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Zoology Department, Faculty of Science, Suez
Canal University, Ismailia, Egypt.
*Correspondence
Corresponding author: Heba A. Hashem
E-mail address: hebahashemm@gmail.com
Abstract
Heba A. Hashem1*, Zohour I. Nabil2, Heba N. Gad EL-Hak3
Original Research
Ameliorative Effect of Ashwagandha (Withania somnifera) Root Extract
on Brain Oxidative Stress and Depression of Diabetic Rats
Evaluation of the ecacy of ashwagandha root extract to ameliorate the oxidative stress and depression result-
ing from diabetes in male rats was conducted in the current study. Thirty-six male albino rats were randomly
grouped into two main groups: normal (n=18) and diabetic (n=18), diabetes was induced by a single dose
of 150 mg/kg BW alloxan injected intraperitoneally. After six weeks, both normal and diabetic groups were
further subdivided into six sub-groups; normal control, 100 & 200 mg/kg BW ashwagandha treated normal,
diabetic control, 100 and 200 mg/kg BW ashwagandha treated diabetic groups, for another six weeks. The
forced swim test was used to assess depression, and serum serotonin levels were measured. In brain tissue
homogenates, the glutathione reduced content, superoxide dismutase, and catalase activity were measured, as
well as the total antioxidant capacity, total oxidative capacity, and malondialdehyde levels. Moreover, histo-
pathological examination of the brain (cerebral cortex and cerebellum) were conducted. The obtained results
revealed that the administration of ashwagandha extract to diabetic rats reduced immobility time during the
forced swim test while increasing the serotonin levels signicantly when compared with the diabetic group.
Similar to this, brain total antioxidant capacity, glutathione reduced content, superoxide dismutase, and cata-
lase activity increased signicantly, while brain total oxidative capacity, oxidative stress index, and malondi-
aldehyde levels decreased signicantly when compared with the diabetic group. Furthermore, the histopatho-
logical changes in brain sections were reversed by ashwagandha root extract. In conclusion, ashwagandha root
extract can be used to ameliorate the brain oxidative stress and depression brought on by diabetes mellitus at
doses of 100 and 200 mg/kg BW.
KEYWORDS
Ashwagandha, Depression, Oxidative stress, Brain, Diabetes
INTRODUCTION
Diabetes mellitus (DM) is dened by elevated blood glucose
levels (hyperglycemia) caused by deciencies in insulin secretion,
action, or both (Ozougwu et al., 2013). As hyperglycemia be-
comes chronic with time, it leads to serious consequences in sev-
eral tissues, especially those that are insulin-insensitive (retina,
neurons, kidneys) (Giri et al., 2018). Diabetes-induced neuronal
damage is most likely caused by oxidative stress (OS) (Vincent
et al., 2004). Reactive oxygen species (ROS) play an essential role
in signal transduction cascades under physiologically typical cir-
cumstances, but when they are present in excess, they become
neurotoxic and cause depression and neurodegeneration (Wink
et al., 2011; Pierzchala et al., 2022). The brain is one of many or-
gan systems aected by DM, and it is thought to be more vulner-
able to OS because It uses a lot of oxygen, has a lot of polyunsat-
urated fatty acids, and has a lot of protective enzymes (Singh et
al., 2019). When there is an increased demand for oxygen, more
ROS are produced (Halliwell, 1991). The increased free radicals
increase neuronal death in several brain regions, and cause DNA
damage, oxidized proteins, and peroxide lipids in membranes
(Pop-Busui et al., 2006). The eects of DM on brain complications
are becoming more commonly recognized (Sima, 2010). Aleem
et al. (2022) observed that DM brought on by alloxan is known to
promote depression. According to Hozayen et al. (2012), DM has
a higher incidence of nervous manifestation due to insulin-in-
duced brain OS that causes disturbances in brain neurotransmit-
ters. Martínez-Tellez et al. (2005) also suggested a connection
between DM and changes in the brain’s structure and function.
In recent years, the medical system has struggled to manage
DM without causing negative side eects. In addition to insulin,
a range of oral synthetic hypoglycemic medications with their
side eects are available for the treatment of DM (Tran et al.,
2015). The demand for natural products that are less expensive
and have antidiabetic activity with fewer side eects is rising as
a result. In India’s Ayurvedic medical system, Withania somnif-
era, also known as ashwagandha, is a shrub in the Solanaceae
family (Visweswari et al., 2013). Numerous studies on this plant
have revealed that it has anti-inammatory, antitumor, antistress,
antioxidant, immunomodulatory, and hematopoietic properties
in addition to its benecial eects on the endocrine system, vas-
cular system, and nervous system. Several animal studies have
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ISSN: 2090-6277/2090-6269/ © 2011-2023 Journal of Advanced Veterinary Research. All rights reserved.
Journal of Advanced Veterinary Research
(2023) Volume 13, Issue 3, 508-514
Received: 03 April 2023, Accepted: 29 April 2023
demonstrated that Ashwagandha is an anxiolytic, antidepressant,
and neuroprotective agent because it improves memory and oth-
er brain and nervous system functions (Singh et al., 2011; Durg
et al., 2015). However, there are only a few studies in the litera-
ture focusing on how ashwagandha can prevent brain damage
caused by DM. Subsequently, the objective of the current study
was to test ashwagandha’s ability to reduce depression and the
OS induced by DM in the brain tissues of diabetic rats.
