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Original Article
EFFICACY OF AN ACTIVE COMPOUND OF THE HERB, ASHWAGANDHA IN PREVENTION OF
STRESS INDUCED HYPERGLYCEMIA
SARJAN H. N., YAJURVEDI H. N.
*
Department of Zoology, University of Mysore, Manasagangotri, Mysore 570006, India
Email: hnyajurvedi@rediffmail.com
Received: 23 Jul 2018 Revised and Accepted: 23 Aug 2018
ABSTRACT
Objective: To find out whether an isolated compound (IC) from the ethanolic extract of roots of ashwagandha prevents stress-induced
hyperglycemia by direct interference with the action of increased concentration of corticosterone on hepatocytes or by preventing hyper-secretion
of corticosterone or both.
Methods: A group of rats served as controls, and those in another group were subjected to restraint (1 h) and forced swimming exercise (15 min),
after a gap of 4 h daily for 4 w. The third group of rats received orally IC (5 mg/kg bw/rat) 1 h prior to exposure to stressors. After the last
treatment period, a blood sample was collected and serum was separated for the estimation of corticosterone and glucose. In in vitro experiment,
hepatocytes were treated with different concentrations of corticosterone (100, 200, 300, 400 and 500 ng/ml). In another set of experiment,
hepatocytes were treated with different doses of IC (1, 10, 100, 1000 and 10 000 μg/ml of medium) along with corticosterone (400ng/ml). The
concentration of glucose and activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were determined after
the treatment.
Results: Stress exposure caused a significant increase in serum concentration of corticosterone and glucose whereas, administration of IC did not
result in similar changes. Further, treatment of corticosterone in in vitro significantly increased the activities of PEPCK and G6Pase and
concentration of glucose in a dose-dependent manner in hepatocytes. However, treatment with IC did not interfere with the corticosterone-induced
an increase in the activities of PEPCK and G6Pase as well as the concentration of glucose in hepatocytes.
Conclusion: The in vivo and in vitro results put together reveal that IC does not directly interfere with the action of corticosterone on hepatocytes.
However, it prevents stress-induced hyperglycemia by suppressing hyper-secretion of corticosterone.
Keywords: Ashwagandha, Corticosterone, G6Pase, Gluconeogenesis, PEPCK
© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2 018v10i10.28717
INTRODUCTION
Stress disturbs the homeostatic equilibrium of the body and makes
individuals susceptible to diseases. Stress affects physiological
energy balance via the activation of hypothalamo-pituitary-
adrenocortical (HPA) and sympathetic-adreno-medullary (SAM)
axes by producing an excess amount of glucocorticoids (GC) and
catecholamines respectively [1]. The Liver is a central organ of
energy metabolism and regulates carbohydrate metabolism in all
vertebrates. A variety of environmental factors viz., fasting, hypoxia,
temperature, stress and seasonality alter the liver metabolism [2].
The hepatocytes are the sites of endogenous glucose synthesis
(gluconeogenesis) from non-carbohydrate sources. The
neuroendocrine response to stress is characterized by excessive
gluconeogenesis, glycogenolysis and insulin resistance. Chronic
stress results in hyperglycemia affecting all the pathways of
carbohydrate metabolism viz. glycolysis, tricaboxylic acid cycle,
gluconeogenesis, hexose monophosphate shunt, glycogenolysis and
glycogenesis [3-5]. Numerous studies reveal stress-induced
alterations in the enzyme activities and concentrations of substrates
of different pathways of carbohydrate metabolism [4, 6-8]. Stress
increases gluconeogenesis by increasing the activities of key
gluconeogenic enzymes viz. phosphoenolpyruvate carboxykinase
(PEPCK), fructose-1, 6-bisphosphatase and glucose-6-phosphatase
(G6Pase) and serves as a mechanism by which the availability of
glucose is maintained. An increase in activities of hepatic G6Pase
and PEPCK under different stressful conditions has been reported
[9-12]. Hyperglycemia due to increased gluconeogenesis has a
negative impact on the body as it results in insulin resistance [13].
