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Medicinal Plant Research 2012, Vol.2, No.2, 6
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in Vero cell line we could not find any inhibition
effect against HSV
-
1 in different dilutions.
To conclude, because of the widespread
Eucalyptus
globulus
consumption in different countries especially
in Iran, the extraction of this plant can be used as an
alternative treatment. For drug production, air spray or
a tropical ointment instead of industrialized drugs
such as acyclovir would be suggested in many cases
and of course in all of these studies, dose of herbs
and the best way of consumption should be tested
before using them to increase their positive effects in
patients.
3 Material and Methods
3.1 Cell culture and Virus
Vero cells (African green monkey kidney cells) were
cultured with Dulbecco’s Modified Eagles’ Medium
(DMEM) supplemented with 10% heat inactivated
Fetal Bovine Serum (FBS), 100 IV/mL penicillin
and l μg/mL streptomycin. The cells were maintained
at 37
℃
in a humidified atmosphere with 5% CO
2
and
were subculture two or three times a week. HSV
-
1
was isolated from patients and identified by specific
monoclonal antibodies. Viruses were quantified in
terms of the 50% tissue culture infective dose
(TCID50) by endpoint dilution, with the infectious
titer determined by the method of Reed and Munch
(14), and stored in small aliquots at
-
70
℃
until use.
3.2 Virus stock
HSV
-
1 was isolated from the lip lesions of a patient
and its genus was confirmed by neutralization test
using guinea pig anti-HSV
-
1 serum and monoclonal
anti-HSV
-
1 antibodies against HSV glycoprotein’s D
and G.
3.3 Plant materials
Eucalyptus globulu
s and
Artemisia draconculus
were
collected from a farmland in Kerman and identified at
the department of Virology of Kerman university of
Medical Sciences. The dried leaves were pulverized
and 200 gr of pulverized sample was extracted with
500 mL of 80% methanol by maceration for 72 h. The
methanol extract was concentrated in a rotary evapo-
rator, lyophilized and thereafter preserved for further
use.
Eucalyptus globulus
and
Artemisia draconculus
stocks were prepared by dissolving 10 mg of each
extract in one mL distilled water. It was then sterilized
by filtration.
For cytotoxicity and antiviral assays, the stock
solutions of
Eucalyptus globulus
and
Artemisia
draconculus
were diluted in the maintenance medium
of Dulbecco’s modified Eagle’s growth medium
(DMEM, Sigma) supplemented with 2% fetal bovine
serum (Gibco, Germany), 0.14% (v/v) sodium bicar-
bonate, 100 U/mL penicillin, 100 μg/mL streptomycin
sulphate, and 0.25 μg/mL amphotericin B.
3.4 Cytotoxicity assay
In order to test the effect of the Eucalyptus globulus
and Artemisia draconculus extract on vero cells, 5×l0
4
cells, (in l mL DMEM, supplemented with 10% FBS)
were seeded in to each well of micro plates, cultured
for 6 hr at 37
℃
, cells were allowed to grow for
additional 48 h in the presence of increasing amounts
of extract (10 μg/mL, 100 μg/mL, 200 μg/mL, 400 μg/mL,
600 μg/mL, 800 μg/mL and 1 000 μg/mL), The
cytotoxicity of the extract was determined on a
conventional hemocytometer using the trypan blue
exclusion method. The 50% cytotoxic concentration
(CC50) was defined. As the concentration, which caused
a 50% reduction in the number of viable cells.
3.5 Incubation of cells with the extract before,
during and after virus infection
Eucalyptus Globulus
and
Artemisia draconculus
hydroalcoholic extract was dissolved in serum free
DMEM and incubated with semi-confluent cell in 24
well tissue culture plates in increasing concentration
from 10 to 1 000 μg/mL for 2 h at 37
℃
. After removal
of the extract, the cells were washed with phosphate
buffered saline (PBS) and then infected with HSV
-
1 at
multiplicity of infection (MOI) 1. After l h incubation,
the unabsorbed virus was removed, the cell monolayer
was washed with PBS and further incubated in
DMEM with 2% FBS. Controls consisted of Vero
cells untreated and Vero cells infected with HSV1.
For determination of antiviral activity of the extract
during and post virus infection the assay was
performed as described above, with the exception that
the extract was added together with the virus and after