Antiviral activity of Ashwagandha extract on Infectious Bursal Disease Virus Replication

Abstract

In the present study Ashwagandha plant was screened for antiviral activity against Infectious Bursal Disease Virus Replication. The hydro-alcoholic extract of Withania somnifera roots showed the inhibition of virus at maximum 99.9 % in its highest nontoxic concentration, 25µg/ml in Cytopathic Effect Reduction Assay. Results indicated that the Withania somnifera root extract has antiviral property against Infectious Bursal Disease virus. Introduction Infectious Bursal Disease is an Office Internationale des Epizooties (OIE) notifiable and highly contagious viral disease of chickens mostly 3-6 weeks of age (OIE, 2004). The causative agent of IBD is Infectious Bursal Disease Virus (IBDV), an nonenveloped ds RNA virus of genus Avibirnavirus and family Birnaviridae (Kibenge et al., 1988). IBD emerges as one of the major threats to the poultry industry as it leads to high morbidity, mortality, marked immunosuppression and decrease in productivity in chickens (Mohantey et al., 1971). Various types of vaccines are available for controlling this infection but vaccination failure generally occurs due to antigenic variation of the virus (Jackwood & Saif, 1987). The antiviral drugs fail to treat the infection due to viral resistance and viral latency which leads to recurrent infection in immunocompromised patients (Field & Biron, 1994). Recent success in using herbs/medicinal plants extract as antiviral agent has raised optimism about phyto-antiviral agents (Jassim & Naji, 2003). Plants like Bergenia ligulata, Nerium indicum and Holoptelia integrifolia showed significant antiviral activities against Influenza virus (RNA) and Herpes Simplex virus (DNA) (Rajbhandari et al., 2001). The antiviral activity of Azadirachta indica (Neem) and Ocimum sanctum (Tulsi) against Newcastle Disease virus (NDV) is well known (Kumar et al., 1997). Ashwagandha /Withania somnifera (WS), a plant well known for its numerous medicinal properties is also used as antiviral herb for the treatment of genital disease caused by Herpes Simplex Virus among African tribes (Kambizi et al., 2007). Therefore the present study was conducted to check the antiviral activity of Withania somnifera against IBD Virus replication. Initially maximum nontoxic concentration of the root extract was calculated using cytotoxity assay. The protective efficacy of the root extract was checked by Cytopathic Effect Reduction Assay.

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Indian Journal of Science and Technology Vol. 5 No. 5 (May 2012) ISSN: 0974- 6846
Research article “Antiviral ashwagandha” M.Pant et al.
Indian Society for Education and Environment (iSee) http://www.indjst.org Indian J.Sci.Technol.
2750
Antiviral activity of Ashwagandha extract on Infectious Bursal Disease Virus Replication
M. Pant1*, T. Ambwani2 and V. Umapathi3
1Dept. of Molecular Biology and Genetic Engineering, G.B.P.U.A&T, Pantnagar-263145, Uttarakhand, India
*Faculty, Department of Biotechnology, Graphic Era University, 266/6 Bell Road, Clement Town, Dehradun-
248001, Uttarakhand, India
2Dept. of Veterinary Biochemistry, College of Veterinary and Animal Science, G.B.P.U.A &T, Pantnagar-
263145 Uttarakhand, India
3Indian Veterinary Research Institute, Regional Station, Palampur-176061, Himachal Pradesh, India
drmanali.biotech@gmail.com*; tambwani@yahoo.com; umapathi_pillai@yahoo.co.in
Abstract
In the present study Ashwagandha plant was screened for antiviral activity against Infectious Bursal Disease Virus
Replication. The hydro- alcoholic extract of Withania somnifera roots showed the inhibition of virus at maximum 99.9
% in its highest nontoxic concentration, 25µg/ml in Cytopathic Effect Reduction Assay. Results indicated that the
Withania somnifera root extract has antiviral property against Infectious Bursal Disease virus.
