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Role of ashwagandha methanolic extract in the regulation of thyroid profile in

hypothyroidism modeled rats

Article  in  Molecular Biology Reports · August 2019

DOI: 10.1007/s11033-019-04721-x

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Molecular Biology Reports (2019) 46:3637–3649
https://doi.org/10.1007/s11033-019-04721-x

ORIGINAL ARTICLE

Role of ashwagandha methanolic extract in the regulation of thyroid

profile in hypothyroidism modeled rats
Khaled G. Abdel‑Wahhab1   · Hagar H. Mourad1 · Fathia A. Mannaa1   · Fatma A. Morsy2   · Laila K. Hassan3   ·
Rehab F. Taher4

Received: 13 December 2018 / Accepted: 27 February 2019 / Published online: 15 June 2019
© Springer Nature B.V. 2019

Abstract
This study aimed to evaluate the anti-hypothyroidism potential of ashwagandha methanolic extract (AME). This target was
performed through induction of animal model of hypothyroidism by propylthiouracil. After 1 month from treatments, blood
samples were collected for biochemical determinations, and liver and kidney were removed for the determination of oxida-
tive stress markers and thyroid gland was removed for histopathological examination. The total phenolic compounds in the
extract and the in vitro radical scavenging activity of extract were also determined. The results revealed that the induction of
hypothyroidism by propylthiouracil induced a significant increase in serum TSH level but it induced significant decreases in
the levels of total T3, free T3, free T4, and total T4 hormones compared with the control values. Also, serum glucose, Il-6,
and body weight gain increased significantly while Il-10 and blood hemoglobin levels showed significant decrease. Induc-
tion of hypothyroidism increased also the levels of hepatic and renal MDA and NO and decreased significantly the values
of GSH, GPx and ­Na+/ ­K+-ATPase. Both AME and the anti-hypothyroidism drug significantly ameliorated the changes
occurred in the levels of the above parameters and improved histological picture of thyroid gland but with different degrees;
where ashwagandha methanolic extract showed the strongest effect. We can conclude that ashwagandha methanolic extract
treatment improves thyroid function by ameliorating thyroid hormones and by preventing oxidative stress.

Keywords  Hypothyroidism · Ashwagandha · Antioxidant · Histopathology

* Khaled G. Abdel‑Wahhab Introduction

kg.el‑deen@nrc.sci.eg; kgm194@yahoo.com
Hagar H. Mourad Thyroid disease constitutes the most common endocrine
hagarhassan@nrc.sci.eg disorder, because it is associated with various metabolic
Fathia A. Mannaa abnormalities. Thyroid hormones, thyroxine (T4), and trii-
fa.elwahid@nrc.sci.eg odothyronine (T3) play an important role in all major meta-
Fatma A. Morsy bolic pathways. Thyroid hormones regulate the basal energy
fa.adly@nrc.sci.eg expense through their direct effect on carbohydrate, protein
Laila K. Hassan and lipid metabolism or through their indirect effect on other
lailakh@nrc.sci.eg regulatory hormones such as insulin or catecholamines [1].
Rehab F. Taher Thyroid hormones affect also all aspects of lipid metabolism
Rf.farid@nrc.sci.eg including synthesis, mobilization and degradation [2, 3].
Hypothyroidism occurs when the thyroid gland fails to
1
Medical Physiology Department, National Research Center, produce a sufficient amount of hormones to meet the meta-
Dokki, Cairo 12622, Egypt
bolic demands of the body. Thyroid hormones regulate glu-
2
Pathology Department, National Research Centre, Dokki, cose homeostasis, and hypothyroidism can have deep effects
Cairo 12622, Egypt
on glucose metabolism and insulin secretion [4, 5]. Hypo-
3
Dairy Department, National Research Centre, Dokki, thyroidism has been reported to be associated with many
Cairo 12622, Egypt
complications such as obesity and its wide disorders in chil-
4
Natural Compounds Chemistry Department, National dren and gestational diabetes in pregnancy [6].
Research Centre, Dokki, Cairo 12622, Egypt

