary syndrome. Although the
exact cause of PCOS in women is unknown, its primary characteristics can be replicated in
a mouse model using DHEA [32–34].
Being a potent anti-inflammatory herbal drug, it was anticipated that TC extracts
would help in improving insulin balance and decrease the risk of developing ovarian
cysts. As an agent for rejuvenation, it may stimulate all the body tissues and improve
metabolism naturally [12]. We examined the effects of TC extracts on body weight, ovarian
weight, insulin sensitivity, and ovarian shape. In this study, DHEA was given at a dose of
6 mg/100 g/day for 21 days to mimic the reproductive characteristics matching the PCOS
condition. Increases in body weight, ovarian weight, glucose, LH, testosterone, and insulin
resistance were all indicators of compromised reproductive health in DHEA-treated groups
compared to controls. It was found that the extracts restored normal body weight, reduced
PCOS-associated dyslipidemia, and reduced insulin resistance. It also regulated the level of
hormones such as LH, FSH, estradiol, progesterone, and testosterone. The morphological
and histopathological studies proved that the extracts inhibit changes in the cellular, tissue,
and organ systems of the body.
In this study, the PCOS group animals’ estrous cycles (consisting of estrous, proestrus,
metestrus, and diestrus phases) were significantly arrested and delayed, with the diestrus
phase lasting significantly longer than the other three [35], similar to our study results
where most vaginal smear cells of negative control groups had leukocytes. The normal
control group (Group I) showed all phases of a normal estrous cycle, i.e., estrous, proestrus,
metestrus, and diestrus. Group III (Metformin) remained in the diestrus phase for a
long period. Groups IV, V, VI, VII, VIII, and IX exhibited the estrous phase of the cycle
having cornified epithelial cells. Restoring normal circulation concentrations of testosterone,
estrogen, progesterone, and gonadotrophins may be related to TC’s restorative impact on
the mouse estrous cycle, as presented in this study. Ovarian functions, such as follicular
maturation and hormonal imbalance, are believed to be normalized by a regular estrous
cycle, which is regulated by these hormones [36].
On day 21, DHEA-treated mice had increased body weight compared to the normal
control group. PCOS and weight gain were associated with DHEA injections. There is a lot
of evidence linking PCOS and being overweight [27]. In this study, the BW of mice treated
with DHEA was significantly increased due to abdominal fat compared to the normal
mice [37]. An increase in growth hormone production from the anterior pituitary gland,
which in turn stimulates the release of insulin-like growth factors (IGFs) I and II, may have
occurred due to the treatment’s effect on the metabolic pathway [38]. This hormone is
essential for promoting the cellular absorption of amino acids, regulating lipolysis, and
promoting skeletal muscle development. This confirms what previous studies found, which
was a considerable rise in female mice’s body weight through increased protein synthesis
and slowed protein breakdown with the help of IGF I and II. In contrast, satva significantly
lowered the BW of mice by the process of restoration when compared to the control group
of animals. The treatment group receiving 400 mg/kg of TC oil and HA extract also lost
significant extra weight that was gained due to DHEA (p < 0.01). After 21 days of therapy,
the mice’s body weight had approximately reached that of the normal mice. This result
agrees with what has been seen before because TC treatment controls cellular homeostasis,
energy metabolism, loss of lipids, and hypoglycemia [13,37].
On day 1, blood glucose levels were normal in all groups. On day 21, the experimental
animals in the DHEA-only-treated group had raised blood glucose levels compared to
the normal control group. This study’s findings that DHEA-induced PCOS mice exhibit
hyperglycemia are in line with those of earlier research [39]. Compared to a DHEA control
Nutrients 2023, 15, 2238 18 of 23
group, mice given TC extracts had significantly reduced blood glucose levels. Treatment
with TC satva and oil resulting in hypoglycemia may be due to the activation of glucose
6-phosphatase and normalizing levels of liver glycogen, suppression of oxidative stress,
inhibition of gluconeogenesis, and inhibition of glycogenolysis [40]. Reported insulin-
mimicking and insulin-releasing actions, insulin secretion, glucose uptake, and suppression
of peripheral glucose release in hyperglycemia have been found in TC’s isoquinoline
alkaloids, including palmatine, jatrorrhizine, and magnoflorine [41,42].
On day 20, 1 h OGTT of TC satva and oil treatment raised glucose levels compared
to 0 h OGTT of TC satva and oil, but it was normal after 2 h OGTT of TC satva and
oil. Moreover, 1 h OGTT of the DHEA-treated group was increased as compared to 0 h
OGTT of the DHEA group, but after 2 h, there was a highly significant rise in blood
glucose levels. This shows that there was an insufficient amount of insulin released upon
loading the glucose. To combat diabetes, decreasing hepatic phosphorylase activity and
increasing glucose absorption by peripheral tissues and organs, including the liver, may be
effective [43], which could be the mechanism of action of TC preparations.
Estradiol and progesterone levels were comparatively lower in the negative control
group compared to normal controls. Low progesterone levels cause anovulation. Higher
plasma androgen and lower estrogen and progesterone concentrations have been observed
in PCOS patients [9]. Hyperandrogenism is caused by the overproduction of ovarian
androgens due to insulin resistance with hyperinsulinemia in PCOS females. It is believed
that IGF-1 receptors on theca and stroma cells are the mechanism through which insulin
regulates ovarian androgen production [44]. The growth hormone IGF-1 promotes granu-
losa cell aromatase production [45] and works in concert with FSH and LH, regulating the
production of aromatase in these cells [24]. In addition, raising androgen levels can increase
FSH receptor levels in PCOS patients, leading to a reduction in FSH serum levels via
negative feedback. Studies have linked PCOS to insulin resistance due to abnormally high
LH or low FSH levels. Both testosterone and LH levels rise when the normal hypothalamic–
pituitary–gonadal axis is disrupted, which is what causes the condition [46]. LH decreases
progesterone levels and increases androgen levels by stimulating testosterone release from
theca cells via the PI3K/Akt pathway. This oversupplies the activity of the 17-a hydroxylase
enzyme, catalyzing the conversion of progesterone to androgens [47]. All TC treatment
groups saw a decrease in serum testosterone, but the highest doses of satva and oil brought
it down to nearly normal levels. Metformin treatment counteracted the estrogenic effects
of DHEA. Maximum satva, oil, and HA extracts significantly increased estradiol levels
relative to the DHEA-treated group. All treatments with TC extracts resulted in a significant
increase in serum progesterone relative to the placebo group. DHEA treatment led to a
greater increase in levels of LH and FSH in mice than in the controls. LH and FSH levels
were found to be lower in mice given TC extracts compared to those given a placebo
for their condition. The LH/FSH ratio of DHEA groups was increased as compared to
normal and TC extract treatment groups. In all TC extract treatment groups, the FSH was
higher than LH. However, metformin treatment slightly significantly reversed the effect
of DHEA. The ratio of LH to FSH dropped significantly after treatment with satva, oil, or
hydroalcoholic extracts. The hormone levels of LH and FSH were restored to normal with
the use of TC extracts.
Regular treatment of mice with DHEA led to an increase in plasma cholesterol and
triglycerides compared to Group I (p < 0.01). Biochemical analysis of PCOS patients reveals
dyslipidemia, characterized by a lipid profile that is out of whack: low HDL, higher
triglycerides (TG), higher total cholesterol, and higher low-density lipoprotein (LDL) [9].
Dyslipidemia, characterized by increased TGs and decreased HDL cholesterol, is commonly
observed in PCOS. Dyslipidemia associated with PCOS does not discriminate by weight.
The syndrome is associated with a considerable increase in the risk of developing type II
DM due to the synergistic effects of obesity and insulin resistance. Patients with PCOS
may experience dyslipidemia due to a variety of factors. IR has a significant role by
increasing lipolysis and altering the production of hepatic lipase and lipoprotein lipase.
Nutrients 2023, 15, 2238 19 of 23
There were significant reductions in blood cholesterol and TG concentrations in the TC