MATERIALS AND METHODS
Plant material
Ashwagandha root extract available as a commercial dietary
supplement capsule (450 mg ashwagandha root extract) was
purchased from Now Foods Company, USA. According to the
manufacturer’s details, each capsule contained min. 2.5% total
withanolides 11 mg, rice our, stearic acid (vegetable source),
and hypromellose (cellulose capsule) found by high-performance
liquid chromatography (HPLC) as per the information provided
by the manufacturer. This product was registered in India as a
traditional herbal medicine. The contents of an ashwagandha
capsule were dissolved in distilled water and administered orally
via an intragastric tube, 100 and 200 mg/kg BW of ashwagandha
were the dosages chosen for this study according to Khan et al.
(2015).
Animals
Thirty-six healthy male albino rats weighing between 140
and 200g were obtained and randomly grouped into two main
groups: normal (n=18), without any treatments, and diabetic
(n=18), which were induced to be diabetic through intraperito-
neal injection of a single dose (150 mg/kg BW) of alloxan (Oxford
Lab Fine Chem LLP, Maharashtra, India). After six weeks, the nor-
mal group was subdivided into three sub-groups; (1) NC, nor-
mal control group without any treatments, (2) ASH100, and (3)
ASH200, normal rats belong to the 2nd and 3rd groups received
100 and 200 mg/kg BW ashwagandha respectively, which was
dissolved in distilled water and administered orally on daily ba-
sis for 6 weeks. The diabetic group was subdivided into three
groups, (4) DC, a diabetic control group without any additional
treatments, (5) DC + ASH100 and (6) DC + ASH200. Rats belong
to the 5th and 6th groups of diabetic rats were treated with 100
and 200 mg/kg BW ashwagandha respectively, which was dis-
solved in distilled water orally on daily manner for 6 weeks.
Ethical statement
Suez Canal University’s research committee gave the go-
ahead for all experimental procedures following the international
standards for the care and use of laboratory animals code (Rec
68/2020).
Depression testing
One of the most popular assays for examining depressive-like
behavior in rodents is the forced swim test (FST). Rats were put
in an impenetrable cylindrical container (Figure 1) lled with tap
water (25±1 °C). Two sessions of the test were held. Rats were rst
made to swim for 15 minutes before the test during the pre-test
session. The process was repeated 24 hours later for a 6-minute
FST session that was recorded on a mobile device. The animal was
initially attempted to ee but eventually became immobile, which
can be seen as a sign of behavioral despair. The total amount of
time spent motionless (measured in seconds) after the rst two
minutes was over four minutes. The rats were deemed to be im-
mobile when they continued to oat with no other movements
than those required to keep their noses above the water. Water
was changed after the sessions and animals were removed and
placed in separate cages to dry before being returned to their
home cages (Yankelevitch-Yahav et al., 2015).
Blood serum collection and brain sampling
Before the beginning of behavioral tests, a suitable amount
of blood was collected into a test tube without any additives us-
ing the retro-orbital technique for serotonin analysis. The blood
was further centrifuged at 3,000 rpm for 10 minutes. After that,
serum was collected and separated with a clean dropper in ster-
ilized tubes, properly labeled, and then frozen at -20°C. At the
end of the experiment, rats were weighed, then sacriced, and
the brain was dissected and washed several times in saline (0.9
% NaCl). Half of each brain was isolated and stored at -20oC for
homogenization. Homogenization was carried out at 20 % (w/v)
in phosphate-buered saline (0.01 M, pH=7.4). Whole homog-
enates were used for the estimation of brain total antioxidant
capacity (TAC), total oxidative capacity (TOC), malondialdehyde
(MDA) level, superoxide dismutase (SOD), catalase (CAT) activity,
and glutathione reduced (GSH) content.
Determination of serum serotonin activity
Determination of Serotonin activity assay using Rat sero-
tonin ELISA kit was purchased from My Biosource, Inc., P.O. Box
153308, San Diego, CA 92195-3308, USA using Sánchez et al.
(2008) method.
Estimation of brain TOC, TAC, MDA, SOD, CAT, and GSH in brain
homogenate
The brain TOC assay (Ma et al., 2010) and brain TAC assay
(Navaie et al., 2018), their kits were obtained from Labor Diag-
nostika Nord GmbH & Co. KG, Nordhorn, Germany; the oxida-
tive stress index (OSI) value was calculated as follows: TOC ÷
TAC (Gunbatar et al., 2020); the brain MDA (Kowalczuk and Stry-
jecka-Zimmer, 2002); brain SOD (Bordet et al., 2000); brain CAT
(Hamby-Mason et al., 1997), their kits were purchased from Cell
Biolabs, Inc., 7758 Arjons Drive San Diego; and brain GSH (Terp-
stra et al., 2003) used GSSG/GSH quantication kit that was ob-
tained from Kamiya Biomedical Co., 12779 Gateway Drive, Seattle
WA 98168.
Histopathological studies of the brain (Cerebral cortex and cere-
bellum)
The second half of each brain from rats was xed in 10% for-
malin, cleaned with distilled water, dried in ethyl alcohol with in-
creasing concentrations, cleared with xylene, and nally embed-
ded in paran wax. For routine histological analysis under a light
microscope, coronal sections were cut using a rotary microtome
and then stained with hematoxylin and counterstained with eosin
(H&E).