13
Vol.:(0123456789)

3638
The identification of new drugs especially from natural
origin represents a worldwide important issue. Ashwagan-
dha (Withania somnifera) is an herbal plant that used in
traditional medicine [7, 8]. It belongings to family Solan-
aceae and has received considerable attention for its ben-
efits in chronic degenerative diseases including diabetes. The
extract has shown potential activities, namely, anticancer
[9], immunomodulating [10, 11], anti-inflammatory [11],
antihyperglycemic [12], and hypolipidemic activities [13].
Ashwagandha aqueous ethanolic extract constitute also an
important succor in the prevention of arthritis [14].
The prevalence of hypothyroidism and its complications
in Egypt is relatively high, especially in children; conse-
quently, there are national strategies for management of the
hypothyroidism that combined with other diseases, as well
as protect against their progressed associated complica-
tions. Therefore, this study was designed to investigate the
safety and therapeutic potential of a variety of ashwagandha
that was recently planted in Egypt and was processed by
an Egyptian company. This target was monitored through
extraction and identification of the active ingredients of the
plant, and using hypothyroidism modeled animals preface
for specialists to treat Egyptian patients.
Materials and methods
Materials
Animals
Male albino rats (their weights raged between 150 and 200 g)
were obtained from the animal colony, National Research
Centre. These animals were maintained on free access to
food and water for a week before starting the experiment for
acclimatization. All animals received human care in compli-
ance with the standard institution’s criteria for the care and
use of experimental animals according to ethical committee
of National Research Centre. This study was approved by
the ethical committee of National Research Centre (FWA
00014747).
Plant materials
Ashwagandha roots powder was obtained from Imtenan
Health Shop, Industrial Area, Obour City, Cairo, Egypt. The
herb was identified by special herbalists at botany depart-
ment, National Research Centre.
Chemical and drugs
Eltroxin™ (Thyroxin tablets) and Thyrocil® (Propylthi-
ouracil Tablets) were obtained from GlaxoSmithKline Co,
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Molecular Biology Reports (2019) 46:3637–3649
Bomerange Building, 46  N 90th Street service Ln, new
Cairo City, Cairo, Egypt. All other chemicals were of the
highest analytical grade available.
Methods
Herb extraction
The herbal root was dried, grinded and soaked in specific
solvents (water or methanol) for extraction process. After
filtration, Rotary apparatus was used for evaporation of the
methanolic-filtrates, while freeze drier was used for lypholy-
zation of the aqueous filtrates; then the yield percentage was
calculated as gram (extract)/100 g (crude powdered herb).
We examined, then, the in vitro the antioxidant activities
of the aqueous and methanolic extracts prepared from the
tested plant.
Determination of total phenolic compounds
Five mg of each extract were dissolved in a 10 ml mixture of
acetone and water mixture (6:4 v/v). Samples (0.2 ml) were
mixed with 1.0 ml of tenfold diluted Folin–Ciocalteu rea-
gent and 0.8 ml of sodium carbonate solution (7.5%). After
30 min, at room temperature, the absorbance was measured
at 765 nm using V-530 UV/visible spectrophotometer. Esti-
mation of phenolic compounds as catechin equivalents was
carried out using standard curve of catechin [15].
Determination of radical scavenging (RSA) activity
by DPPH assay
Certain amount from each crude extract was dissolved in
methanol to obtain a concentration of 200 ppm. A volume of
0.2 ml of this solution was completed to 4 ml by methanol;
then 1 ml DPPH solution (6.09 × 10−5 mol/l, dissolved in
the same solvent) was added. The absorbance of the mixture
was measured at 516 nm after 10 min standing. Also, the
absorbance of the reference sample or blank (1 ml of DPPH
solution and 4 ml methanol) was measured. Triplicate meas-
urements were made and the radical scavenging activity was
calculated by the percentage of DPPH that was scavenged
according to Nogala-Kalucka et al. [16].
Based on the highest free radical scavenging activity, the
best extract, the methnolic one was used in current study.
Chemical composition identification
of the methnolic extract
LC/MS/MS,4000 Qtrap Applied Biosystems,(Quadrupole/
linear ion trap mass spectrometer), and the liquid chro-
matography (Agilent Technologies, Palo Alto, CA, USA)
Molecular Biology Reports (2019) 46:3637–3649 3639
system coupled with an electrospray ionization–mass spec-
trometer (ESI–MS/MS) detector were employed to identify
the chemical composition of the methnolic extract [17,
18].
Experimental design
Hypothyroidism was induced by giving the animal 0.05%
(w/v) propylthiouracil in drinking water for 6 weeks as
described by Sahoo et al. [19]. After induction of hypo-
thyroidism model, all animals (hypothyroidism-modeled
rats and healthy ones) were randomly grouped into five
groups (ten rats each), weighed and treated with either
ashwagandha methanolic extract or anti-hypothyroidism
pharmaceutical drug (Eltroxin™) as following:
Group 1 is comprised of healthy animals that fed free
without any treatments and acting as normal control.
Group 2 is comprised of healthy animals that were
subjected to oral administration of ashwagandha meth-
anolic extract at a dose of 500 mg/kg/day [20] for a
period of one month to study its toxicological and phys-
iological effects.
Group 3 is comprised of hypothyroidism-modeled ani-
mals that fed free without any treatments and acting as
positive control.
Group 4 is comprised of hypothyroidism-modeled ani-
mals that treated with Eltroxin™ or thyroxin drug (20 µg/
kg) by gavage [21] for one month and fed free.
Group 5 is comprised of hypothyroidism-modeled ani-
mals that treated with ashwagandha methanolic extract
(500 mg/kg/day) for a similar period and fed free.
At the end of the experimental period, the animals were
weighed then fasted overnight and following diethyl ether
anesthesia, blood samples were withdrawn from the retro-
orbital venous plexus. Each blood sample was divided into
2 portions: the smaller portion was taken on heparin and
the whole blood was used for the determination of hemo-
globin while the other portion was centrifuged at 3000 rpm
for 15 min at 4 °C where the clear serum was separated and
stored at − 70 until biochemical determinations.
After blood collection, each animal was rapidly sacrificed,
and the liver and kidney were dissected out and washed with
saline, dried on filter paper, weighed and homogenized in
50 mmol/l phosphate buffer (ice-cold) solution (pH 7.4)
for the determination of the oxidative stress markers. Also,
thyroid gland was dissected out and immediately kept in
10% buffered formalin–saline solution for a later histopatho-
logical examination where 5 μm thick paraffin sections were
stained with haematoxylin and eosin [22] and investigated
by light microscope.
Biochemical determination
Thyroid stimulating hormone (TSH), total triiodothyronin
(T3) and thyoxine (T4) levels were determined in serum
by ELISA technique using rat ELISA kits purchased from
MyBiosource Co, San Diego, CA 92195-3308, USA. Serum
free triiodothyronine (FT3) and free thyroxine (FT4) lev-
els were determined by ELISA technique using rat ELISA
KIT Cat. 1650 for quantitative determination purchased
from ALPHA DIAGNOSTIC international, 6203 Wood-
lake Center Drive, San Antonio, Texas78244, USA. Malon-
dialdehyde (MDA) in liver and kidney homogenates was
determined chemically according to the method described
by Ruiz-Larnea et al. [23]. Nitric oxide (NO) was deter-
mined spectrophotometrically in the tissue homogenates
using Biodiagnostic Kit No. NO 25 33 (Biodiagnostic Co.,
Egypt). Estimation of reduced glutathione (GSH) level and
glutathione peroxidase (GPx) activity in tissue homogen-
ates were carried out using the kits supplied by Biodiagnos-
tic Company (Egypt). ­Na+/K+-ATPase activity in liver and
kidney homogenates was assayed according to the modified
chemical method that described by Tsakiris et al. [24]. Total
hemoglobin (Hb) concentration in whole blood and serum
glucose were measured colorimetrically using kits produced
by Biodiagnostics Co. (Cairo, Egypt). Serum interleukin-6
(IL-6) was determined by enzyme-linked immuno-sorbent
assay (ELISA) technique using a kit manufactured by Assay-
pro Co., USA while interleukin-10 (IL-10) was determined
by enzyme-linked immuno-sorbent assay (ELISA) technique
using a kit manufactured by RayBiotech, Inc. ELISA Kit,
USA. An enzymatic procedure was used to determine serum
urea level and serum creatinine concentration was kineti-
cally evaluated using a kit obtained from Biodiagnostic Co.
(Egypt). Serum alanine aminotransferase (ALAT) and aspar-
tate aminotransferase (ASAT) activities were determined
using reagent kits purchased from Human Gesell Schaft fur
Biochemical und Diagnosticamb H, Germany.
Statistical analysis
All the obtained data are presented as means ± SE and sub-
jected to One-way analysis of variance (ANOVA) followed
by post hoc test (Duncan). This statistical analysis was car-
ried out using SAS software program to analyze the interac-
tion between the different groups. All statements of signifi-
cance were based on a probability of P ≤ 0.05.
Results
Table  1 illustrates the yields, total phenolics content
(TPC) and radical scavenging activity (RSA) of metha-
nolic and aqueous extracts of ashwagandha roots (Withania
13