treatment animals compared to the DHEA control group. The most interesting result was
the normalization of TG and cholesterol levels in the animals treated with TC. Previous
research has revealed that the lipid-lowering characteristics of TC’s active components,
including its alkaloids, glycosides, flavonoids, saponins, and tannins, may be responsible
for the regulated BW and lipid profile [13].
DHEA-treated mice exhibited hyperinsulinemia, a marker of insulin resistance, in
contrast to the normal control group. Similar to the hyperinsulinemia/hyperandrogenism
combination seen in PCOS, insulin resistance can significantly raise levels of circulating
proinflammatory cytokines [48]. The symptoms of PCOS, including elevated levels of
androgens and irregular or absent menstrual cycles, are likely caused by insulin resistance,
which also raises the risk for hyperlipidemia, diabetes mellitus, and cardiovascular dis-
ease [49]. Hence, decreasing insulin resistance could be the most important step, and it
may also improve reproductive health [24]. In mice treated with DHEA, the administration
of TC resulted in a significant decline in blood insulin levels, demonstrating a reversal of
insulin resistance. DHEA caused a rise in the HOMA-IR index, which was then reversed
by TC therapy. A highly significant reduction in serum insulin levels was observed after
TC treatment in the DHEA-pretreated group of mice. Similarly, the HOMA-IR index was
reduced by TC treatment in mice. A dose-independent effect of TC on DHEA-induced
QUICKI was observed with HA extract, oil, and satva compared to the negative control.
However, the lower doses of TC satva, oil, and HA extracts significantly improved QUICKI.
The mechanism whereby the active components of TC increase muscle glucose absorption
is supported by the Insulin Sensitivity Index QUICKI. Treatment with TC oil and HA
extracts demonstrated a dose-dependent improvement in the activity of beta cells, reflected
in the HOMA-Beta values [50]. These findings are consistent with earlier findings that TC
reduced fructose-induced increases in blood sugar, insulin, and triglycerides. The berberine
alkaloid, discovered in the TC plant stem, has been shown to significantly enhance glucose
tolerance. Treatment with berberine significantly reduces fasting plasma glucose, improves
insulin sensitivity, and decreases LDL-C in type II diabetic rats, demonstrating its potent
antihyperglycemic and hypolipidemic effects. The expression of GLUT4 in tissues was
significantly increased after being exposed to berberine. Decreased expression of muscle
and adipose GLUT4 in diabetic persons is one of the most important reasons for IR, since
it is well known that the absorption of glucose into cells depends on the help of mem-
brane GLUT4. In IR animal models, berberine was found to improve insulin sensitivity by
activating AMP-activated protein kinase [51].
In silico investigations found that the alkaloids syringin, berberine, and rumphioside-I
significantly inhibited IRS1 and IRS2 receptors by acting as antagonistic ligands. Isoquino-
line alkaloids (such as berberine and palmatine) play significant roles in warding off a
wide range of illnesses. Virtual screening has been used to evaluate the efficacy of an
alkaloid extracted from TC stems for the treatment of PCOS [14]. Berberine improved IR
and blood hormone levels in PCOS. Berberine therapy also restored ovarian apoptosis and
morphological abnormalities. Berberine’s effects on the proliferation of granulosa cells and
death were mitigated by blocking the PI3K/AKT pathway [15]. After extensive animal
and human studies, it is possible that these drugs may be beneficial in the management of
PCOS [16].
In this study, we used the right ovary of each mouse for morphological examination
and the left ovary for histopathological study. The diameter of the ovaries of DHEA-treated
mice was higher than that of the control group. This shows that DHEA treatment induces
hypertrophy of ovaries. Mice given TC oil that had PCOS induced by DHEA appeared to
recover to normal health. After treating PCOS mice with TC, follicle numbers were restored
and the ovaries were protected from oxidative stress, as was previously described [52].
The weight of the ovary was also computed in this study. DHEA treatment increased
the weight of the ovary compared to the normal control group. Consistent with prior
observations, a rat model of PCOS induced by DHEA showed that both the animals and
Nutrients 2023, 15, 2238 20 of 23
the ovaries grew in size [7]. Ovary weight was nearly twice that of the control group after
DHEA therapy [36]. Treatment of TC for 21 days did not allow the weight of the ovary