Statistical analysis
The statistical package SPSS 20.0 was used for data analy-
sis (SPSS Inc., Chicago, IL, USA). The data were analyzed using
Heba A. Hashem et al. /Journal of Advanced Veterinary Research (2023) Volume 13, Issue 3,
509
descriptive statistics., and the results were shown as the mean
and standard error of the mean (Mean±SE). One-way analysis of
variance (ANOVA) was used to analyze the data, and P < 0.05 was
accepted as statistically signicant. Duncan’s multiple compari-
sons were then used as post hoc multiple comparisons.
RESULTS
Eect of ashwagandha on immobility duration during FST
The DC group showed a signicant increase (P < 0.05 vs.
NC) in immobility duration during FST when compared with the
NC group. On the other hand, both D+ASH100 and D+ASH200
groups exhibited a signicant decrease (P < 0.05 vs. DC) in immo-
bility duration during FST when compared with the DC group. No
signicant change (P < 0.05 vs. NC) in immobility duration during
FST was observed in both ASH100 and ASH200 groups during
FST when compared with the NC group (Table 1).
Eect of ashwagandha on serotonin levels in serum
The present DC group manifested a signicant decrease (P
< 0.05 vs. NC) in serotonin levels when compared with the NC
group, while both D+ASH100 and D+ASH200 groups displayed
a signicant increase (P< 0.05 vs. DC) in serum serotonin lev-
els when compared with the DC group. A signicant increase (P
< 0.05 vs. NC) in serum serotonin levels was observed in both
ASH100 and ASH200 groups when compared with the NC group
(Table 1).
Eect of ashwagandha on oxidative / anti-oxidative parameters
in the brain
The present DC group exhibited a signicant increase (P <
0.05 vs. NC) in brain TOC, OSI, and MDA levels while there was
a signicant decrease in brain TAC, GSH content, SOD, and CAT
activity when compared with the NC group. Conversely, both
D+ASH100 and D+ASH200 groups displayed a signicant de-
crease (P < 0.05 vs. DC) in brain TOC, OSI, and MDA levels, while
there was a signicant increase (P < 0.05 vs. DC) in brain TAC,
GSH content, SOD, and CAT activity when compared with the DC
group. The ASH200 group showed a signicant decrease (P <
0.05 vs. NC) in brain TOC and MDA levels while there was a signif-
icant increase (P < 0.05 vs. NC) in brain TAC, GSH content, SOD,
and CAT activity when compared with the NC group. The ASH100
group showed a signicant increase (P < 0.05 vs. NC) in brain
SOD and GSH levels while there was no signicant change (P <
0.05 vs. NC) in brain TOC, TAC, MDA levels, and CAT activity when
compared with the NC group (Tables 2 and 3).
Heba A. Hashem et al. /Journal of Advanced Veterinary Research (2023) Volume 13, Issue 3, 508-514
Groups Immobility Duration (s) FST (%) Serum Serotonin (mmol/L) (%)
NC 120±3.7d------- 295±2.74b-------
ASH100 117±3.1d-3% 301±1.32a,b 2%
ASH200 114±4.1d-5% 304±1.67a3%
DC 181±3.9a51% 153±4.49e-48%
D+ASH 100 151±2.6b26% 202±2.01d-32%
D+ASH 200 140±4.0c17% 253±2.03c-14%
Table 1. e immobility duration during forced swimming test (FST) and serum serotonin level in normal control and dierent treated groups.
Values are expressed as Mean ± (SE), (n= 6/group). Values with dierent letters dier, P < 0.05 using Duncan’s test; one way ANOVA. (NC: normal control, ASH100 & ASH200: 100 & 200
mg/kg ashwagandha, DC: diabetic control, D+ASH100 & D+ASH200: 100 & 200 mg/kg ashwagandha-treated diabetic groups).
Groups Brain TOC
(µmol H2O2 Eq/g p) (%) Brain TAC
(µmol Trolox Eq/g p) (%) Brain OSI
(arbitrary unit) (%)
NC 0.36±0.007d------- 2.40±0.01b------- 0.15±0.004d-------
ASH100 0.35±0.004d,e -3% 2.41±0.004a,b 0% 0.15±0.002d0%
ASH200 0.34±0.004e-6% 2.43±0.002a1% 0.14±0.000d-7%
DC 0.70±0.002a94% 0.93±0.02e-61% 0.76±0.01a407%
D+ASH 100 0.55±0.002b53% 1.42±0.01d-41% 0.39±0.004b160%
D+ASH 200 0.40±0.004c11 % 1.88±0.02c-22% 0.21±0.004c40%
Table 2. e brain total oxidative capacity (TOC), brain total anti-oxidative capacity (TAC), and brain OSI values in normal control and dierent treated groups.
Values are expressed as Mean ± (SE), (n= 6/group). Values with dierent letters dier, P < 0.05 using Duncan’s test; one way ANOVA. (NC: normal control, ASH100 & ASH200: 100 & 200
mg/kg ashwagandha, DC: diabetic control, D+ASH100 & D+ASH200: 100 & 200 mg/kg ashwagandha-treated diabetic groups).