3640
Table 1  In vitro determination of yield, total phenolic content (TPC)
and radical scavenging activity (RSA) of methanolic and aqueous
extracts of ashwagandha (Withania somnifera)
AME AAE
Yield (g %) 8.45 ± 0.65 8.33 ± 0.92
TPC (mg/g) 1.12 ± 0.43 0.69 ± 0,13
RSA (%) 56.36 ± 4.73 48.76 ± 4.12
Values are represented as mean ± standard error mean for three repli-
cates measurements
AME ashwagandha methanolic extract, AAE ashwagandha aqueous
extract
somnifera). The data show that methanolic extract possess
the higher values of yield, TPC and RSA.
As shown in Table 2 21 compounds were identified in the
methanolic extract of ashwagandha roots using LC/MS. The
compounds identified were found to include withanolides,
alkaloids and fatty acids and organic acids.
The data in Table 3 show that the induction of hypothy-
roidism by propylthiouracil induced a significant increase in
serum TSH level but it induced significant decreases in the
levels of total T3, free T3, free T4, and total T4 level com-
pared with the control values. Normal animals that received
ashwagandha methanolic extract (AME) recorded insignifi-
cant changes in levels of these hormones compared with
that of the normal control. Both ashwagandha extract and
the anti-hypothyroidism drug Eltroxin™ (thyroxine) signifi-
cantly ameliorated the changes occurred in the levels of the
above hormones but with different degrees.
Data in Tables 4 and 5 show the effects of different treat-
ments on hepatic and renal oxidative stress markers of the
animals. On comparison with the control group, propylthi-
ouracil significantly increased the levels of hepatic and
renal MDA and NO and decreased significantly the values
of GSH, GPx and ATPase. AME intake alone had no signifi-
cant effect on these parameters. Thyroxine treatment failed
to restore the changes in hepatic oxidative stress markers
induced by propylthiouracil but it could to offers little pro-
tection in renal tissue. AME administration succeeded to
improve the levels of these markers towards the normal val-
ues in both liver and kidney tissues. These results reflect the
antioxidant potential of AME.
Blood hemoglobin decreased significantly in hypothy-
roidism modeled rats. Administration of thyroxine to hypo-
thyroidism modeled rats failed to induce any significant pro-
tection against blood hemoglobin decrements while AME
administration succeeded to ameliorate significantly the
changes in the mentioned parameter (Fig. 1).
Hypothyroidism induction resulted in a significant
increase in BWG (%) and serum glucose when compared
to control rats (Table 6). Administration of AMA alone to
normal rats did not affect BWG or serum glucose. On the
13
Molecular Biology Reports (2019) 46:3637–3649
other hand, thyroxine and AME administration to hypo-
thyroidism modeled rats caused a significant decrease in
BWG and serum glucose.
In Fig. 2, IL-6 level increased significantly but IL-10
decreased significantly in hypothyroidism modeled rats.
Thyroxine or AME administration to hypothyroidism mod-
eled rats succeeded to restore significantly the induced
changes in the mentioned parameters.
Table 7 comprises the selected specialized serum mark-
ers of liver (AST and ALT) and kidney (urea and creati-
nine) functions among the different groups. It is clearly
indicated that ashwagandha methanolic extract (AME)
and the reference drug thyroxine had no effects on these
markers indicating the safety of ashwagandha extract and
thyroxine.
Histopathological examination showed that sections of
thyroid glands from the control rat revealed impacted folli-
cles separated by thin connective tissue septa. The thyroid
follicles were variable in size and epithelial lining; the
central follicles had a smaller diameter and lined by single
layer of cubical epithelium with rounded nuclei whereas,
the peripheral ones were larger in size and delimited with
flat or low cubical epithelium. The follicular lumen was
filled with an acidophilic colloid (Fig. 3a and b). Histo-
logical examination of H&E-stained sections of thyroid
glands of rat treated with a ashwagandha extract showed
some follicles are lined by more than one layer of follicular
cells (Fig. 3c).
Histological alterations in the thyroid glands in the case
of hypothyroidism revealed disintegration and disorganiza-
tion of thyroid follicles .The follicles with variable in size
and were filled with vacuolated irregular colloid. Prolif-
eration or multiple layers of follicular cells and fibrosis
were seen. Additionally some follicular cells with dark
stained nuclei and congested of blood vessel were seen
(Fig. 4a and b).
Sections of the thyroid gland, of hypothyroidism mod-
eled rats treated with thyroxin, induced some improve-
ment in pathological changes in the form of no fibrosis.
Examination of thyroid gland demonstrated variable size
of thyroid follicle and most of follicle with normal col-
loid, and others with partial colloidal and vacuolation.
Some follicle with proliferating follicular cells are clearly
noted and others that are lined by vacuolated follicular
cells. Presence of dilated and congested blood vessel was
recorded (Fig. 4c and d).
Microscopic examination of thyroid sections in the group
of hypothyroidism modeled rats treated with ashwagandha
extract demonstrated more improvement in pathological
changes as compared to group of hypothyroidism in the form
of the thyroid follicles nearly normal that lined with flattened
or low cuboidal epithelium. There are some follicles which
exhibited colloidal vacuolation (Fig. 5).
Molecular Biology Reports (2019) 46:3637–3649 3641

