Clinical Study by Dabur Mediclub- Neuroprotective Effects of Withania somnifera Dunn. in Hippocampal Sub-regions of Female Albino Rat. For more details visit:http://www.daburmediclub.com/clinicalreport/reports.aspx

1. SHORT COMMUNICATION
Neuroprotective Effects of Withania somnifera
Dunn. in Hippocampal Sub-regions of Female
Albino Rat
Sushma Jain,1
Sunil Dutt Shukla,1
Kanika Sharma2
and Maheep Bhatnagar1
*
1
Department of Zoology, University College of Science, M.L. Sukhadia University, Udaipur 313001, India
2
Department of Botany, University College of Science, M.L. Sukhadia University, Udaipur 313001, India
The neuroprotective effects of W. somnifera were studied on stressed adult female Swiss albino rats.
Experimental rats were subjected to immobilization stress for 14 h and were treated with a root powder
extract of W. somnifera available as Stresscom capsules (Dabur India Ltd). Control rats were maintained
in completely, non stressed conditions. Thionin stained serial coronal sections (7mm) of brain passing
through the hippocampal region of stressed rats (E1 group) demonstrated 85% degenerating cells (dark
cells and pyknotic cells) in the CA2 and CA3 sub-areas. Treatment with W. somnifera root powder extract
significantly reduced (80%) the number of degenerating cells in both the areas. The study thus demon-
strates the antistress neuroprotective effects of W. somnifera. Copyright # 2001 John Wiley & Sons, Ltd.
Keywords: W. somnifera; root powder; hippocampus; cell degeneration; immobilization stress; rat.
INTRODUCTION
Withania somnifera Dunn. (Family Solanaceae) com-
monly known as ‘Ashwagandha’ is used as ‘Medhya
Rasayana’ for the treatment of mental diseases and
anxiety states, in the traditional Indian medicinal system
(Chatterjee and Prakesh, 1995). It is an erect shrub 0.5–2
m high and grows throughout the dry and sub-tropical
parts of India. The drug reduces the levels of acetylcho-
line and catecholamine and increases the levels of
serotonin and histamine in brain tissue (Handa, 1993).
W.somnifera has also been shown to possess both
depressant and anticonvulsant properties (Kulkarni and
Verma, 1993; Kulkarni et al., 1993; Kulkarni and George,
1996). Ziauddin et al., (1996) studied its adaptogenic,
antistress, anticonvulsant and cognitivedysfunction prop-
erties. The antioxidative effects of the plant extract have
been demonstrated (Bhattacharya et al., 1997; Panda and
Kar, 1997). Cytoprotective effects of W. somnifera have
also been demonstrated in cancer (Uma Devi, 1996).
The present study was undertaken to investigate the
antistress and neuroprotective effects of W. somnifera
root extract on neuron cell bodies in hippocampal sub-
regions of the adult female rat brain.
MATERIALS AND METHODS
Ten adult female Swiss albino rats, body weight 60 Æ 5g,
age 2 months, were maintained in the animal room at a
controlled temperature (26° Æ 2°C) and a light and dark
cycle (12 h light and 12 h dark) for 7 days and were
provided with food and water ad libitum. Rats were
divided into two groups: control and experimental
groups.
Control group. Rats were placed in the animal room
(n = 3 per cage). It was locked for 24 h. The next morning
the animal room was opened and the rats were killed
immediately by decapitation.
Experimental group. Experimental animals were di-
vided into five groups E1–E5. Animals in these groups
were subjected to stress for specific periods and treated
with the experimental drug.
Stress protocol. Rats were placed in tightly fitting
ventilated plastic boxes(10 Â 5cm) for a specific stress
period (14 h) every day. The animals were unable to
move in these boxes and thus became stressed. Stress was
given for 30 days. The stress period was selected on the
basis of a pilot study to obtain the maximum percentage
cell degeneration in the CA1-Dg region. Although
habituation was observed in the general behaviour, cell
degeneration was complete.
Drug preparation. An extract of W.somnifera Dunn.
available as a commercial preparation Stresscom capsule
(Dabur India Ltd), was used. The capsule consisted of a
hydroalcoholic extract of Ashwagandha root powder. This
extract was standardized for withanolids and withnoles.
