Characterization of Anti-Coxsackie Virus B3 Constituents ... 1492.4. Virus Strain
Coxsakievirus B 3 Nancy strain (kindly provided by Pr. Bruno Pozzetto,
Laboratory of Bacteriology-Virology, Saint-Etienne, France) was propagated
in Vero cells. In brief, 100 μL of the virus suspension were used to infect a
confluent monolayer of Vero cells in 75 cm^2 culture flask and adsorbed for 1 h
to allow viruses entry into the cells. Non-adherent particles were washed off
using 2% RPMI 1640 medium and the infected cells overlaid with 20 mL of
2% RPMI 1640 and incubated again until full cytopathic effect was observed
in five to six days. When a cytopathic effect CPE in the virus-infected cells
was observed microscopically, TCID 50 (the 50% tissue culture infective dose)
was determined by the method of Reed and Muench [28]. The harvested virus
was stored at −70°C until used.
2.5. Chemical Analysis: Bioassay Guided Fractionation
The bioguided chromatographic fractionation process began with the
evaluation of anti-coxsackie B 3 screening of these sixteen samples. The roots
EtOAc fraction which has the best anti-coxsackie B 3 , was then submitted to
chromatographic separation processes. This fraction (4.2 g) was
chromatographed on silica gel column (4 cm x 80 cm) and eluted with ethyl
acetate: methanol gradient mixture starting from 100% ethyl acetate and
increasing the polarity up to 100% methanol to afford 216 fractions (25 mL).
The fractions were monitored by thin layer chromatography (TLC silica gel 60
F 254 ) using ethyl acetate/methanol as eluent. After drying, the plates were
visualized under UV light at 254 and 365 nm and then were sprayed with 10%
sulfuric acid in methanol, and those with similar profiles were combined
resulting in 4 subfractions (SR 1 - SR 4 ), which were analyzed by RP-HPLC.
2.6. Cytotoxicity Assay
The evaluation of the cytotoxic effect of samples is based on the reduction
of MTT (3[4, 5-dimethylthiazol- 2 - yl]-2, 5-diphenyl tetrazolium bromide), by
the mitochondrial dehydrogenase of viable cells, to give a blue formazan
product which can be measured spectrophotometrically at 540 nm.
The MTT colorimetric assay was performed in 96-well plates [29]. Cells
were seeded in 96-well plates at a concentration of 5 × 10^4 cells per well and
incubated for 24 h at 37 °C in a 5% CO 2 humidified atmosphere. After
treatment with various concentrations of the test compound (15.6, 31.25, 62.5,