Table 2  Lc/ms/ms analysis of methanolic extract of ashwagandha (Withania somnifera) roots

No Retention time/ [M + H]+ Compound Structure
min

1 0.787 104.2 N-methyl-alanine

2 0.787 141.7 Tropine

3 0.787 176.5 6-Hydroxy, 4-methyl, 2-benzopyrone

4 0.787 181.2 Caffeic acid

5 0.787 184.1 Epinephrine

6 0.050 219 l-β-Homotryptophan

7 0.050 224.1 6-Bromo-2(1H) quinolinone

8 0.050 226.1 6-Benzylaminopurine

9 0.050 239.1 3,4,5-Trimethoxycinnamic acid

10 0.050 262.1 Tryptophylglycine

11 0.017 312.5 Caftaric acid

12 0.017 364.9 l-5-Hydroxytryptophan

13 0.017 381.1 15-Tetracosenoic acid methylester (z)

14 0.369 472.0 27-Hydroxy withanolide B (W 6)Or 5,7 α epoxy-6α, 20α-dihydroxy-1-
oxowitha-2, 24-dienolide (W 7)Or 5α, 17 β dihydroxy-6α, 7α-epoxy-1-
oxo-with a 2, 24-dienolide (W8)

15 0.369 474.0 l-Hydroxyproline

16 0.369 488.6 Β-hydroxy-2,3-dihydro-withanolide F (W1)

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3642 Molecular Biology Reports (2019) 46:3637–3649