Soya lectins, bees wax and arachis oil was used as a base
for medicament and packed in soft gelatin capsules. The
capsule was dissolved in water and given orally, daily
(20mg/kg body weight) for 30 days. Treatment was
performed everyday between 9 am and 10 am.
PHYTOTHERAPY RESEARCH
Phytother. Res. 15, 544–548 (2001)
DOI: 10.1002/ptr.802
Copyright # 2001 John Wiley & Sons, Ltd.
* Correspondence to: Dr M. Bhatnagar, Associate Professor, Department of
Zoology, University College of Science, M.L. Sukhadia University, Udaipur
313001, India.
E-mail: mbhatnagar@yahoo.com
Received 4 November 1998
Accepted 10 August 2000

2. Figure 1. (1) Photomicrograph of CA2 hippocampal region of the control animal (C) showing
normal cell bodies (→) (Â40). (2) Photomicrograph demonstrating CA2 region of stress (E1)
group totally dark cell (") with few pyknotic nuclei (A) (Â40). (3) Photomicrograph
demonstrating CA2 region of dose (E2) group. Cells showing normal cell morphology (Â40).
(4) Photomicrograph demonstrating CA2 region of dose ‡ stress (E3). Normal cells with few
pyknotic nuclei (Â40).
Figure 2. (1) Photomicrograph showing CA3 hippocampal sub-region of control (C). Normal
cell bodies (→) present (Â40). (2) Photomicrograph demonstrating CA3 region of stress (E1)
group showing dark cells (") with pyknotic nuclei (A) (Â40). (3) Photomicrograph showing
CA3 region of dose ‡ stress (E3) group. All are normal cell bodies (2) (Â40). (4)
Photomicrograph showing CA3 of dose ‡ stress (E3) group demonstrating normal cell (→)
(Â40).
NEUROPROTECTIVE EFFECTS OF WITHANIA SOMNIFERA 545
Copyright # 2001 John Wiley & Sons, Ltd. Phytother. Res. 15, 544–548 (2001)

3. Treatment Schedule. Group E1: Rats were subjected to
14h stress every day for 30 days and killed on day 31.
Group E2: Normal rats were given the drug daily for 30
days.
Group E3: Rats were given the drug daily for 30 days
and from day 31 onwards were subjected to stress for 30
days.
Group E4: Rats were subjected to stress for 30 days and
from day 31 onwards given the drug for 30 days.
Group E5: Rats were given the drug and stress daily for
30 days.
Parameters of study. Daily body weight, food and water
consumption was recorded. After completion of the
experiments, the rats were killed by decapitation, and
then the brain was dissected out. The ascorbic acid level
(n = 4) was measured (Natelson, 1971), in the whole fore
brain homogenate to confirm the stress effects.
The remaining brain tissue (n = 6) was rinsed in
distilled water and fixed for 12 h in 10% neutral chilled
formalin. After fixation the brains were dehydrated in
alcohol series, embedded in paraffin wax and 7mm thick
serial coronal sections were cut. Sections were stained
with thionin stain (Clark and Sperry, 1945) for demon-
stration of nerve cells.
RESULTS
Hippocampal sub-regions CA1-Dg were studied in
the control (C) and experimental (E1–E5) groups.
Identification of the hippocampal sub-regions was based
on a rat brain atlas by Paxinos and Watson (1986).
Photomicrographs of C, E1, E2 and E3 groups are
included only.
CA2 hippocampal sub-regions
In the control group (Fig. 1.1) the cell bodies were 5–
6 mm in size, and multipolar in shape. All the cells in the
region demonstrated a large and distinct nucleus, a
centrally located nucleolus and darkly stained cytoplasm.
In group E1 (Fig. 1.2) the thionin stained preparation
showed degenerating cells, (85%) darkly stained cells
and pyknotic nuclei in CA2 (Fig. 3). Cell bodies were
3–5mm in size and irregular in shape. A few apoptotic
bodies were observed. The cells were compactly arranged
in a tier of 3–5 cells.
In the E2 group (Fig. 1.3) the cells were large (5–6mm),
with a large centrally located nucleus. The cytoplasm was
darkly stained. No degenerative characteristics were
observed. In the E3 group (Fig. 1.4) the cell bodies were
large, the nucleus and nucleolus were distinct in most of
the cell bodies. The number of dark cell bodies, pyknotic
nuclei or apoptotic bodies were reduced significantly. In
the E4 and E5 groups darkly stained cells, pyknotic nuclei
and vacuolated spaces were observed, similar to the E1
group.