Glucocorticoids have long been known to regulate glucose
homeostasis and have a vital role in gluconeogenesis. Metabolic
effects of GC include an increase in blood glucose concentration
through the activation of key enzymes involved in hepatic
gluconeogenesis and inhibition of glucose uptake in peripheral
tissues such as skeletal muscles [14]. Studies have shown that the
primary effect of GC is exerted on existing enzymes to increase their
activities. Numerous in vivo studies have shown the effects of a
higher level of corticosterone on the activities of hepatic
gluconeogenic enzymes in animal models under stressful condition
[4, 10, 12, 15]. However, there are no reports on the effect of
physiological concentration as well as stress level concentration of
corticosterone on the activities of gluconeogenic enzymes of
hepatocytes in in vitro.
It is reported that stress-induced higher levels of GC affect different
pathways of carbohydrate metabolism which might lead to
metabolic syndrome like diabetes [16]. Since stress cannot be
avoidable in the modern day society, the effects of stress can be
controlled or prevented with suitable remedies. Though, numerous
anti-stress synthetic compounds are available to prevent stress
effects, because of their undesirable side effects there is a need for
better compounds that can prevent stress effects and maintain
normoglycemia despite experiencing stressful conditions. The herb
ashwagandha (Withania somnifera), also knew as Indian Ginseng or
Winter cherry has been well documented in the Ayruveda the
traditional Indian medicine system and has multiple biological
properties viz. antioxidant, adaptogenic, aphrodisiac, astringent and
antiulcer [17-20]. Root extracts of ashwagandha are known to
prevent stress-induced hyperglycemia, alterations in hepatic
enzyme activities and glucose intolerance in in vivo [21-23]. It is also
known that a root extract of ashwagandha prevents a rise in stress-
induced glucocorticoid levels [22, 23] in in vivo. However, whether
the normoglycemia maintained by ashwagandha under stress
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 10, Issue 10, 2018
Yajurvedi et al.
Int J Pharm Pharm Sci, Vol 10, Issue 10, 44-49
45
exposure is due to the prevention of excess secretion of GC alone or
due to direct interference with gluconeogenic enzymes in
hepatocytes or both is not known. Hence, in the present study effects
of an isolated compound (IC) from root extract of ashwagandha on
corticosterone-induced alterations in activities of gluconeogenic
enzymes in hepatocytes in vitro were studied to understand whether
or not IC acts directly on hepatocytes.
MATERIALS AND METHODS
Chemicals
Corticosterone, Krebs-Ringer-Hepes (KRH), glucose 6 phosphate,
dithiothreitol, adenosine diphosphate (ADP), 3-phosphoglycerate,
phosphoenolpyruvate, 3-phosphoglycerate phosphokinase, and
glyceraldehyde 3-phosphate dehydrogenase were purchased from
Sigma Aldrich (United Kingdom). Tris-hydrochloric acid (HCL),
trichloroacetic acid, sodium bicarbonate (NaHCO3), magnesium
chloride (MgCl2) and BIS-TRIS buffer were obtained from Merks
specialties, Pvt. Ltd (Mumbai, India). Serum glucose was measured by
using the kit supplied by Span Diagnostics Ltd. (Gujarat, India). The
ELISA kit for the estimation of serum concentration of corticosterone
was supplied by Demeditec Diagnostics GmbH, Germany). All the other
reagents and chemicals used were of analytical grade.
Animals
Adult male albino Wistar rats weighing 200-220 g were obtained from
the inbred colony of the central animal facility of University of Mysore.
The rats were provided a standard rat chow and water ad libitum and
were kept in temperature 27±2˚C, under 12 h: 12 h light: dark cycle
(light on 07:00-19:00 h). The experimental design was approved by
Institutional Animal Ethics Committee of University of Mysore, India
(Reference number UOM/IAEC/ 07/2016) and guidelines of the
committee was followed for care and treatment of the rats.
Isolation of active compound from ethanolic extract (EE) of
ashwagandha root
Earlier in vivo and in vitro studies showed the potential anti-stress
activity of EE in roots of ashwagandha [22]. Hence, active compound
was isolated from the EE as per the procedure of Nirupama et al.