Table 2  (continued)
No Retention time/ [M + H]+ Compound Structure
min

17 0.369 493.0 Succinic acid

18 0.017 509.3 Fumaric acid

19 0.017 543.2 Methyl ethyl glycol

20 0.017 596.3 Octadecanoic acid

21 0.067 992.3 Withanolide sulfoxide (W11)

Table 3  Effect of ashwagandha TSH Total T3 Free T3 Total T4 Free T4

extract and Eltroxin™ on
thyroid hormones in serum of (ng/ml) (ng/ml) (pg/ml) (µg/dl) (ng/ml)
hypothyroidism modeled rats
Control 1.26 ± 0.12 65.5 ± 2.9 2.22 ± 0.11 5.62 ± 0.15 1.49 ± 0.12
AME 1.25 ± 0.06 62 ± 1.37 2.92 ± 0.06 6.03 ± 0.16 1.54 ± 0.11
Hypo (positive) 14.86 ± 0.33* 34.4 ± 1.67* 1.59 ± 0.11* 3.91 ± 0.15* 0.58 ± 0.02*
Hypo + Eltroxin™ 1.51 ± 0.14# 72.4 ± 5.46# 2.91 ± 0.18# 7.32 ± 0.52# 1.16 ± 0.06#
Hypo + AME 1.21 ± 0.13# 41.3 ± 1.14# 2.91 ± 0.12# 6.44 ± 0.42# 1.11 ± 0.07#

Data are presented as mean ± standard error mean

AME ashwagandha methanolic extract, Hypo hypothyroidism model, Eltroxin™ anti-hypothyroidism phar-
maceutical drug (Thyroxine 100 mg), TSH thyroid stimulating hormone, T3 triiodothyronin, T4 thyoxine
*P ≤ 0.05 from control group; #P ≤ 0.05 from hypothyroidism group

Table 4  Levels of liver MDA NO GSH GPx ATPase

homogenate oxidative
stress markers of control; µmol/g µmol/g nmol/g U/g µmol pi/h/g
hypothyroidism and AME
Control 59 ± 3 11.4 ± 3.5 6.29 ± 0.2 4826 ± 125 272 ± 39
treated animals groups
AME 62 ± 5 10.7 ± 1.42 6.24 ± 0.31 5012 ± 133 271 ± 37
Hypo (positive) 100 ± 12* 17.6 ± 3.6* 4.94 ± 0.04* 3069 ± 70* 163 ± 21*
Hypo + Eltroxin™ 111 ± 18 18.2 ± 4.42 4.61 ± 0.07 3444 ± 76 203 ± 36#
Hypo + AME 82 ± 6.1 6.46 ± 1.27# 5.06 ± 0.07# 4509 ± 111# 265 ± 19#

Data are presented as mean ± standard error mean

AME ashwagandha methanolic extract, Hypo hypothyroidism model, Eltroxin™ anti-hypothyroidism phar-
maceutical drug (Thyroxine 100 mg), MDA malondialdehyde, NO nitric oxide, GSH reduced glutathione,
GPx glutathione peroxidase, ATPase sodium–potassium adenosine triphosphatase
*P ≤ 0.05 from control group; #P ≤ 0.05 from hypothyroidism group

13
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Table 5  Levels of kidney MDA NO GSH GPx ATPase

homogenate oxidative
stress markers of control, µmol/g µmol/g nmol/g U/g µmol pi /h/g
hypothyroidism and AME
Control 180 ± 12 16.3 ± 1.49 2.95 ± 0.06 5159 ± 143 217 ± 10.8
treated animals groups
AME 183 ± 8 14.4 ± 2.34 3.12 ± 0.04 5421 ± 140 214 ± 24.4
Hypo (positive) 203 ± 18* 33.2 ± 1.56* 1.81 ± 0.03* 3766 ± 103* 178 ± 9.04*
Hypo + Eltroxin™ 241 ± 23 # 38.8 ± 2.35 1.91 ± 0.02# 3869 ± 151 193 ± 17.6#
Hypo + AME 191 ± 12 29.3 ± 1.67# 1.95 ± 0.03# 4236 ± 179# 205 ± 9.20#

Data are presented as mean ± standard error mean

AME ashwagandha methanolic extract, Hypo hypothyroidism model, Eltroxin™ anti-hypothyroidism phar-
maceutical drug (Thyroxine 100 mg), MDA malondialdehyde, NO nitric oxide, GSH glutathione, GPx glu-
tathione peroxidase, ATPase sodium–potassium adenosine triphosphatase
*P ≤ 0.05 from control group; #P ≤ 0.05 from hypothyroidism group