Groups Brain MDA
(nmol/g p) (%) Brain SOD
(U/mg p) (%) Brain CAT
(U/mg p) (%) Brain GSH
(μmol/g p) (%)
NC 0.12±0.004d------- 7.17±0.05c------- 5.38±0.05b------- 12.46±0.07b-------
ASH100 0.13±0.002d8% 7.32±0.02b2% 5.40±0.03a,b 0% 12.60±0.03a1%
ASH200 0.11±0.002e-8% 7.42±0.01a4% 5.48±0.04a2% 12.70±0.02a2%
DC 0.29±0.002a142% 4.66±0.03f-35% 3.94±0.03e-27% 8.09±0.06e-35%
D+ASH 100 0.24±0.002b100% 5.41±0.03e-25% 4.60±0.03d-15% 9.38±0.03d-25%
D+ASH 200 0.17±0.004c42% 6.64±0.03d-7% 4.91±0.02c-9% 10.53±0.05c-16%
Table 3. e brain malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) in control and dierent treated groups.
Values are expressed as Mean ± (SE), (n= 6/group). Values with dierent letters dier, P < 0.05 using Duncan’s test; one way ANOVA. (NC: normal control, ASH100 & ASH200: 100 & 200
mg/kg ashwagandha, DC: diabetic control, D+ASH100 & D+ASH200: 100 & 200 mg/kg ashwagandha-treated diabetic groups).
510
Histopathological studies
Light microscopic examination of the cerebral cortex sections
of NC, ASH100, and ASH200 groups was the same. Six layers of
gray matter were found to be ordered and well-organized from
outside to inside as follows: outer molecular layer, external gran-
ular layer, external pyramidal cell layer, internal granular layer,
internal pyramidal, and polymorphic cell layer (Figure 2a, b & c).
The DC group’s cerebral cortex sections revealed loss of organi-
zation of layers, vacuolation of nerve cells, and aggregation of in-
ammatory cells (Figure 2d). Examination of cerebral cortex sec-
tions from D+ASH100 and D+ASH200 groups revealed organized
and regularly arranged six layers of gray matter (Figure 2e & f).
Light microscopic examination of the cerebellum of NC, ASH100,
and ASH200 groups showed the same results. Sections revealed
organized and regularly arranged three layers. These layers were
the Molecular layer (outer), Purkinje layer (middle), and Granular
layer (inner) as shown in Figure 3a, b & c. Cerebellum sections
from the DC group revealed degeneration and vacuoles in the
molecular layer (Figure 3d). Examination of cerebellum sections
of D+ASH100 and D+ASH200 groups revealed organized and
regularly arranged three layers (Figure 3e & f).
DISCUSSION
The current research shows that ashwagandha consumption
in rats protected their brains from the oxidative damage brought
on by DM. The current investigation examined the depression
tests, lipid peroxidation and antioxidant defense mechanisms,
and the histopathological organization of the cerebral cortex
and cerebellum. In the current study, a modied FST model of
depression is used to characterize the eects of ashwagandha
in diabetic rats. When forced to swim, the diabetic rats treated
with 100 & 200 mg/kg BW ashwagandha performed signicantly
better than the normal control group. The antidepressant eect
of ashwagandha agreed with Zahiruddin et al. (2020). The pres-
ence of the active constituent, withanolides, has been linked to
ashwagandha’s antidepressant action (Mk et al., 2017; Speers et
al., 2021). Depression and other neuropsychological complica-
tions are frequently linked to DM because of altered neurotrans-
mitter function. Due to altered physiological processes, such as
increased glucose oxidation and insulin deciency, DM has a de-
pression prevalence rate of 24–30% (Roriz-Filho et al., 2009). To
conrm the FST results, the levels of serotonin, a neurotransmitter
involved in the pathophysiology of depression, were measured
(Abomosallam et al., 2023). In the current study, serum serotonin
levels of the DC group signicantly decreased when compared
with the NC group. This agreed with Gupta et al. (2014) who ob-
served that serotonin levels were found to be lower in diabetic
animal models. Manjarrez-Gutiérrez and Hernández-Rodríguez
(2016) demonstrated a specic change in the serotonergic sys-
tem during DM, consisting of a decrease in serotonin biosyn-
thesis owing to a decrease in free fractions of L-tryptophan in
plasma and the brain. On the other hand, diabetic groups that
received 100 & 200 mg/kg BW of ashwagandha demonstrated
a signicant rise in serum serotonin levels when compared with
the DC group. This is in line with an earlier study by Bansal and
Banerjee (2016) who found that chronic ashwagandha adminis-
tration signicantly increased serotonin levels. Also, Priyanka et
al. (2020) pointed to the ability of ashwagandha in increasing se-
rotonin concentrations. This suggests that the serotonergic sys-
tem may play a role in the antidepressant eects of ashwagandha
(Jahanbakhsh et al., 2016).
Figure 1. Rat was placed in a glass container lled with tap water during the
forced swim test.
Figure 2. Photomicrographs of sections in the cerebral cortex of the frontal lobe of adult albino rats from (a) the control group showed well organized regularly ar-
ranged six layers from outer to the inner surface: (1) Molecular layer, (2) external granular, (3) external pyramidal, (4) internal granular, (5) internal pyramidal and (6)
polymorphic layer. (b &C) ASH100 and ASH200 showed well-organized regularly arranged six layers. (d) DC group revealed loss of organization of layers, vacuoles of
nerve cells (V), and aggregation of inammatory cells (IF) (circle). (e&f) D+ASH 100 and D+ASH 200 groups revealed organized and regularly arranged six layers
of gray matter (H.&E., × 100).