Keywords: Infectious Bursal Disease Virus, Ashwagandha, hydro-alcoholic extract, Antiviral, Withania somnifera
Introduction
Infectious Bursal Disease is an Office Internationale
des Epizooties (OIE) notifiable and highly contagious viral
disease of chickens mostly 3-6 weeks of age (OIE, 2004).
The causative agent of IBD is Infectious Bursal Disease
Virus (IBDV), an nonenveloped ds RNA virus of genus
Avibirnavirus and family Birnaviridae (Kibenge et al.,
1988). IBD emerges as one of the major threats to the
poultry industry as it leads to high morbidity, mortality,
marked immunosuppression and decrease in productivity
in chickens (Mohantey et al., 1971).
Various types of vaccines are available for controlling
this infection but vaccination failure generally occurs due
to antigenic variation of the virus (Jackwood & Saif,
1987). The antiviral drugs fail to treat the infection due to
viral resistance and viral latency which leads to recurrent
infection in immunocompromised patients (Field & Biron,
1994). Recent success in using herbs/medicinal plants
extract as antiviral agent has raised optimism about
phyto-antiviral agents (Jassim & Naji, 2003). Plants like
Bergenia ligulata, Nerium indicum and Holoptelia
integrifolia showed significant antiviral activities against
Influenza virus (RNA) and Herpes Simplex virus (DNA)
(Rajbhandari et al., 2001).
The antiviral activity of Azadirachta indica (Neem)
and Ocimum sanctum (Tulsi) against Newcastle Disease
virus (NDV) is well known (Kumar et al., 1997).
Ashwagandha /Withania somnifera (WS), a plant well
known for its numerous medicinal properties is also used
as antiviral herb for the treatment of genital disease
caused by Herpes Simplex Virus among African tribes
(Kambizi et al., 2007). Therefore the present study was
conducted to check the antiviral activity of Withania
somnifera against IBD Virus replication. Initially
maximum nontoxic concentration of the root extract was
calculated using cytotoxity assay. The protective efficacy
of the root extract was checked by Cytopathic Effect
Reduction Assay.
Materials and methods
Preparation of extract
To prepare the hydro-alcoholic root extract of
Withania somnifera, the roots were cut into smaller
pieces, dried under hot circulating air at 40ºC for 3-4
days, and ground into a fine powder. The powder was
allowed to soak in 50% ethanol (v/v) for 48 hours under
continuous agitation at 40ºC in a shaking incubator. The
mixture was first filtered through muslin cloth, then
through filter paper. The final hydro-alcoholic extract was
obtained by drying the filtrate under hot circulating air at
40ºC.
Cells and virus
IBD Virus UABz-2 strain (passage 10) which was
adapted in Chicken embryo fibroblast (CEF) cell cultures
and maintained at Veterinary Biochemistry lab of
G.B.P.U.A&T, Pantnagar was used.
Preparation of chicken embryo fibroblast (CEF) Culture:
For the propagation of virus primary CEF cell cultures
were prepared using 9-11 days old chicken embryos
according to the method described with slight modifications
(Cunningham, 1973).
MTT assay for cytotoxicity testing
To investigate cellular toxicity of the extract, CEF
cells were grown in 96- well plate for 24 hours to obtain
confluence. After 24 h, the monolayer was washed with
EMEM and different concentrations of Withania somnifera
extract prepared in EMEM were added and the plate was
sealed and kept at 37°C in an atmosphere of 5% CO2 for
24 h. Afterwards, Withania somnifera solution was
removed from the flask. 50-μl of MTT solution (4mg/ml in
EMEM) was added to each well in a 96-well plate, and
incubated at 37°C for 4 h. MTT solution was removed and
200 μl of DMSO was added to dissolve MTT- formazan
crystals. Then 25 μl of glycine buffer was added and
absorbance was recorded at 570 nm immediately with the
help of a micro plate reader. The rate of growth inhibition
was calculated as formula in which percent cytotoxicity is

Indian Journal of Science and Technology Vol. 5 No. 5 (May 2012) ISSN: 0974- 6846
Research article “Antiviral ashwagandha” M.Pant et al.