[23]. The EE was subjected to thin layer chromatography and was
further fractionated in column chromatography. The extract was
first run on thin layer chromatography plate to detect the number of
compounds. The solvent system used contained chloroform and
methanol. The same solvent was used for column chromatography
to isolate a single compound. The compound isolated was subjected
to nuclear magnetic resonance, infrared spectroscopy and liquid
chromatography-mass spectrometry studies for the characterization
of its chemical nature.
In vivo experiment
Adult male rats were divided into three groups of 5 animals each
(n=5). First group rats were treated as controls and were maintained
in a normal condition without any disturbance. Animals in the second
group were exposed to two stressors viz., restraint (1 h) followed by a
gap of 4 h to force swimming exercise (15 min) for 4 w. Each rat in the
third group was orally treated with IC (5 mg/kg bw/rat) 1 h prior to
exposure to stressors similar to rats in the second group. The fasting
blood glucose concentration was measured in animals of all groups at
weekly intervals. After the treatment period, the serum concentration
of corticosterone was estimated by ELISA using kit and methods of the
manufacturer (Demeditec Diagnostics GmbH, Germany).
In vitro experiment
Two sets of in vitro experiments were conducted using the
hepatocytes. In the first experiment dose-dependent effect of
corticosterone, if any on the activity of hepatic G6Pase and PEPCK
were studied. The effect of IC on the action of corticosterone on the
activities of gluconeogenic enzymes in hepatocytes was studied in
the second experiment. Three replicates were used for each dose.
Preparation of the liver tissue slice system
The liver for these studies was collected from control rats used for in
vivo experiment. The liver lobes were processed following the
procedure of Wormser et al. [24]. Briefly, the liver lobes were placed
on a glass surface and sliced into small pieces of about 0.5 x 0.5 x 0.5
mm. Slices were incubated with KRH medium for 1 h and washed
every 10 min with this medium. The slices were then divided into
small portions (100-120 mg wet weight each) and were incubated in
glass tubes containing 2 ml KRH medium at 37 ˚C for l h before
experimentation.
Effect of corticosterone on the activities of key enzymes of
gluconeogenesis
The liver slices were incubated with different concentrations of
corticosterone (100, 200, 300, 400 and 500 ng/ml of medium) at 37
˚C for 2.5 h. At the end of incubation, the tissue homogenate was
used for the estimation of the activities of key gluconeogenic
enzymes, G6Pase and PEPCK and concentration of glucose.
Effect of IC on corticosterone-induced alterations in the
activities of key enzymes of gluconeogenesis
The liver slices were incubated with the different concentrations of
IC (1, 10, 100, 1000 and 10 000 μg/ml of medium) and 400 ng
corticosterone at 37 ˚C for 2.5 h. At the end of incubation, the tissue
homogenate was used for the estimation of activities of G6Pase and
PEPCK and concentration of glucose.
Activity of G6Pase
The activity of G6Pase was estimated following the method of
Zhu et al. [25]. Liver slices were homogenized in 0. 25 M sucrose
solution and centrifuged at 3000 rpm for 15 min. The
supernatant containing enzyme source (1 µl) was mixed with
100 mmol BI S-TRIS buffer and 200 mmol of glucose 6 phosphate
(subs trat e). The reaction mixture was inc ubated at 37 ˚C for 5
min. The reaction was stoppe d by addi ng 20 % trichloroacetic
acid, incubated for 5 m in at 25 ˚C and centrifuged at 4000 rpm
for 10 min to remove the precipitate. The supernatant (1 µl) was
mixed with Taussky shorr color reagent for the development of
color . The mixture was incubated at 25 ˚C for 5 min a nd t he
optic al density was measured at 660 nm. The specific activity
was e xpressed as μmol/mg/min.
Activity of PEPCK
The activity of PEPCK was estimated following the method of Kin et
al. [26]. The enzyme sample was prepared in 5 ml of 100 mmol tris-
HCL at 4 ˚C. The sample (200 µl) was added to the reaction medium
containing 500 mmol tris-HCl (pH 6.6), 350 mmol NaHCO3, 160
mmol MgCl2, 6 mmol NADH, 20 mmol dithiothreitols, 0.2 M ADP, 36
mmol 3-phosphoglycerate, 50 mmol phosphoenolpyruvate, 3-
phosphoglycerate phosphokinase and glyceraldehyde 3-phosphate
dehydrogenase. The absorbance was read at 340 nm for 60 seconds
at room temperature using a UV-Visible spectrophotometer.