to increase and it maintained almost a normal weight. TC treatment reduced the DHEA-
induced ovarian hypertrophy and ovarian weight. These are in line with the study in
which supplementation with TC extracts has been shown to significantly reduce the risk of
morphological abnormalities in the ovaries due to radiation exposure [52]. Hypertrophy, as
well as the formation of black patches and granular alterations, were observed in previous
research, and these may be seen with the naked eye after DHEA treatment [50]. Treatment
with metformin reduced the black spots and granulation but not as well as in TC-treated
groups. Treatment of mice with DHEA-induced PCOS using TC satva and oil resulted in
normal ovaries. Treatment with TC HA extract also significantly reduced the alteration in
the appearance of ovaries. These results are in line with the study in which supplementation
with TC extract significantly reduced the risk of morphological abnormalities in the ovaries
due to radiation exposure [52].
Cystic follicles, as opposed to growing follicles and the corpus lutea, are more prevalent
in the ovaries of the PCOS group, suggesting that physiological activity in the ovaries is
being inhibited in this population. There was a marked improvement in cyst reduction,
and the number of Graafian follicles and corpus lutea were considerably decreased in
the disease control group of mice compared to the normal group. The Graafian follicular
numbers were considerably higher than the PCOS group, suggesting enhanced ovarian
function. The rate of corpus luteum formation was considerably greater for the TC group.
Primitive follicle counts in the ovaries of the therapy groups were, thus, considerably
greater than in the PCOS group. Finally, the number of oocytes was considerably reduced
in the PCOS group. The total number of oocytes produced followed a generally rising trend
across all treatment groups. Polycystic ovarian syndrome is associated with anovulation
due to increased androgen levels. Follicular atresia is thought to be caused by androgens
binding to cell receptors and causing cell death. The oocytes in the follicles degenerate as a
result of androgens, which increase the number of pyknotic granulosa cells.
5. Conclusions
The results of the study show that mice with DHEA-induced PCOS treated with TC
preparations as nutritional supplements maintained a normal estrous cycle and gained a
normal amount of weight. TC satva and oil were found to be the most effective in lowering
blood sugar levels, OGTT, testosterone, LH/FSH ratio, lipid profile, insulin resistance, and
ovary diameter and weight, and reducing granular changes and disease severity. Treatment
of PCOS mice with TC satva and oil increased the levels of estrogen and progesterone, the
number of follicles, insulin sensitivity, and FSH. Based on these results, we conclude that
TC preparations as nutritional supplements may have beneficial effects in the treatment
and management of PCOS-associated conditions and complications. Further studies may
be required to establish the molecular mechanism of action of TC extracts for generalization
and possible clinical applications.
Author Contributions: Conceptualization, H.R.C. and R.R.; methodology, H.R.C., R.R., N.K. and P.P.;
software, R.R. and N.K.; validation, R.R. and H.R.C.; formal analysis, R.R. and H.R.C.; investigation,
H.R.C., R.R., N.K. and P.P.; resources, H.R.C.; data curation, R.R.; writing—original draft preparation,
R.R. and H.R.C.; writing—review and editing, writing—review and editing, B.A.A.-W., M.M.K.,
M.S.H. and M.B.B.; visualization, H.R.C.; supervision, H.R.C.; project administration, H.R.C.; funding
acquisition, B.A.A.-W., M.M.K. and M.S.H. All authors have read and agreed to the published version
of the manuscript.
Funding: The APC was funded by Najran University, Najran, Saudi Arabia (NU/RG/MRC/12/13).
Institutional Review Board Statement: The research protocol was approved by the Institutional
Animal Ethics Committee (DITU/IAEC/21-22/07-06, 10 July 2021).
Informed Consent Statement: Not applicable.
Nutrients 2023, 15, 2238 21 of 23
Data Availability Statement: All data generated in the study are provided in the manuscript. No
extra data are available.
Acknowledgments: The authors are thankful to the Deanship of Scientific Research at Najran Uni-
versity for funding this work under the Research Groups Funding program (Grant code NU/RG/
MRC/12/13).
Conflicts of Interest: The authors declare no conflict of interest.
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Nutrients 2023, 15, 2238. https://doi.org/10.3390/nu15102238 https://www.mdpi.com/journal/nutrients