Indian Society for Education and Environment (iSee) http://www.indjst.org Indian J.Sci.Technol.
2751
equal to the 100 – [OD extract treated/ OD of Control]) in
percentage.
Propagation of virus in CEF culture - To propagate the IBDV,
culture flasks with 80-90% confluency were taken and the
spent media was decanted and then inoculated with 1ml of
previously CEF adapted virus per 25 cm2 flasks. These
flasks were then incubated for 45 min at 37ºC for adsorption
of the virus. The flasks were regularly tilted every 6-8 min.
Then the inocula were decanted and the fresh maintenance
medium (EMEM with 2% FBS) was added to the flasks and
kept in incubator at 37oC and 5% CO2 for development of
cytopathic effects characterized by small round refractile
cells for 3-4 days.
Viral inhibition assays
Cytopathic Effect Reduction Assay: Antiviral activity of
WS root extract was determined by reduction of virus titer
using TCID50 determinations (Kibenge et al., 1988). For this,
culture supernatant collected at 10th passage level from 25
cm2 culture flasks and were serially diluted (10 fold) from 101
to 108 dilutions. 100μl of diluted virus was serially added to
confluent monolayer CEF cells from well no. 1 to 8, each
dilution in 8 rows. Well no. 9 and 10; 11and12 were kept for
virus and cell controls respectively. Antiviral activity of the
extract was determined by treating the virus infected CEF
cells with Withania somnifera extract (i.e. 45 min of PI) in 96
well plates keeping controls. Afterwards Plates were
properly sealed and kept at 37o C in 5%CO2. Development of
cytopathic effects was observed after every 24 hrs interval
for three days. The reading of CPE was recorded and the
titer was calculated (Reed & Muench, 1938).
Table 1. Virus titer with and without Withania somnifera
treatment in different time intervals
Time
Interval
(hrs)
WS
extract
untreated CEF
WS
extract
treated CEF
% decrease
in virus titer
TCID
50
/ ml
TCID
50
/ ml
24
1 x 10
4
3.16x10
3
68.4
48
1.46 x 10
7
2.1 x 10
4
99.9
72
1x 10
7
3.1x 10
5
96.6
Results
In the MTT cytotoxic assay 25, 50 and 75 μg of Withania
somnifera hydro- alcoholic root extract showed 100 %, 91%
and 90% survival respectively. Therefore maximum nontoxic
concentration i.e. 25 μg of Withania somnifera hydro-
alcoholic root extract was used to treat the CEF cells. Viral
inhibition effect of WS extract was shown in TCID50 in which
virus titer i.e. 1.0 × 104 TCID50/ml in 24 hrs, 1.46 × 107
TCID50/ml in 48 hrs & 1 × 107 TCID50/ml in 72 hrs was
reduced to 3.16 × 103 TCID50/ml in 24 hrs, 2.1 × 104
TCID50/ml in 48 hrs and 3.1 × 105 TCID50/ml in 72 hrs
respectively as seen in Table1. This shows the antiviral
activity of the WS root extract.
Discussion
The present study was carried out to test the antiviral
activity of hydro-alcoholic root extract of Withania somnifera
/Ashwagandha against Infectious Bursal Disease Virus using
Cytopathic Effect Reduction Assay. Antiviral activity of
Withania somnifera extract has been reported earlier on
Herpes Simplex Virus Type-1 (Kambizi et al., 2007). The
inhibitory action of Withaferin A, a steroidal compound
present in Withania somnifera against Herpes Simplex Virus
has also been reported (Grover et al., 2011). In an in-vivo
experiment polyherbal drug (Immu-25), containing Withania
somnifera has shown antiretroviral activity against HIV
infection (Usha et al., 2003). In this study, a well known
medicinal plant i.e. Withania somnifera showed antiviral
properties against IBD Virus. In conclusion, this is a
preliminary report on antiviral activity of Withania somnifera,
a well known medicinal plant against IBDV. Further studies
are required to know the mechanism of action.
Acknowledgements
This research work was funded by Department of
Biotechnology, India.
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