Vascular Pharmacology 44 (2006) 406 – 410

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Immunomodulatory role of Withania somnifera root powder on experimental

induced inflammation: An in vivo and in vitro study
M. Rasool a,⁎, P. Varalakshmi b
a
Department of Biosciences, Vellore Institute of Technology, Deemed University, Vellore-632 014, India
b
Department of Medical Biochemistry, Dr ALMPGIBMS, University of Madras, Taramani Campus, Chennai-113, India
Received 1 August 2005; accepted 1 January 2006

Abstract

The aqueous suspension of Withania somnifera root powder was investigated for their in vivo and in vitro immunomodulatory properties. W.
somnifera showed potent inhibitory activity towards the complement system, mitogen induced lymphocyte proliferation and delayed-type
hypersensitivity reaction. Administration of W. somnifera root powder did not have a significant effect on humoral immune response in rats. Our
results report immunosuppressive effect of W. somnifera root powder, thus it could be a candidate for developing as an immunosuppressive drug
for the inflammatory diseases.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Withania somnifera root powder; Humoral immunity; Mitogen induced lymphocyte proliferation; Delayed-type hypersensitivity

1. Introduction anti-inflammatory properties (Buddhiraja and Sudhir, 1987). It

Withania somnifera (L) Dunal (Solanaceae), commonly
called Ashwagandha (Sanskrit) is an Ayurvedic Indian
medicinal plant, which has been widely used as a home remedy
for several ailments. W. somnifera is mentioned in Indian sys-
tems of medicine as a herbal tonic and health food. It is an
official drug and is mentioned in the Indian Pharmacopoeia. Its
root is regarded as a tonic, aphrodisiac and is used in con-
sumption, emaciation, debility, dyspepsia and rheumatism. The
plant is used in treating syphilis and a decoction of the root bark
is administered for asthma (Nadkarni, 1954). Of all parts of this
plant, the root has been considered to be the most active for
therapeutic purposes (Tripathy et al., 1996).
W. somnifera possesses antisertogenic, anticancer, anabolic
activity and beneficial effects in the treatment of arthritis,
geriatric problems and stress (Asthana and Raina, 1989).
Various withanolides have been isolated from W. somnifera.
Withaferin A and 3-β-hydroxy-2, 3 dihydro withanolide F show
promising antibacterial, antitumour, immunomodulating and
⁎ Corresponding author. Fax: +91 416 2243092, 2240411.
E-mail address: mkr474@rediffmail.com (M. Rasool).
1537-1891/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.vph.2006.01.015
also possesses adaptogenic, cardiotropic, cardioprotective and
anticoagulant properties (Dhuley, 2000).
Inflammatory diseases of the skin, gut, respiratory tract,
joints and central organs, as well as infectious diseases, are
now primarily considered immunological disorders. Modula-
tion of immune responses to alleviate the diseases has been of
interest for many years. Apart from being specifically stim-
ulatory or suppressive, certain agents have been shown to
possess activities to normalize or modulate pathophysilogical
process and hence are called immunomodulatory reagents.
There are very few reports on the immunosuppressive activity
of W. somnifera (Agarwal et al., 1999). Our previous studies
confirms its anti-inflammatory and lysosomal membrane
stabilizing effect on adjuvant-induced arthritis (Rasool et al.,
2000). Therefore, in the present study, the effect of W.
somnifera root powder on certain immune parameters was
studied, which is closely related to inflammatory processes i.e.
complement activity, T-lymphocytes proliferation, humoral
immune response and the delayed-type hypersensitivity
(DTH). This study can also help in understanding the mech-
anism of action of this drug and its possible use as an immu-
nomodulator in arthritis.
 M. Rasool, P. Varalakshmi / Vascular Pharmacology 44 (2006) 406–410
2. Methods
2.1. Animals
Albino Wistar strain rats of either sex weighing about 130±
20 g obtained from Veterinary College, Chennai, India were
used for the experiments. Animals were acclimatized to the
animal house conditions (12:12 h light and dark cycle) for a
week. The rats were fed with commercial pelleted rat feed (Gold
Mohur — Hindustan Lever Ltd., Mumbai, India) and water was
given ad libitum.
2.2. Drug
The commercially available powdered root of W. somnifera
was obtained from Indian Medical Practitioners Co-operative
Pharmacy and Stores (IMPCOPS), Adyar, Chennai, India and
its aqueous suspension in 2% gum acacia was used.
2.3. Anti-complement activity
2.3.1. Induction of arthritis
Two groups each containing five rats, were inoculated with
0.1 ml of Complete Freund's Adjuvant (CFA) in the right hind
paw (Rasool et al., 2000). The adjuvant (Tuberculosis Research