18 Discussion
16 #
14 * Propylthiouracil (PTU) is a thioamide antithyroid drug. It
Blood Hb level (g/dl)

12 has been used in the management of hyperthyroidism for

10 more than half a century. PTU induces a hypothyroidism
8 status [25]. So it has been frequently used to create an ani-
mal model of hypothyroidism [26, 27]. Umezu et al. [28]
6
observed that serum total thyroxine (T4) level and free
4
triiodothyronine (FT3) were significantly decreased, and
2
serum thyroid-stimulating hormone (TSH) concentration
0
was markedly increased in hypothyroid modeled rats than
in normal rats and these results are go with the results of
our study. Fumarola et al. [29] reported that PTU inhibits
the oxidation of iodine and ionization of the monoiodoty-
rosine. It prevents also the coupling stage in the process of
Fig. 1  Shows the effect of ashwagandha extract, in compare to
Eltroxin™, on blood hemoglobin (Hb) of hypothyroidism modeled
thyroxin production, and inhibits the peripheral conversion
rats. AME ashwagandha methanolic extract, Hypo hypothyroidism of thyroxin (T4) into triiodothyronine (T3). So, it sup-
model; Eltroxin™ is anti-hypothyroidism pharmaceutical drug (Thy- presses thyroid hormones synthesis by blocking thyroid
roxine 100 mg); *P ≤ 0.05 from control group; #P ≤ 0.05 from hypo- peroxidase activity [30].
thyroidism group
Hypothyroidism is a state of increased oxidative stress
and associated with increased oxidative stress response [31].
MDA is a marker of lipid peroxidation, to measure oxidative
stress. MDA level was found to be higher in oxidative stress
Table 6  Effect of ashwagandha extracts and Eltroxin™ on body
weight gain and serum glucose in hypothyroidism modeled rats in hypothyroid subjects [32, 33]. Excess TSH is known to
directly produce oxidative stress; these findings supported
Body weight gain Glucose
our results that show a marked increase in liver and kidney
(g/100 g) (mg/dl)
MDA level in hypothyroid modeled rats and also go with the
Control 25.5 98 ± 5.2 results of Haribabu et al. [34].
AME 51.9 106 ± 8.3 Antioxidant enzymes such as catalase (CAT), superoxide
Hypo (positive) 56.8* 120 ± 3.3* dismutase (SOD), and glutathione peroxidase (GPx), and
Hypor + Eltroxin™ 33.2# 105 ± 4.1# transition-metal binding proteins, such as transferrin, fer-
Hypor + AME 36.8# 90 ± 4.7# ritin (that considered as iorn carrier), prevent the production
of or rapidly inactivate free radicals. GPx detoxifies lipid
Data are presented as mean ± standard error mean
peroxides by transforming them in the corresponding alco-
AME ashwagandha methanolic extract, Hypo hypothyroidism model,
Eltroxin™, anti-hypothyroidism pharmaceutical drug (Thyroxine
hols, so GPx level and other antioxidants decreased during
100 mg) hypothyroidism as antioxidants [35]. This may asserts our
*P ≤ 0.05 from control group; #P ≤ 0.05 from hypothyroidism group results that showed the increase in the levels of liver and

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3644 Molecular Biology Reports (2019) 46:3637–3649

Fig. 2  Shows the effect of 12 16

ashwagandha extract, in * 14 # #

Serum IL-6 level (ng/ml)

10

Serum IL-10 level (ng/ml)

compare to Eltroxin™, on
serum interleukin-6 (IL-6) # 12
8 # *
and interleukin-10 (IL-10) in 10
hypothyroidism modeled rats. 6 8
AME ashwagandha methanolic
extract, Hypo hypothyroidism 4 6
model; Eltroxin™ is anti-hypo-
2 4
thyroidism pharmaceutical drug 2
(Thyroxine 100 mg); *P ≤ 0.05 0 0
from control group; #P ≤ 0.05
from hypothyroidism group

Table 7  Markers of liver (ASAT ASAT ALAT Urea Creatinine

and ALAT) and kidney (urea
and creatinine) functions in (U/L) (U/L) (mg/dl) (mg/dl)
different studied animal groups
Control 26.6 ± 2.6 27 ± 1.62 31.7 ± 3.1 0.98 ± 0.12
AME 29 ± 2.47 25 ± 1.94 33.8 ± 2.01 0.85 ± 0.11
Hypo (positive) 33 ± 1.38 26 ± 1.97 21.8 ± 1.33 0.81 ± 0.02
Hypo + Eltroxin™ 30.77 ± 1.11 29.2 ± 1.45 28.4 ± 1.9 0.69 ± 0.06
Hypo + AME 30.1 ± 6.95 30.6 ± 1.56 27.2 ± 1.98 0.76 ± 0.06

Data are presented as mean ± standard error mean

AME ashwagandha methanolic extract, Hypo hypothyroidism model, Eltroxin™ anti-hypothyroidism phar-
maceutical drug (Thyroxine 100 mg), ASAT aspartate aminotransferase, ALAT alanine aminotransferase
*P ≤ 0.05 from control group; #P ≤ 0.05 from hypothyroidism group