Heba A. Hashem et al. /Journal of Advanced Veterinary Research (2023) Volume 13, Issue 3, 508-514
511
Diabetic-induced neuronal damage is likely caused by OS
(Tan et al., 2022). The levels of oxidant/antioxidant parameters
were investigated to better evaluate OS in brain tissues. In the
current study, the rats in the DC group showed a signicant in-
crease in brain TOC, OSI, and MDA levels along with a signicant
decrease in brain TAC, GSH content, SOD, and CAT activity when
compared with the NC group, indicating that they were experi-
encing OS, which can lead to neurological disorders. DM is fre-
quently accompanied by an increase in MDA production, which
raises the levels of MDA in diabetic tissues and blood (Tiwari et
al., 2013). The elevated MDA levels in DC rats imply that lipid per-
oxidation (LPO) is being boosted because of an increase in ROS
generation and that this increase tended to the OS produced
by hyperglycemia. A decrease in enzymatic and non-enzymatic
antioxidants of the defense system in diabetic rats may also be
reected in the rise in LPO (De M. Bandeira et al., 2013). The pres-
ent results agreed with Uzar et al. (2012) who reported that after
21 days of treatment, diabetic rats’ brain tissues had signicantly
higher TOC levels and OSI values, but signicantly lower TAC lev-
els when compared to control rats. Okutan et al. (2005) indicated
that in the hippocampus of diabetic rats, LPO levels signicantly
increased while GSH content, SOD, and CAT activity signicantly
decreased. Similar to this, a prior study found that after 15 days
of treatment, the cerebrum, cerebellum, and midbrain of diabetic
rats compared to non-diabetic rats showed a signicant increase
in MDA levels and a signicant decrease in GSH levels, CAT, and
GPx activities (Kapoor et al., 2009). Sudhakara et al. (2012) and
Samarghandian et al. (2015) also reported that STZ-induced DM
in rats increased brain MDA levels while GSH content, SOD, and
CAT activity had decreased.
On the other hand, diabetic groups treated with 100 and 200
mg/kg BW of ashwagandha showed a signicant decline in brain
TOC, OSI, and MDA levels, whereas brain TAC, GSH levels, SOD,
and CAT activity signicantly increased when compared with DC
group. According to Singh et al. (2011), ashwagandha has strong
antioxidant properties, which aid in the prevention of cellular
damage caused by free radicals. The antioxidant capacity of ash-
wagandha is directly correlated with the total phenolic content
(Alam et al., 2011). The administration of 200 mg/kg BW ashwa-
gandha was the most eective in enhancing the overall state of
antioxidants in the brains of normal rats in the present study, as it
was able to signicantly increase the brain TAC, GSH levels, SOD,
and CAT activity whereas the administration of 100 mg/kg ash-
wagandha showed a signicant increase in the brain GSH con-
tent and SOD activity only. This is following Parihar et al. (2016)
who reported that giving ashwagandha to STZ-treated rats via
oral administration resulted in a signicant drop in MDA and a
signicant rise in GSH levels. Also, Ahmed et al. (2013) reported
that administering ashwagandha extract orally after receiving STZ
infusion resulted in a decrease in LPO level and an increase in an-
tioxidant enzyme activities as well as a rise in GSH level in various
regions of brain tissue. In like manner, John (2014) found that
ashwagandha administration for 28 days tends to bring the MDA,
SOD, and CAT values in a parkinsonism-induced mice model to
near-normal levels and the ashwagandha is known to modulate
brain OS markers, such as LPO, SOD, CAT, GPx, and GSH in anoth-
er context. Conversely, Hosny et al. (2021) reported that a 30-day
ashwagandha treatment did not aect the decrease in CAT and
SOD activity in the hippocampus caused by induced hypothy-
roidism.
The current investigation looked at the histological changes
caused by ashwagandha in the cerebral cortex and cerebellum of
the brain of adult male albino rats with DM. In the current study,
inammatory cells were agglomerated, and the cortical layers
of diabetic rats’ cerebral cortex showed marked disorganization
where they had lost their normal shape. The molecular layer of
the cerebellum displayed vacuoles. These ndings are consistent
with those of Malik et al. (2011) who noted neuronal damage
brought on by DM. These results coincide with Sharma et al.
(2005) and Gad El-Hak and Mobarak (2019) who claimed that
exposure to free radicals changed the histology of their brain. A
disturbance in the balance of ROS production and antioxidant
protection caused by excessive exposure to free radicals is known
as OS (Birben et al., 2012). Because of its high oxygen intake and
limited antioxidant content, the brain is highly susceptible to OS
damage (Floyd, 1999). Protein modications brought on by OS
can result in a drop in enzymatic activity and a loss of function
(Burton and Jauniaux, 2011). In this study, after the ashwagand-
ha treatment, the cortical layer and cerebellum of the diabetic
rats signicantly improved. Further research can be carried out to
dene the active constituents responsible for the mechanism of
action of ashwagandha root plants. Practical information on the
Figure 3. Photomicrographs of sections in the cerebellum of adult albino rats from (a) the control group showed well-organized regularly arranged three layers (b
&C) ASH100 and ASH200 showed well organized regularly arranged three layers. (d) DC group revealed degeneration and vacuoles (arrow) in the molecular layer.
(e&f) D+ASH 100 and D+ASH 200 groups revealed organized and regularly arranged three layers. Molecular layer (ML), Purkinje layer (PCL), and Granular layer
(GCL) (H.&E., × 100).