Concentration of glucose
The concentration of glucose was estimated by glucose oxidase and
peroxidase method using a kit manufactured by Arkray Healthcare,
India and the procedure of the manufacturer was adopted. The
sample (10 µl) was added to 1 ml reagent and incubated at 37 ˚C for
10 min. Optical density was read at 505 nm and the concentration of
glucose was expressed as mg/dl.
Statistical analysis
Mean±SE of each parameter was computed considering the data on
at least 5 rats per group (N=5) and 3 replicates for each dose of in
vivo and in vitro experiments respectively. The mean values of each
parameter of different groups were compared using one way ANOVA
followed by Duncan's multiple range test and judged significant if
P<0.05.
RESULTS
In vivo experiment
A significant increase in the serum concentration of corticosterone
was observed in stressed rats compared to controls, whereas that
of IC pretreated rats exposed to stressors was similar to controls
(fig. 1).
Yajurvedi et al.
Int J Pharm Pharm Sci, Vol 10, Issue 10, 44-49
46
Fig. 1: All values are mean±SE. Vertical bars to show serum concentration of corticosterone in control, stress and IC pretreated rats
exposed to stress. Note the higher level of corticosterone in stressed rats compared with controls and IC pretreated rats exposed to stress.
Bars with the same superscript letters do not significantly differ among themselves whereas those with different superscript letters
significantly differ. IC-isolated compound from ashwagandha (n=5)
There was a significant increase in the fasting blood glucose
concentration in stress group rats from 1
st
w through 4
th
w,
compared to controls. The IC pretreated rats exposed to stress did
not differ from controls (fig. 2).
Fig. 2: All values are mean±SE. Fasting blood glucose concentrations in control, stress and IC pretreated rats exposed to stress. Note the
higher level of glucose in stressed rats compared with controls and IC pretreated rats exposed to stress. Groups with the same superscript
letters at each weekly interval do not significantly differ among themselves whereas those with different superscript letters significantly
differ. IC-isolated compound from ashwagandha (n=5)
In vitro experiment
Activities of G6Pase and PEPCK and concentration of glucose in
hepatocytes
There was a significant dose-dependent increase in the activities of
G6Pase and PEPCK following treatment with increasing doses of
corticosterone (100, 200, 300, 400 and 500 ng/ml) compared to
control and vehicle control. However, G6Pase activity after the
treatment of 300 ng did not significantly differ either from that of 200
ng or 400 ng and that of PEPCK after the treatment of 400 ng did not
significantly differ either from that of 300 ng or 500 ng (table 1).
The concentration of glucose in the hepatocytes showed a dose-
dependent significant increase following treatment with increasing
doses of corticosterone (100, 200, 400 and 500 ng/ml) compared to
control and vehicle controls.
However, glucose concentration after treatment of 300 ng did not
significantly differ either from that of 200 ng or 400 ng (table 1).
Table 1: Effect of corticosterone on the activities of key enzymes of gluconeogenesis and concentration of glucose in hepatocytes in vitro
Groups
Glucose 6 phosphatase (μmol/mg/min)
Phosphoenolpyruvate
carboxykinase
(U/mg protein)
Glucose mg/dl
Control
2.74
±
0.0
4
a
2.29
±
0.08
a
29.35
±
0.87
a
Control
+
0.1 %
DMSO
2.72
±
0.08
a
2.25
±
0.05
a
29.43
±
0.62
a
100 ng corticosterone
3.30
±
0.09
b
4.40
±
0.10
b
33.32
±
0.73
b
200 ng corticosterone
3.76
±
0.21
c
4.86
±
0.04
c
38.17
±
0.76
c
300 ng corticosterone
4.07
±
0.15
cd
5.50
±
0.06
d
40.32
±
1.69
cd
4
00 ng corticosterone
4.43
±
0.12
d
5.88
±
0.03
de
42.13
±
1.47
d
500 ng corticosterone
5.47
±
0.20
e
6.06
±
0.14
e
46.16
±
1.74
e
ANOVA F
Value
(df = 6, 28)
25.56
P<0.001
312.52
P<0.001
21.77
P<0.001
Note: All values are mean±SE, Groups with the same superscript letters do not significantly differ among themselves, whereas groups with different
superscript letters significantly (P<0.05) differ as judged by ANOVA followed by Duncan’s test. df: degree of freedom. DMSO-dimethyl sulfoxide
(n=3).