Figure 3. (1) Photomicrograph showing cellular condensation (Â100). (2) Photomicrograph
showing pyknotic cell (Â100). (3) Photomicrograph showing cellular vacualization and swelling
(Â100). Photomicrograph showing dark cell (Â100).
546 S. JAIN ET AL.
Copyright # 2001 John Wiley & Sons, Ltd. Phytother. Res. 15, 544–548 (2001)

4. CA3 hippocampal sub region
In the control group (C, Fig. 2.1), the cells were large
(7–8mm) and multipolar in shape. All the cells demon-
strated a large, distinct nucleus, centrally located
nucleolus nissel material in the cytoplasm was intensely
stained. Cell bodies were arranged compactly in 4–6 tiers
of cells.
In group E1 (Fig. 2.2) the thionin stained preparation
showed degenerating cells, darkly stained cells and
pyknotic nuclei (85%). Pyknotic cell bodies were
comparatively small (4–5 mm) and irregular in shape.
Cell bodies with condensed cytoplasm, apoptotic bodies
and vacuolar spaces were also observed.
In group E2 (Fig. 2.3) the pattern of cell distribution
was similar to the controls. The cells were large with a
distinct nucleus and nucleolus. All the cells were
arranged compactly in the layer. Vacuolar spaces,
pyknotic nuclei etc. were not observed. In group E3
(Fig. 2.4) the cell bodies were large with a distinct and
centrally placed nucleus. The number of vacuolar
spaces, pyknotic nuclei, cells with condensed or
shrunken cytoplasm were drastically reduced. In the E4
and E5 groups darkly stained cells, pyknotic nuclei and
vacuolated spaces were observed, similar to the E1
group.
No significant change in body weight was observed in
the rats of the control or experimental group in the
present study.
The study of ascorbic acid level in the fore brain
homogenate of the control and experimental groups
showed a significant reduction in ascorbic acid level after
stress commensurate with the drug treatment (Fig. 4).
DISCUSSION
The results of the present study demonstrates that
Aswagandha (W. Somnifera Dunn.) produces neuropro-
tective effects and reduces the stress induced changes in
neuron cell bodies in the CA2 and CA3 sub-area of the
hippocampus. Stress caused a significant degeneration in
the cell bodies of these areas showing as dark cells,
pyknotic cells, cells with condensed nuclei and cyto-
plasm. Vacuolar spaces in the cell cytoplasm were
observed in thionin stained serial sections. In sections
demonstrating histochemical localization of Acpase,
passing through the region. After treatment with
Aswagandha root powder extract, a significant reduction
in the number of these degenerated cells was also
observed. Dark cells, cells with shrunken nucleus and
cytoplasm, and vacuolar spaces were significantly
reduced in both CA2 and CA3. Interestingly animals of
group E3 (drug treatment for atleast 30 days given prior
to subjecting to stress) showed more cytoprotective
effects than the post or simultaneous treatment.
Although the mechanism of degeneration and cyto-
protective effects of the drug can not be explained on
basis of these experiments it is possible that an increase in
corticosteroid level during stress might be associated
with the cell degeneration. Several authors have reported
the neurotoxicological effects of corticosterone on
hippocampal cells (Sapolasky et al. 1985, 1991; Gould
et al., 1990). A significant decrease in the ascorbic acid
level in the brain homogenate of the stressed group and
restoration to the normal level after treatment with the
herbal drug substantiate our observation.
The cytoprotective properties of W. somnifera and
several other plants have been studied earlier. Al-Harbi et
al. (1997) showed anti-ulcer and gastric cytoprotective
effects of Commifora mol mol. Zhu et al. (1997) showed
such effects using Cyperus rotundus in rats. Dhuley and
Naik (1997) demonstrated the protective effects of a
Rhinax herbal formulation on CCl4 induced liver injury.
Uma Devi et al. (1993) demonstrated cytoprotective
andradiosenitization effects of W.somnifera.
The present study is the first to report the cytoprotec-
tive antistress effects of W.somnifera on hippocampal
cell bodies.
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