Heba A. Hashem et al. /Journal of Advanced Veterinary Research (2023) Volume 13, Issue 3, 508-514
512
safety of ashwagandha root plants should be made in combina-
tion with chemical diabetic drugs.
CONCLUSION
Based on the available information, it is possible to draw the
conclusion that ashwagandha supplementation protects against
alloxan-induced diabetic neuropathy. By reducing the oxidative
stress that diabetes causes, this eect was mediated. Considering
the ndings, it has been hypothesized that ashwagandha sup-
plements have strong anti-depressant eects by enhancing FST
behavior.
CONFLICT OF INTEREST
The authors declare that they have no conict of interest.
REFERENCES
Abomosallam, M., Hendam, B.M., Abdallah, A. A., Refaat, R., Elshatory, A.,
Gad El Hak, H.N., 2023. Neuroprotective eect of piracetam-load-
ed magnetic chitosan nanoparticles against thiacloprid-induced
neurotoxicity in albino rats. Inammopharmacology 31, 943-965.
Ahmed, M.E., Javed, H., Khan, M.M., Vaibhav, K., Ahmad, A., Khan, A., Ta-
bassum, R., Islam, F., Safhi, M.M., Islam, F., 2013. Attenuation of
oxidative damage-associated cognitive decline by Withania som-
nifera in rat model of streptozotocin-induced cognitive impair-
ment. Protoplasma 250, 1067-1078.
Alam, N., Hossain, M., Khalil, M., Moniruzzaman, M., Sulaiman, S., Gan, S.,
2011. High catechin concentrations detected in Withania somnif-
era (ashwagandha) by high performance liquid chromatography
analysis. BMC Complementary and Alternative Medicine 11, 65.
Aleem, A., Shahnaz, S., Javaid, S., Ashraf, W., Rasool, M.F., Ahmad, T.,
Alotaibi, A.F., Albeshri, K.S., Alqahtani, F., Imran, I., 2022. Chron-
ically administered Agave americana var. marginata extract ame-
liorates diabetes mellitus, associated behavioral comorbidities
and biochemical parameters in alloxan-induced diabetic rats.
Saudi Pharmaceutical Journal 30, 1373–1386.
Bansal, P., Banerjee, S., 2016. Eect of Withinia Somnifera and Shilajit on
Alcohol Addiction in Mice. Pharmacogn Mag. 12, S121-128.
Birben, E., Sahiner, U.M., Sackesen, C., Erzurum, S., Kalayci, O., 2012. Ox-
idative Stress and Antioxidant Defense. World Allergy Organ. J.
5, 9-19.
Bordet, R., Deplanque, D., Maboudou, P., Puisieux, F., Pu, Q., Robin, E.,
Martin, A., Bastide, M., Leys, D., Lhermitte, M., 2000. Increase in
endogenous brain superoxide dismutase as a potential mecha-
nism of lipopolysaccharide-induced brain ischemic tolerance. J.
Cerebral Blood Flow Metabol. 20, 1190-1196.
Burton, G.J., Jauniaux, E., 2011. Oxidative stress. Best Practice and Re-
search Clinical Obstetrics and Gynaecol. 25, 287-299.
De M. Bandeira, S., Da Fonseca, L.J.S., Da S. Guedes, G., Rabelo, L.A., Gou-
lart, M.O.F., Vasconcelos, S.M.L., 2013. Oxidative Stress as an Un-
derlying Contributor in the Development of Chronic Complica-
tions in Diabetes Mellitus. Int. J. Mol Sci. 14, 3265-3284.
Durg, S., Dhadde, S.B., Vandal, R., Shivakumar, B.S., Charan, C.S., 2015. W
ithania somnifera (Ashwagandha) in neurobehavioural disorders
induced by brain oxidative stress in rodents: A systematic review
and meta-analysis. J. Pharm. Pharmacol. 67, 879-899.
Floyd, R.A., 1999. Antioxidants, Oxidative Stress, and Degenerative Neu-
rological Disorders. Proceedings of the Society for Experimental
Biology and Medicine 222, 236-245.
Gad El-Hak, H.N., Mobarak, Y.M., 2019. The neurotoxic impact of sub-
chronic exposure of male rats to copper oxychloride. J. Trace Ele-
men. Med. Biol. 52, 186-191.
Giri, B., Dey, S., Das, T., Sarkar, M., Banerjee, J., and Dash, S.K., 2018. Chron-
ic hyperglycemia mediated physiological alteration and metabol-
ic distortion leads to organ dysfunction, infection, cancer pro-
gression and other pathophysiological consequences: an update
on glucose toxicity. Biomed. Pharmacother. 107, 306-328.
Gunbatar, H., Kaplan, H., Yildiz, S., 2020. Is there a correlation between
obstructive sleep-apnea syndrome severity and prolidase activity
as an oxidative stress marker? Nigerian J. Clin. Pract. 23, 252-252.
Gupta, D., Kurhe, Y., Radhakrishnan, M., 2014. Antidepressant eects of
insulin in streptozotocin induced diabetic mice: Modulation of
brain serotonin system. Physiol. Behavior. 129, 73-78.
Halliwell, B., 1991. Reactive oxygen species in living systems: source, bio-
chemistry, and role in human disease. Amer. J. Med. 91, S14-S22.
Hamby-Mason, R., Chen, J.J., Schenker, S., Perez, A., Henderson, G.I., 1997.
Catalase mediates acetaldehyde formation from ethanol in fetal
and neonatal rat brain. Alcoholism: Clin. Exper. Res. 21, 1063-
1072.