Center, Chennai) contained dry heat killed Mycobacterium
tuberculosis in sterile paraffin oil (10 mg/ml). W. somnifera root
was orally administered, 1 h before induction of adjuvant, then
daily for 5 days to the one group of rats and the other group of
five rats served as control.
2.3.2. Anti-inflammatory test
The thickness of the right hind foot was measured just before
and over a period of five days for the adjuvant challenge, using
sliding vernier scale.
2.3.3. Complement test
Rats were bled immediately before and after injection of test
samples and adjuvant, and thereafter, bled daily for five days.
Complement activity and immunohaemolysing effect of test
samples via the classical pathway were determined spectropho-
tometrically (Kapil and Moza, 1992).
Veronal buffer (25 mM, pH 7.3, containing 0.15 mM Ca2+
and 0.5 mM Mg2+) was used as diluent in the complement assay
and rat serum was used as a source of complement. Sensitized
sheep erythrocytes were incubated with the complement of
treated and control rats, respectively. Degree of haemolysis was
determined spectrophotometrically at 413 mm and then
compared with the measured thickness of the rat's right hind
paws, respectively.
2.4. Cell proliferation assay
Human mononuclear cell proliferation was assayed accord-
ing to the method described by Lie-chwen Lin et al. (1999).
Heparinized human peripheral blood (20 ml) was obtained from
a healthy donor. The blood was centrifuged at 2500 rpm, 4 °C for
407
10 min to remove plasma. Blood cells were then diluted with
PBS buffer and centrifuged in a Ficoll-Hypaque discontinuous
gradient at 1500 rpm in room temperature for 30 min. Human
mononuclear cells (HMNC) layer was collected and washed
with cold distilled H2O and 10× Hank's buffer saline solution to
remove red blood cells. The density of HMNC was adjusted to
5 × 106 cells/ml before use. Cell suspension solution (100 μl)
introduced into each well of a 96-well flat bottomed plate with or
without W. somnifera root powder (50 μg, 100 μg/ml) and 10 μg/
ml phytohaemagglutinin (PHA) (Sigma, St. Louis, USA) were
co-cultured with the cells. The plates were put aside in 5% CO2-
air humidified atmosphere at 37 °C for 3 days. Subsequently, 3H-
Thymidine (1 μCi/well) (BARC, Mumbai, India) was added into
each well. After a further 16-h incubation, the cells were
harvested on glass fiber filters by an automatic harvester
(Dentate, MultiMate 2000, Billinghurst, UK). Radioactivity in
the filter was measured by a liquid scintillation counter.
2.5. Humoral antibody response
Rats were intraperitoneally immunized with 0.2 ml of SRBC
(5 × 109 cells/ml) on day zero according do the technique described
by Subramoniam et al. (2000). W. somnifera root powder
(1000 mg/kg b.wt) was administered orally on days −3, −2, −1,
0, +1, +2 and +3. Blood samples were collected from individual
animals from the orbital plexus on day 7. Two fold dilutions of sera
were performed in 0.15 phosphate buffered saline (pH 7.2) and
50 μl of each dilution was aliquoted into 96-well microtitre plates.
A 25 μ1 of fresh 1% SRBC suspension in the above buffer was
dispersed into each well and mixed. The plates were incubated at
37 °C for 2 h and examined visually for agglutination. The value of
the highest serum dilution causing haemagglutination was taken as
the antibody titre. Antibody titres were expressed in a graded
manner, the minimum dilution (1/2) being ranked as 1, and the
mean ranks of different groups were compared for statistical sig-
nificance. Each experimental group contains 6 animals.
2.6. Delayed-type hypersensitivity response (DTH)
The effect of the W. somnifera root powder on the antigen
specific cellular immune response in experimental animals was
measured by determining the degree of DTH response using the
foot paw swelling test (Benencia et al., 2000). Rats were in-
jected intraperitoneally with a suspension containing 1 × 106
SRBC in 0.2 ml of phosphate buffered saline (PBS) on day zero
sensitization. Seven days later (day +7), the same animals were
injected subcutaneously with 1 × 106 SRBC suspended in 50 μl
of PBS into the right hind foot pad for elicitation of the DTH
reaction. The left hind foot pad was injected with 50 μl of PBS
as control. Foot pad swelling was measured on day + 8 with a
caliper. The difference between the means of right and left hind
footpad thickness gave a degree of foot pad swelling which was
used for group comparisons. To establish the effect of W. som-
nifera root powder on this immune response, a daily dose of W.
somnifera root powder (1000 mg/kg b.wt) was administered at
the induction phase (+ 4 to +7 days). Simultaneously, another
group of animals (controls) was inoculated in the same
408 M. Rasool, P. Varalakshmi / Vascular Pharmacology 44 (2006) 406–410