Yajurvedi et al.
Int J Pharm Pharm Sci, Vol 10, Issue 10, 44-49
47
The activities of hepatic G6Pase and PEPCK and concentration of
glucose were significantly increased in hepatocytes following
treatment with corticosterone alone as well as cortico-
sterone+different concentrations of IC (1, 10, 100, 1000 and 10 000
μg/ml) compared to controls and vehicle controls (1 % carboxy
methyl cellulose) (table 2).
Table 2: Effect of a compound isolated from ashwagandha root on corticosterone-induced alterations in the activities of key enzymes of
gluconeogenesis and concentration of glucose in hepatocytes in vitro
Groups
Glucose 6 phosphatase
(μmol/mg/min)
Phosphoenol pyruvate
carboxy kinase
(U/mg
protein)
Glucose
mg/dl
Control
2.80
±
0.08
a
2.44
±
0.05
a
29.83
±
0.71
a
Control
+
1
%
CMC
2.73
±
0.08
a
2.41
±
0.07
a
29.91
±
0.52
a
400 ng c
orticosterone
4.12
±
0.09
b
5.61
±
0.06
b
41.66
±
0.65
b
400 ng corticosterone
+
1
μg IC
4.01
±
0.11
b
5.53
±
0.09
b
40.14
±
0.42
b
400 ng corticosterone
+
10
μg IC
4.09
±
0.23
b
5.60
±
0.10
b
39.49
±
0.90
b
400 ng corticosterone
+
100
μg IC
4.07
±
0.08
b
5.47
±
0.05
b
40.69
±
1.25
b
400 ng co
rticosterone
+
1000
μg IC
4.41
±
0.09
b
5.46
±
0.06
b
40.65
±
1.26
b
400 ng corticosterone
+
10
000
μg IC
4.20
±
0.13
b
5.51
±
0.05
b
40.77
±
0.48
b
ANOVA F
Value
(df=7, 32)
26.65
P<0.001
406.92
P<0.001
34.51
P<0.001
Note: All values are mean±SE, Groups with the same superscript letters do not significantly differ among themselves, whereas groups with different
superscript letters significantly (P<0.05) differ as judged by ANOVA followed by Duncan’s test. df: degree of freedom. CMC-carboxy methylcellulose,
IC-isolated compound from ashwagandha (n=3).
DISCUSSION
Metabolic stress responses are mediated by hormones of the adrenal
gland [27] and GC have long been known to regulate glucose
homeostasis [28]. In the present study, increased concentration of
corticosterone in stressed rats accompanied with higher blood
glucose level indicates that stress-induced excess production of GC
affected glucose metabolism. Blood glucose level is an important
indicator of stress responses in animal [29] and a higher level of
glucose in stressed rats is due to enhanced hepatic gluconeogenesis
[30]. This was further supported by the present in vitro study
wherein treatment of different doses of corticosterone significantly
increased the activities of gluconeogenic enzymes, G6Pase and
PEPCK of hepatocytes in a dose-dependent manner. Although most
steps in gluconeogenesis are the reverse of those found in glycolysis,
PEPCK and G6Pase regulate the rate process of gluconeogenesis by
converting oxaloacetate to phosphoenolpyruvate and glucose-6-
phosphate to glucose respectively [31]. The over-expression of these
enzymes in hepatocytes results in a reduction of glycogen synthesis
and an increase in the production of glucose [32]. Hence, studies
related to carbohydrate metabolism mainly focus on changes in the
activities of these hepatic enzymes.