kidney MDA and NO due to hypothyroidism induced oxida-
tive stress and the decreases in the values of, GPx, GSH as
antioxidants due to free radical scavenging.
Thyroid gland also has a crucial effect on erythropoiesis
by activation of erythropoietin release and proliferation of
erythroid progenitors. The level of blood hemoglobin was
significantly decreased in in the hypothyroid rats when it was
compared with that of control rats; this finding correlates
with the present study of Erdogan et al. [36] who reported
that anemia was found to increase in overt and subclinical
hypothyroid patients, and suggested that thyroid hormones
stimulate, directly or indirectly, growth of erythroid colo-
nies through erythropoietin. Otherwise, treatment of hypo-
thyroid-rats with ashwagandha methanolic extract tends to
bring hemoglobin level near to normal. It was found that
ashwagandha increased both the RBC count and hemoglobin
level that, in its turn, increases the capacity of the blood to
transport oxygen directly to all tissues, thereby would pro-
vide conclusive evidence regarding the mechanism of the
ergogenic effect of Ashwagandha [37].
Na+/K+-ATPase activity was also decreased in hyopthy-
rodism group. N ­ a+/K+-ATPase is a cell membrane located
enzyme [38], and its function is the active transport of
sodium and potassium across the cell membrane [39]. ­Na+/
K+-ATPase depends on lipids, Therefore, lipid peroxidation
of cell membrane influences the activity of this enzyme [40];
hence propylthiouracil leads to lipid peroxidation that dis-
turbs the lipid structure of cell membranes causing decrease
in ­Na+/K+-ATPase activity.
Primary hypothyroidism is considered as an inflammatory
condition. High levels of cytokines such as tumor necro-
sis factor-alpha, interleukin-6 (IL-6) and C-reactive protein
are found in hypothyroidism condition [41]. The increased
production of interleukins due to inflammation can reduce
the expression of deiodinases, inducing a low-T3 level that
leads to hypothyroidism, so it causes an increase in serum
concentration of proinflammatory cytokines such as IL-6
and decreased the anti-inflammatory cytokine as IL-10
[42]. In addition, Iyer et al. [43] stated that hypothyroid-
ism is associated with raised secretion of proinflammatory
cytokines such as IL-6 and decreased release of adipokines
that down regulate inflammation, and their results are agreed
with our results that showed increase in the level of IL-6 and
decreased level of IL-10.
The thyroid gland has an important role in the synthesis
of fatty acids and the degradation of lipids and influences
the major metabolic pathways that associated with obesity
and metabolic syndrome. Alternatively, thyroid disorder is
associated with low basal metabolic rate that affects weight
gain [44]. Thyroid hormones affect the metabolism of

13
Molecular Biology Reports (2019) 46:3637–3649 3645
Fig. 3  Section of the thyroid
gland of rats. a Control showing
thyroid follicles of different
sizes; their cavities contain
acidophilic colloid. Blood
capillaries are seen between
thyroid follicles (H&E × 40),
b higher power of the control
thyroid gland (H&E × 400), c
a rat treated with ashwaganda
extract only demonstrated most
of thyroid follicles are lined by
cubical cells. Few of them are
lined by more than one layer of
follicular cells (H&E × 400)
carbohydrates and fats and influence the synthesis, mobi-
lization and degradation of fats, which is often linked with
increased lipoprotein and lipase activity [45]. Therefore,
hypothyroidism causes a body weight gain and elevated
glucose levels [28]. Our results are agreed with that results
which indicate an increase in body weight and glucose val-
ues [46].
In our study, hypothyroidism did not affect the values
of serum AST and ALT. However, these results are in con-
flict with other studies which found a significant increase in
AST and ALT values by PTU therapy [47, 48]. Our results
showed also that PTU insignificantly changed the markers
of kidney function (urea and creatinine) which is in con-
trary with the study performed by Sarandol et al. [49] who
reported that chronic administration with PTU is associated
with renal toxicity. The marked variability in the reports
about liver and kidney functions might be attributed to dif-
ferences in study design, species or PTU dose.
In the recent years, importance is being given to poly-
herbal plants due to their effective therapeutic action and
minimal side effects. Withania somnifera (Ashwagandha) is
considered to be one of the most important medicinal plants
of the medicinal system for over 3000 years. W. somnifera
has several pharmacological activities, such as antioxidant,
antimicrobial, antifungal, adaptogenic, cardioprotective,
anticancer, neuroprotective, anticonvulsant, immunomodu-
latory, apoptic, diuretic, hepatoprotective and anti-inflam-
matory [50].
Ashwagandha contains a variety of nutrients and phyto-
chemicals and also used as a dietary supplement. As shown
by LC/MS analysis in the current study, ashwagandha extract
contains 21 compounds include withanolides, alkaloids and
fatty acids and organic acids. It has been reported that the
roots, fruits and leaves of W. somnifera provide potential
benefits for human health which attributed to their high con-
tents of polyphenol and flavonoids [51]. Many studies have
demonstrated the effectiveness of phenolics and flavonoids
as antitumor, anti-inflammatory agent or in reducing the risk
of many diseases [52].
Withanolides, which are known as anticancer agents are
considered the most active components of genus Withania;
the flavonoids Withaferin A, Withanolide A and Withanone
are the major withanolides present in genus Withania [53,
54]. Withaferin A is the most important one in terms of its
concentration and spectrum of activity [55]. Withaferin A
has anti-inflammatory, anticancer [56–58], immunomodula-
tion [59], cardio protective [60] and neuroprotective effects
[61].
Ashwagandha methanolic extract has been reported
to elevate the level of circulating thyroid hormones and
13