Hosny, E.N., El-Gizawy, M.M., Sawie, H.G., Abdel-Wahhab, K.G., Khadrawy,
Y.A., 2021. Neuroprotective Eect of Ashwagandha Extract
against the Neurochemical Changes Induced in Rat Model of Hy-
pothyroidism. J. Dietary Suppl. 18, 72-91.
Hozayen, W.G., Mahmoud, S.S., Amin, K.A., Ahmed, R.R., 2012. Modulato-
ry Eects of Grape Seed Extract on Brain Neurotransmitters and
Oxidative Stress in Alloxan Diabetic Rats. J. Amer. Sci. 8, 12.
Jahanbakhsh, S.P., Manteghi, A.A., Emami, S.A., Mahyari, S., Gholampour,
B., Mohammadpour, A.H., Sahebkar, A., 2016. Evaluation of the
ecacy of Withania somnifera (Ashwagandha) root extract in pa-
tients with obsessive-compulsive disorder: A randomized dou-
ble-blind placebo-controlled trial. Complem. Therap. Med. 27,
25-29.
John, J., 2014. Therapeutic potential of Withania somnifera: a report on
phyto-pharmacological properties. Inter. J. Pharm. Sci. Res. 5,
2131-2148.
Kapoor, R., Srivastava, S., Kakkar, P., 2009. Bacopa monnieri modulates
antioxidant responses in brain and kidney of diabetic rats. Envi-
ron. Toxicol. Pharmacol. 27, 62-69.
Khan, M.A., Subramaneyaan, M., Arora, V.K., Banerjee, B.D., Ahmed, R.S.,
2015. Eect of Withania somnifera (Ashwagandha) root extract
on amelioration of oxidative stress and autoantibodies produc-
tion in collagen-induced arthritic rats. J. Compl. Integr. Med. 12,
117-125.
Kowalczuk, K., Stryjecka-Zimmer, M., 2002. The inuence of oxidative
stress on the level of malondialdehyde (MDA) in dierent areas of
the rabbit brain, In: Annales Universitatis Mariae Curie-Sklodows-
ka. Sectio D: Medicina, pp. 160-164.
Ma, L., Liu, J., Li, N., Wang, J., Duan, Y., Yan, J., Liu, H., Wang, H., Hong, F.,
2010. Oxidative stress in the brain of mice caused by translocated
nanoparticulate TiO2 delivered to the abdominal cavity. Bioma-
terials 31, 99-105.
Malik, Z.A., Singh, M., Sharma, P.L., 2011. Neuroprotective eect of Mo-
mordica charantia in global cerebral ischemia and reperfusion
induced neuronal damage in diabetic mice. J. Ethnopharmacol.
133, 729-734.
Manjarrez-Gutiérrez, G., Hernández-Rodríguez, J., 2016. Clinical and basic
studies on diabetes and brain serotonin. Int. J. Endocrinol. Metab.
Disord. 2, 1-6.
Martínez-Tellez, R., de Jesús Gómez-Villalobos, M., Flores, G., 2005. Al-
teration in dendritic morphology of cortical neurons in rats with
diabetes mellitus induced by streptozotocin. Brain Res. 1048,
108-115.
MK, J., Prathima, C., Huralikuppi, J., Suresha, R., Murali, D., 2012. An-
ti-depressant eects of Withania somnifera fat (Ashwagandha
Ghrutha) extract in experimental mice. IJPBS 3, 33-42.
Navaie, F., Hassanzadeh, G., Mahakizadeh, S., Mehrannia, K., Alizamir, T.,
Dashti, N., Poorhassan, M., Mokhtari, T., Akbari, M., 2018. Anti-ox-
idative and neuroprotective eects of supplementary axseed on
oxidative damage in the hippocampus area of a rat model of hy-
poxia. Arch. Neurosci. 5, e60193.
Okutan, H., Ozcelik, N., Ramazan Yilmaz, H., Uz, E., 2005. Eects of caeic
acid phenethyl ester on lipid peroxidation and antioxidant en-
zymes in diabetic rat heart. Clin. Biochem. 38, 191-196.
Ozougwu, J., Obimba, K., Belonwu, C., Unakalamba, C., 2013. The patho-
genesis and pathophysiology of type 1 and type 2 diabetes mel-
litus. J. Physiol. Pathophysiol. 4, 46-57.
Parihar, P., Shetty, R., Ghafourifar, P., Parihar, M.S., 2016. Increase in oxi-
dative stress and mitochondrial impairment in hypothalamus of
streptozotocin treated diabetic rat: Antioxidative eect of Witha-
nia somnifera. Cell. Mol. Biol. 62, 77-83.
Pierzchala, K., Simicic, D., Sienkiewicz, A., Sessa, D., Mitrea, S., Braissant, O.,
McLin, V., Gruetter, R., Cudalbu, C., 2022. Central nervous system
and systemic oxidative stress interplay with inammation in a bile
duct ligation rat model of type C hepatic encephalopathy. Free
Rad. Biol. Med. 178, 295-307.
Pop-Busui, R., Sima, A., Stevens, M., 2006. Diabetic neuropathy and oxida-
tive stress. Diabetes/metabolism Res. Rev. 22, 257-273.
Priyanka, G., Anil Kumar, B., Lakshman, M., Manvitha, V., Kala Kumar, B.,
2020. Adaptogenic and Immunomodulatory Activity of Ashwa-
gandha Root Extract: An Experimental Study in an Equine Model.
