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in vitro antiviral potential of Ocimum sanctum leaves extract against New Castle Disease Virus of poultry,”

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Ocimum sanctum is well known for its antimicrobial properties in Indian traditional medicinal system in Ayurveda. Different preparations of O. sanctum plants and parts of it have been reported throughout the world for its medicinal properties including antiviral effects. Thus in present study hot aqueous extract of O. sanctum leaves was used to study the antiviral activity against the New Castle Disease Virus of poultry chicken embryo fibroblast monolayer culture. Before performing the study nontoxic dose of the extract was also decided for the chicken fibroblast culture and the concentrations of 10mg/ml or less of hot aqueous extract of O. sanctum leaves in basal media (RPMI 1640) appeared to be non toxic. As cytopathic effects of NCD virus on chicken embryo fibroblast monolayer culture are well established so these was used to detect the antiviral activity of O. sanctum along with Haemaggulitation test to get an idea of viral concentration in culture. Absence of cytopathic effects in monolayer and lower the HA titer were considered as the indicative of antiviral activity of extract of O. sanctum leaves. The concentrations of 10mg/ml or less of hot aqueous extract of O. sanctum leaves prevented the cytopathic effects and growth of NCD virus in chicken fibroblast monolayer.
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International Journal of Microbiology and Immunology Research Vol.2(7), pp. 051-055, July, 2013
Available online at http://www.apexjournal.org
ISSN 2315-8743 ©2013 Apex Journal International
Full Length Research Paper
In vitro antiviral potential of Ocimum sanctum leaves
extract against New Castle Disease Virus of poultry
Jayati1, A.K.Bhatia2, Amit Kumar1*, A.Goel2, Sandeep Gupta3, Anu Rahal4
1Department of Veterinary Microbiology, U.P. Pt. Deen Dayal Uphadhayay Veterinary University Evam Go Anusandhan
Sansthan, Mathura, UP, India, 281001.
2Department of Microbiology, GLA University, Mathura, UP, India.
3Animal Husbandry Department, University, Mathura, UP, India.
4Department of Veterinary Pharmacology and Toxicology, University, Mathura, UP, India.
Accepted 14 June, 2013
Ocimum sanctum is well known for its antimicrobial properties in Indian traditional medicinal system in
Ayurveda. Different preparations of O. sanctum plants and parts of it have been reported throughout
the world for its medicinal properties including antiviral effects. Thus in present study hot aqueous
extract of O. sanctum leaves was used to study the antiviral activity against the New Castle Disease
Virus of poultry chicken embryo fibroblast monolayer culture. Before performing the study nontoxic
dose of the extract was also decided for the chicken fibroblast culture and the concentrations of
10mg/ml or less of hot aqueous extract of O. sanctum leaves in basal media (RPMI 1640) appeared to be
non toxic. As cytopathic effects of NCD virus on chicken embryo fibroblast monolayer culture are well
established so these was used to detect the antiviral activity of O. sanctum along with
Haemaggulitation test to get an idea of viral concentration in culture. Absence of cytopathic effects in
monolayer and lower the HA titer were considered as the indicative of antiviral activity of extract of O.
sanctum leaves. The concentrations of 10mg/ml or less of hot aqueous extract of O. sanctum leaves
prevented the cytopathic effects and growth of NCD virus in chicken fibroblast monolayer.
Key words: Ocimum sanctum, antiviral activity, New Castle Disease Virus, chicken embryo fibroblast
monolayer culture
INTRODUCTION
Over centuries several important traditional medicinal
systems such as Greek, Chinese, Tibetian, Indian,
Siddha and Mediterranean have been evolved and
established all over the world with the use of naturally
present active principles of plants in the form of various
preparations for the relief of human and veterinary
diseases (Kumar et al., 2013). In Ayurveda, an Indian
system of Medicine, about 2500 plants have been prized
for their medicinal abilities and these are the true treasure
of Indian biodiversity (Uphadhayay et al., 2013). Among
*Corresponding author. Email: balyan74@gmail.com. Tel.:
+919412120813.