condition with 0.1 ml of PBS. Each experimental group Table 1

contains 6 animals. Effect of Withania somnifera root powder (1000 mg/kg b.wt) on complement
activity in adjuvant-induced arthritis

2.7. Statistical analysis Time Group I (Arthritis) Group II (Arthritis + Withania

(h) somnifera root powder)

The statistical significance of the data was determined by Complement activity % Complement activity %
(absorbance) (absorbance)
Student's t-test. p value at 0.05 was taken as significant.
0 0.45 ± 0.03 0.45 ± 0.02
24 0.68 ± 0.05 51 0.59 ± 0.05 31
3. Results
48 0.74 ± 0.06 64 0.52 ± 0.05 16
72 0.84 ± 0.07 86 0.50 ± 0.04 11
3.1. Complement activity 76 0.74 ± 0.07 64 0.49 ± 0.02 9
The comparisons are made with control groups.
In arthritic rats (Group I), the foot pad thickness was The symbols represent statistical significance at: p b 0.05.
increased by 135% after 72 h of adjuvant injection with a gradual
decrease (Fig. 1). The oral administration of W. somnifera root
powder once daily for five days to arthritic rats, produced 62.5% hand W. somnifera root powder (1000 mg/kg b.wt) treatment
increase in foot pad thickness at 72 h with a further decrease in was able to diminish the inflammation occurring during the
inflammation at 96 h. In arthritic rats (Group I), the complement DTH response in rats (Fig. 4).
activity was increased by 86% at 72 h when compared with 0 h
and there was a decrease at 96 h (Table 1). In Group II drug 4. Discussion
treated arthritic rats, the complement activity was increased by
11% at 72 h and 9% rise in 96 h respectively. The immunological models selected for the screening of
modulatory activity of W. somnifera root powder deal with
3.2. Lymphocyte proliferation assay inflammatory diseases i.e. complement activity, T-lymphocyte
proliferation, humoral antibody response and cell mediated
A significant inhibition in cellular proliferation was observed immune response (DTH). The data observed in this study shows
at both concentration of W. somnifera root powder tested that all these immune functions are inhibited by W. somnifera
(p b 0.05) in these experiments (Fig. 2), W. somnifera was added root powder.
along with PHA at 0 h. In order to ascertain that W. somnifera The complement system is an important source of pro-
itself was not cytotoxic to the cells, trypan blue dye exclusion inflammatory mediators especially in autoimmune disorders
test was carried out and it was shown that W. somnifera by itself where the cascade is generally triggered by antigen–antibody
does not alter cell viability. complexes (Morgan, 1990) and through the release of
anaphylatoxins (Silva Da Dias and Lepow, 1967). Complement
3.3. Humoral antibody and delayed-type hypersensitivity activation products elicit the synthesis or secretion of numerous
response other inflammatory mediators, such as synthesis and secretion
of pro-inflammatory cytokines by macrophages (Bacle et al.,
W. somnifera root powder (1000 mg/kg b.wt) did not have a 1990). A strong correlation between complement inhibitory and
significant effect on humoral response (Fig. 3). On the other anti-inflammatory activity in experiment models has been