Front. Vet. Sci. 7, 541112.
Roriz-Filho, J.S., Sá-Roriz, T.M., Rosset, I., Camozzato, A.L., Santos, A.C.,
Chaves, M.L.F., Moriguti, J.C., Roriz-Cruz, M., 2009. (Pre) diabetes,
brain aging, and cognition. Biochimica et Biophysica Acta (BBA) -
Mol. Basis Dis. 1792, 432-443.
Heba A. Hashem et al. /Journal of Advanced Veterinary Research (2023) Volume 13, Issue 3, 508-514
513
Samarghandian, S., Azimi-Nezhad, M., Samini, F., Farkhondeh, T., 2015.
Chrysin treatment improves diabetes and its complications in liv-
er, brain, and pancreas in streptozotocin-induced diabetic rats.
Can. J. Physiol. Pharmacol. 94, 388-393.
Sánchez, S., Sánchez, C., Paredes, S.D., Cubero, J., Rodríguez, A.B., Barriga,
C., 2008. Circadian variations of serotonin in plasma and dierent
brain regions of rats. Mol. Cell. Biochem. 317, 105-111.
Sharma, Y., Bashir, S., Irshad, M., Nag, T.C., Dogra, T.D., 2005. Dimetho-
ate-induced eects on antioxidant status of liver and brain of rats
following subchronic exposure. Toxicology 215, 173-181.
Sima, A.A., 2010. Encephalopathies: the emerging diabetic complications.
Acta Diabetologica 47, 279-293.
Singh, N., Bhalla, M., de Jager, P., Gilca, M., 2011. An overview on ashwa-
gandha: a Rasayana (rejuvenator) of Ayurveda. African Journal of
Traditional, Complementary and Alternative Medicines: AJTCAM
8(5 Suppl), 208–213.
Singh, A., Kukreti, R., Saso, L., Kukreti, S., 2019. Oxidative stress: a key
modulator in neurodegenerative diseases. Molecules 24, 1583.
Speers, A.B., Cabey, K.A., Soumyanath, A., Wright, K.M., 2021. Eects of
Withania somnifera (ashwagandha) on stress and the stress-relat-
ed neuropsychiatric disorders anxiety, depression, and insomnia.
Curr. Neuropharmacol. 19, 1468.
Sudhakara, G., Ramesh, B., Mallaiah, P., Sreenivasulu, N., Saralakumari, D.,
2012. Protective eect of ethanolic extract of Commiphora mukul
gum resin against oxidative stress in the brain of streptozotocin
induced diabetic Wistar male rats. Excli. J. 11, 576-592.
Tan, S.W., Xie, T., Malik, T.H., Gao, Y., 2022. . Advances of neurovascular
protective potential of 3-N-butylphthalide and its derivatives in
diabetic related diseases. J. Diab. Complications 36, 108335.
Terpstra, M., Henry, P.G., Gruetter, R., 2003. Measurement of reduced glu-
tathione (GSH) in human brain using LCModel analysis of dier-
ence-edited spectra. Magnetic Resonance in Medicine: An O-
cial Journal of the International Society for Magnetic Resonance
in Medicine 50, 19-23.
Tiwari, B.K., Pandey, K.B., Abidi, A.B., Rizvi, S.I., 2013. Markers of Oxidative
Stress during Diabetes Mellitus. J. Biomarkers 2013, 378790.
Tran, L., Zielinski, A., Roach, A.H., Jende, J.A., Householder, A.M., Cole, E.E.,
Atway, S. A., Amornyard, M., Accursi, M.L., Shieh, S.W., Thompson,
E.E., 2015. Pharmacologic treatment of type 2 diabetes: oral med-
ications. Annals Pharmacother. 49, 540–556.
Uzar, E., Alp, H., Cevik, M.U., Fırat, U., Evliyaoglu, O., Tufek, A., Altun, Y.,
2012. Ellagic acid attenuates oxidative stress on brain and sciatic
nerve and improves histopathology of brain in streptozotocin-in-
duced diabetic rats. Neur. Sci. 33, 567-574.
Vincent, A., Russell, J., Low, P., Feldman, E.L., 2004. Oxidative stress in the
pathogenesis of diabetic neuropathy. Endocrine Rev. 25, 612-628.
Visweswari, G., Christopher, R., Rajendra, W., 2013. Phytochemical screen-
ing of active secondary metabolites present in Withania somnif-
era root: role in traditional medicine. Inter. J. Pharm. Sci. Res. 4,
2770-2776.
Wink, D., Hines, H., Cheng, R., Switzer, C., Flores-Santana, W., Vitek, M.,
Ridnour, L., Colton, C., 2011. Nitric oxide and redox mechanisms
in the immune response. J. Leukocyte Biol. 89, 873-891.
Yankelevitch-Yahav, R., Franko, M., Huly, A., Doron, R., 2015. The forced
swim test as a model of depressive-like behavior. JoVE 97, 52587.
Zahiruddin, S., Basist, P., Parveen, A., Parveen, R., Khan, W., Gaurav, Ah-
mad, S., 2020. Ashwagandha in brain disorders: A review of recent
developments. J. Ethnopharmacol. 257, 112876.
Heba A. Hashem et al. /Journal of Advanced Veterinary Research (2023) Volume 13, Issue 3, 508-514
514