these plants O. sanctum, distributed mainly in the tropical
and subtropical region of the world, is considered to be
highly scared, medicinal application in the indigenous
system of medicine of many Asian, African and South
American countries (Kumar et al., 2011, 2013). Since
long in India the practitioner of traditional system of
medicine present in villages and towns have been using
O. sanctum for curing various ailments of microbial origin,
a rational approach to this traditional medicinal practice
with modern system of medicine is, however, not much
available. In order to establish the scientific based
therapeutic use of O. sanctum in modern medicine,
several Indian scientists and researchers (Prakash and
Gupta, 2005; Sood et al., 2006; Bhartiya et al., 2006;
Goel et al., 2008; Kumar et al., 2011, 2013) have studied
052 Int. J. Microbiol. Immunol. Res.
the pharmacological effects of various part of this plant.
However, there is little information available about its
application as antiviral in poultry health and thus it
appears to be the need of today as in Indian scenario
poultry industry is most rapidly growing veterinary sector
(Mahima et al., 2012b) and viral diseases are causing
most serious losses to this industry. Among these viral
diseases new castle disease that is also known as
ranikhet disease is very much prevalent in Indian
subcontinent and causing losses in millions every year.
Henceforth, in this study in vitro antiviral activity was
assessed on chicken embryo fibroblast culture with the
objective to establish the antiviral properties of leaves of
O. sanctum against New Castle Disease Virus (NCDV).
MATERIALS AND METHODS
Collection of leaves of Ocimum sanctum: Leaves of
O. sanctum were collected from the campus of UP Pt.
Deen Dayal Upadhayay Pashu Chikitsa Vigyan
Vishwavidyalaya Evam Go Anusandhan Sansthan,
Mathura and dried under shade. The leaves were verified
by Department of Botany, BSA College Mathura and
preserved in Department of Microbiology. Dried leaves
were used for preparation hot aqueous extract.
Viral isolate: For antiviral effect of extract of O. sanctum
leaves, New Castle Disease virus (NDV) was used as a
challenge to the chicken embryo fibroblast culture. The
virus was procured from the Department of Avian
Diseases, Indian Veterinary Research Institute,
Izatnagar, UP, India.
Extraction: Hot aqueous extract (HAE) of O. sanctum
leaves was prepared by the method of Goel et al. (2008).
In this method the 50 gram of dried powder of O.
sanctum leaves was placed in a porous cellulose thimble.
The thimble was then placed in an extraction chamber,
above a collection flask containing the 750 ml solvent
(triple glass distilled water). The flask was heated and the
solvent was allowed to evaporate. Temperature was
adjusted according to boiling temperature of the solvent
(100°C). The extraction process lasted 6-8 hours and
then flask containing the solvent and extract were
removed. The solvent in the flask was allowed to
evaporate and finally the remaining material was
collected and weighed.
Cultivation of New castle Disease virus: The New
Castle Disease virus was cultivated in embryonated
chicken eggs by allantoic cavity route method as
described by Cunningham (1973). The NDV was taken
from deep freezer and thawed at room temperature. The
eggs were inoculated by allantoic cavity route using 0.1
ml of virus inoculum per egg. Eggs were incubated at
37°C in egg incubator under moisture and examined daily
by candling. Eggs dying after 2nd day were removed from
incubator and allantoic fluid was collected and preserved
in deep freeze (- 20°C) for further use.
Titration of New castle Disease virus: The virus was
titrated by haemagglutination test (HA test) as per the
procedure advocated by Cunningham (1973). Two-fold
serial dilutions of the virus was made in normal saline
beginning with 1:2 dilution in 1st well through 1:1024
dilution in 10th well. 0.5 ml RBCs suspension (1%) was
added in all the wells. The plate was shaken to mix the
contents and incubated at room temperature for
haemagglutination. Plate was examined every 15
minutes upto 1 h. for uniform haemagglutination covering
the bottom of plate. Button formation was taken as
negative.