Fig. 1. Effect of Withania somnifera on foot thickness in adjuvant arthritis.

 M. Rasool, P. Varalakshmi / Vascular Pharmacology 44 (2006) 406–410
Fig. 2. Effect of Withania somnifera on human lymphocyte proliferation in response to PHA stimulation.
observed (Geetha and Varalakshmi, 1999). It is evident that
complement plays a key role in inflammation (Sell, 1980). It
would be predicated that inhibition of complement activity
might be a possible mechanism for inhibiting inflammatory
increase. Thus, our results indicate that W. somnifera root
powder may offer promise for the therapy of inflammation and
other disorders associated with complement activation.
The effect of W. somnifera root powder on the proliferative
response of T-lymphocytes to the mitogen phytohaemagglutinin
(PHA) was investigated. It is well known that T cells respond to
stimulation in vitro by non-specific polyclonal activators like
mitogen such as phytohaemagglutinin (PHA), concavilin A,
pokeweed mitogen etc., and enter into cell division. The
mitogen acts first by binding to cell surface receptors which in
turn, initiates a cascade of biochemical reactions leading to cell
proliferation (Talwar, 1983). This situation mimics the unre-
stricted proliferation of T cells in vivo, in disease conditions
such as arthritis. Thus the observations made in this study on
human PBMNCs showed that W. somnifera is an inhibitor of T-
Fig. 3. Effect of Withania somnifera on SRBC-induced humoral antibody titres
in rats.
409
cell proliferation and might be useful as an immunosuppressive
or anti-inflammatory agent with special relevance to arthritis.
Taking into account the results obtained in vitro, we decided
to evaluate the effect of drug on different in vivo immune
responses such as antibody production and the delayed-type
hypersensitivity response in which it is known that lymphocytes
play a major role. W. somnifera root powder did not have a
significant effect on humoral response, but a previous report
(Ziauddin et al., 1996) states that W. somnifera root extract has
an immunostimulatory effect on immunosuppressed animals. In
the DTH model, sensitized animals when challenged with the
same allergen, resulted in a significant increase in paw oedema
when compared with right paw (receiving SRBC) and left paw
(receiving PBS as control) establishing the validity of the
model. The interaction of sensitized T cells with presented
antigen in known to be associated with the release of mediators
such as histamine, products of arachidonic acid metabolism and
eventually interferon-γ leading to DTH (Mungantiwar et al.,
Fig. 4. Effect of Withania somnifera on DTH response.
410 M. Rasool, P. Varalakshmi / Vascular Pharmacology 44 (2006) 406–410
1999). Therefore the inhibitory action could be due to an
influence of W. somnifera root powder on the biological
mediators. W. somnifera in herbal formulations has been proved
to be effective in rheumatoid arthritis (Chopra et al., 1996),
suggesting its role in control of autoimmune disorder by im-
munomodulation. Results of the present study also support
these findings. Thus, W. somnifera root powder has immuno-
modulator principles, specially affecting inflammatory re-
sponses. Its immunosuppressive action might be because of
the presence of withanolides, steroidal lactones and a few
flavanoids (Kamath et al., 1999). Further work is in progress
with the active principle isolated from the W. somnifera root
powder to evaluate its exact mechanism of action.
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