2. Materials and Methods

2.2. Reagents and Chemicals

2.4. Preparation of Hydroalcoholic Extract

2.5. Formulation of Guduchi Satva

2.6. Isolation of Essential Oil

2.7. Animal Handling and Groups

2.8. Establishment of PCOS Model

2.9. Estrous Cycle Monitoring and Vaginal Smear Test

2.10. Change in Body Weight

2.12. Blood Collection, Collection of Plasma, and Detection of Biochemical Indexes

2.13. Measurement of Weight, Size, and Morphological Changes in the Ovaries

2.14. Histological Examination

2.15. Severity of PCOS

PCOS Severity Score = ( Presence or Absence o f Oocyte + Number o f Cystic f ollicles)×

where the presence of oocytes = 0 and the absence of oocytes = 1.

2.16. Statistical Analysis

3.3. Fasting Blood Glucose Level

Nutrients 2023, 15, 2238 8 of 23

3.3. Fasting Blood Glucose Level

Normal control DHEA Control DHEA +Met (250 mg/kg)

3.6. LH and FSH Level

Nutrients 2023, 15, 2238 11 of 23

3.8. Insulin Profile

Nutrients 2023, 15, x FOR PEER REVIEW 13 of 23

Nutrients 2023, 15, x FOR PEER REVIEW 16 of 23

Figure 8. Effects of TC extracts on histopathology of DHEA-induced PCOS ovaries in mice. Com-

There were significant reductions in blood cholesterol and TG concentrations in the TC

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