Preparation of chicken embryo fibroblast (CEF)
monolayer: The chicken embryo fibroblast monolayer
was prepared as per the method described by
Cunningham (1973). The embryo was taken out in
petridish containing PBS. The embryo was washed with
PBS and the head, appendages and viscera were
removed. The embryonic tissues were cut into small
pieces and washed thoroughly and transferred into a
flask containing 0.25% trypsin solution (pH 7.6-7.8) in
PBS for trypsinization for 1 hrs on magnetic stirrer. The
contents of flasks were filtered using muslin cloth.
Filtered cell suspension was centrifuged at 1500 RPM for
15 min. and supernatant was discarded. Cells pellets
were resuspended with culture medium and centrifuged
at 1500 RPM for 10 min. Supernatant were discarded
and repeated washing was done at same RPM and time.
Cells were resuspended in medium and adjusted to1x107
cells/ml. 1ml of cells suspension was taken in all the wells
of a six wells tissue culture plate and incubated at 37°C in
an atmosphere of 80-85% humidity and 5% CO2.
Determination of nontoxic concentration for CEF
monolayer culture: Before conducting the antiviral
properties, maximum nontoxic concentration of extract
was determined in 24 hr grown monolayer CEF culture.
The extract was diluted so as to contain 100, 50, 20, 10,
5, 2.5, and 1.25 mg/ml of extract in maintenance medium.
1 ml of each dilution was inoculated to CEF culture in a
six wells culture plate and incubated at 37°C in the
presence of 5% CO2. The toxic effect of each
concentration was observed under microscope at 12 hr
intervals up to 48 hrs. Highest dilution showing any
degenerative changes/CPE in cell culture was considered
as cytotoxic concentration of the extract.
Antiviral activity of O. sanctum extract: Three different
concentrations less than the non toxic concentration i.e.
2.5 mg/ml, 5.0 mg/ml and 10.0 mg/ml were used to
determine the antiviral effect of extract of O. sanctum
leaves. The monolayer cultures were challenged with
Jayati et al 053
(a) (b)
(c)
Figure 1. Effect of Ocimum sanctum extract on New castle Disease Virus replication in chicken embryo fibroblast culture. (a) Normal growth
Pattern -Control (b) Cytopathic effects produced by NCD Virus (c) Protective effect of extract on NCD virus infected cells.
NCD virus having 0.512 HA units mixed with
maintenance medium. The HA titer of virus in the culture
supernatant was estimated. Growth of fibroblasts was
monitored and supernatant was collected at different
intervals viz: 12, 24, 36, 48, 60 and 72hrs.
RESULTS AND DISCUSSION
In viral pathogens related problems various preparations
have been attempted as ethanolic extracts
(Direkbusarakom et al., 1996; Parida et al., 1997, Chiang
et al., 2005; Balasubramanian, et al., 2007;
Bhanuprakash et al., 2008a,b), acetone extracts (Deepthi
et al., 2007), aqueous extracts (Parida et al., 1997;
Chiang et al., 2005; Balasubramanian et al., 2007;
Bhanuprakash et al., 2007, 2008) and petroleum ether,
benzene, diethyl ether, chloroform, ethyl acetate,
methanol and ethanol extracts (Balasubramanian et al.,
2007). Depending upon the type of extracts, the antiviral
activity of O. sanctum has been assessed against many
important viral agents as fish pathogenic viruses viz.
Infectious hematopoietic necrosis virus (IHNV);
Oncorhynchus masou virus (OMV); Infectious pancreatic
necrosis virus (IPNV) (Direkbusarakom et al., 1996), polio
virus type-3 (Parida et al., 1997), herpes viruses (HSV),
adenoviruses (ADV), hepatitis B virus and RNA viruses
viz. coxsackievirus B1 (CVB1), enterovirus 71 (EV71)
(Chiang et al., 2005), white spot syndrome virus (WSSV)
in shrimp (Balasubramanian, et al., 2007),) Buffalo pox
virus (GTPV) (Bhanuprakash et al., 2007) and infectious
bovine rhinotracheitis virus (IBR) (Sharma et al., 2011).