3646
Fig. 4  Slide a illustrates a section of the thyroid gland of rats with
hypothyroidism and shows disorganization of thyroid follicles that
are filled with vacuolated irregular colloid (V); proliferation or mul-
tiple layers of follicular cells and fibrosis are seen (F); some follicles
are lined by vacuolated follicular cells (black line); Congestion of
blood vessel is noted (H&E × 400). Slide b also illustrates a section
of the thyroid gland of rats with hypothyroidism and shows the fol-
licles with variable size; some follicles appeared dilated and others
with narrowing lumen as compared to control (H&E × 400). Slide
Fig. 5  Section of the thyroid gland, of hypothyroidism rats treated
with ashwagandha extract, shows that the thyroid follicles are nearly
normal and lined with flattened or low cuboidal epithelium follicles;
some follicles exhibited colloidal vacuolation (V) (H&E × 400)
13
Molecular Biology Reports (2019) 46:3637–3649
c illustrates a section of the thyroid gland of hypothyroid lesions
treated with thyroxin demonstrating variable size of thyroid follicle,
and most of follicle with partial colloid and congestion of blood ves-
sel (star) (H&E × 40). Slide d another filed, of thyroid gland section
of hypothyroid rats treated with thyroxin, shows colloidal vacuolation
(V); some follicle with proliferating follicular cells are clearly noted
(arrow head) and others that are lined by vacuolated follicular cells
(H&E × 400)
regulates glucose metabolism and oxidative stress [62].
Panda and Kar [63] reported also that administration of root
extract of ashwaganda to mice stimulates thyroidal activity
and also enhances the antiperoxidation of hepatic tissue. Our
results are agree with the results of Jatwa and Kar [64] who
suggested that thyroid stimulatory effect of the ashwagandha
extract is an outcome of the direct action of its active ingre-
dients—Withaferin-A—at molecular level.
Withaferin A exerts its antitumor activity by inducing
apoptosis through inhibiting the nuclear factor-κB (NF-κB)
activation [65, 66]. So, ashwagandha methanolic extract
decrease serum IL-6 and ameliorate serum IL-10 that is an
anti-inflammatory cytokine down regulates the expression
of Th1 cytokines and also block NF-κB [67] in ashwagan-
dha treated groups when compared with normal groups. Our
results are going with the results of Mosser and Zhang [67].
Molecular Biology Reports (2019) 46:3637–3649 3647
The oxidative stress due to hypothyroidism may be
exerted through free radicals metabolites. Free radicals react
with oxygen to produce superoxide anions and other reac-
tive oxygen species (ROS) that affect antioxidant enzymes
[68]. In our study, significantly lower values of GSH and
GPx were detected in the liver and kidney tissues of hypo-
thyroid rats when compared to control rats. The observed
decreases in the values of these antioxidants may be due to
their increased utilization for scavenging ROS [69]. Treat-
ment with ashwagandha methanolic extract improved GSH
and GPx by scavenging superoxide and H ­ 2O2 produced due
to propylthiouracil induced hypothyroidism. GSH and GPx
values were also higher in ashwagandha methanolic extract
alone treated group when compared with the control group.
This indicates that ashwagandha methanolic extract not only
scavenges the oxidative stress but also increases the activ-
ity of the antioxidant enzymes during normal physiological
conditions. Our findings are coping with the results of Alam
et al. [70] and Ahmed et al. [71]. Oral administration of
methanolic W. somnifera extract in turn significantly lowered
the elevated level of MDA due to its antioxidant scavenging
activity and in turn restoring ­Na+/K+-ATPase activity .
In our study, the histopathological changes in thyroid
gland confirmed the biochemical results and revealed that
thyroid gland in the case of hypothyroidism showed severe
histological changes as shown in the histopathological pic-
tures. However, animals treated with Ashwaganda extract
or the reference drug (thyroxin) resulted in a significant
improvement in thyroid gland tissue as compared to group
of hypothyroidism, as ashwagandha extract demonstrated
the more improvement in pathological changes. The results
from the current study indicate that ashwagandha metha-
nolic extract treatment improves thyroid function by ame-
liorating thyroid hormones and by preventing oxidative
stress; these effects could be due the higher contents of
antioxidant components.
Acknowledgements  The authors would like to thank the authorities
of the National Research Centre, Giza, Egypt, for providing the facili-
ties and supporting by the fund (Project Code: 11010331) to carry out
this work.
Funding  This study was funded by the National Research Centre, Giza,
Egypt (Grant Number: 11010331).
Compliance with ethical standards  Res Int 36(2):117–122
Conflict of interest  Khaled G. Abdel-Wahhab has received research a
grant from National Research Centre,Cairo, Egypt. The authors de-
clare that they have no conflict of interest.
Ethical approval  All animals received human care in compliance with
the standard institutional criteria for the care and use of experimental
animals according to the National Research Centre ethical committee
(FWA 00014747).
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