An increase in the activities of gluconeogenic enzymes due to higher
level of endogenous GC under stressful condition has been observed
in in vivo system [10, 12, 15]. GC increase the synthesis of PEPCK
and G6Pase by increasing the expression of their transcription
factors [33, 34]. In addition to transcriptional regulation, studies
have shown that GC directly enhance the activities of gluconeogenic
enzymes under stressful condition [35-37]. Stress-induced increased
gluconeogenesis results in hyperglycemia. Present in vivo and in
vitro studies support this view as there was a significant increase in
serum glucose concentration in in vivo as well as in hepatocytes in in
vitro system accompanied with increased activities of PEPCK and
G6Pase of hepatocytes in vitro following corticosterone treatment.
The physiological concentration of corticosterone varies from 50
to100 ng/ml and during stressful condition it rises from 120 to 425
ng/ml. In the present study, corticosterone at the dose in the range
of stress-induced alterations (100-500 ng/ml) significantly
increased the activities of key enzymes of gluconeogenesis and
glucose concentration in a dose-dependent manner. Thus action
resembled the action of endogenous GC on glucose metabolism
during stress. The severe implication of GC induced chronic
hyperglycemia is insulin resistance and glucose intolerance [38]
which may lead to type 2 diabetes mellitus [29]. Since stress induced
higher level of GC is the main cause of hyperglycemia, it is logical
that, either suppression of hypersecretion of GC or interference with
action of corticosterone on hepatocytes under stressful conditions
could prevent hyperglycemia and insulin resistance and consequent
pathological implications.
Interestingly, pretreatment of IC in rats exposed to stressors
maintained normoglycemia and normal blood corticosterone levels as
blood glucose level and corticosterone level of IC pretreated rats
exposed to stressors were similar to controls in contrast to a
hyperglycemic condition in rats exposed to stressors alone. In
contrast, in in vitro system, treatment of IC did not prevent
corticosterone induced enhanced activity of G6Pase and PEPCK as well
as the concentration of glucose in hepatocytes as there was no
difference in the activities of these enzymes and glucose concentration
in corticosterone alone and corticosterone+IC treated hepatocytes.
The fact that IC maintained normal blood concentration of glucose and
corticosterone in stressed rats in in vivo and it did not prevent
corticosterone induced increase in activities of gluconeogenic enzymes
and glucose concentration in hepatocytes in in vitro, indicates that
normoglycemic effect of IC during stressful condition in vivo is by
suppressing the HPA activation and subsequent excess production of
GC, whereas it does not directly interfere with the action of
corticosterone on hepatocytes. This view is further supported by a
report of Nirupama et al. [23] wherein treatment of IC from EE of
ashwagandha root normalized the adrenal activity as well as the
activities of gluconeogenic enzymes under stressful condition.
Ashwagandha is known to stimulate the central nervous system (CNS)
[17, 39] by modulating different neurotransmitter receptors
particularly gamma-aminobutyric acid (GABA) [40]. The GABAergic
neurons inhibit HPA activity by reducing the secretion of
adrenocorticotropic hormone [41, 42]. However, under stressful
condition there is depletion of GABA receptor binding in the CNS [43,
44] which leads to the activation of the HPA axis. Studies have shown
that extracts and active compounds of ashwagandha have GABA-like
activity and also increase the production of GABA in brain [45-47].
Therefore, it is suggested that IC might induce higher levels of GABA or
exert GABA like action in brain under the stressful condition and
thereby suppress the activation of the HPA axis and the subsequent
increase in serum level of corticosterone which is to be substantiated
by future studies.
CONCLUSION
The in vivo and in vitro studies put together reveal that IC does not
interfere with the action of corticosterone on hepatocytes, whereas,
it prevents stress-induced hyperglycemia by preventing hyper-
secretion of corticosterone.
ACKNOWLEDGMENT
The first author acknowledges the Department of Science and
Technology, New Delhi, India, for the award of a fellowship under
INSPIRE scheme. The work was supported by a financial grant from
Yajurvedi et al.
Int J Pharm Pharm Sci, Vol 10, Issue 10, 44-49
48
University Grants Commission, New Delhi, under the Centre for
Advanced Studies Scheme [F.4-20/2015/CAS-I SAP-II].
AUTHORS CONTRIBUTIONS
H N Sarjan performed the experiments and Dr. H N Yajurvedi
designed the study and wrote the paper.
CONFLICT OF INTERESTS
The authors declare that they have no conflicts of interest to
disclose.
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