On the basis of the effects observed in all these findings
hot aqueous extract of O. sanctum leaves were selected
as absence of any other substance as ether, methanol,
ethanol also interfere in virus growth. NCD virus is an
important poultry pathogen with substantial economic
losses to poultry industry. Initially the nontoxic dose of
extract was assessed and lowest dose showing the
cytopathic changes in chicken embryo fibroblast cell
culture was 20.00 mg/ml in the RPMI medium. Thus all
the three concentrations of 10, 5 and 2.5 mg/ml which
were less than 20mg/ml were considered non toxic
concentrations and were used for anti viral activity. These
doses are almost in the concurrence of the non toxic
range of 22.5 to 0.175 mg/ml concentration of ethanolic
extract of basil observed in VERO cells (Parida et al.,
1997). The antiviral effects were assessed on the basis of
occurrence of changes in the CEF monolayer culture
(Figure 1c) and the HA titers of NCD virus in the
054 Int. J. Microbiol. Immunol. Res.
Table1. HA titer in fibroblast cell culture supernatant.
Groups (Conc. of
extract)
HA titer at different intervals (unit/ml)
12 h 24 h 36 h 48 h 60 h 72 h
Control 32 32 128 512 1024 1024
2.5mg/ml 0 8 64 512 512 512
5.0mg/ml 0 8 8 128 128 128
10mg/ml 0 0 0 16 16 16
32
000
32
880
128
64 80
512 512
128
16
1024
512
128
16
1024
512
128
16
0
200
400
600
800
1000
1200
12hrs 24hrs 36hrs 48hrs 60hrs 72hrs
HA titre at different intervals (Unit/ml)
HA titres at different intervals in fibroblast cell
culture supernatant
Control O. sanctum 2.5mg/ml
O. sanctum5.0mg/ml O. sanctum10mg/ml
Figure 2. HA titers at different intervals in chicken embryo fibroblast culture.
supernatant of culture. The HA titer was found significant
at 16 with 10.0 mg/ml of HAE of O. sanctum leaves
(Figure 1c) as compared to 1024 with virus control.
Simultaneously it also prevented the CPE of the NCD
virus (Table 1 and Figure 1b, c). The virus infection
produces cytopathic effects on chicken embryo fibroblast
mono-layer culture (Figure 1a,b). Any reduction in the
changes or absence of CPE is supposed to be protective
effects of extract. At the same time the reduced HA titer
is also indicative of inhibition of viral growth. The
investigation revealed that antiviral effect as evidenced
on the basis of HA titer was also concentration
dependent and higher concentration of extract inhibited
the NCD Virus replication in corresponding higher extent
(Figure 2). Sikader et al. (1998) and Kumar et al. (1997)
reported the antiviral effect of different extracts on
infectious bursal disease and new castle disease viruses.
In present time use of herbs in poultry industry has a
significant market and herbs are being added as a
compulsory feed ingredient in poultry feed. The results of
the study ar very promising and support the use of these
as feed additive to protect birds from viruses like NCD.
Moreover, in the present study all the three
concentrations of extract prevented the CPE with the
lowest HA titer in the 10 mg/ml con-centration (Figure 2).
Thus the dose of 10.0 mg/ml conc. of extract of O.
sanctum leaves prevented the virus to multiply and can
be used to get protection against NCD virus. As the role
of Ocimum sanctum as immunomodulator has been
thoroughly studied and well established (Godhwani et al.,
1988; Bhartiya et al., 2006, Mahima et al., 2012b) along
with antibacterial activity against common bacterial
pathogens of animals (Kumar et al., 2011, 2013) thus it
can be a sole source with multiple activity in poultry feed.
ACKNOWLEDGEMENTS
Authors are highly thankful to Dean, College of Veterinary
Sciences and Hon’ble Vice Chancellor of DUVASU,
Mathura for providing all kind of support and facilities to
conduct the study.
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