Characterization of Anti-Coxsackie Virus B3 Constituents ... 1512.8. Analysis of Individual Phenolic Compounds by Analytical
RP-HPLC
The dried subfractions from the more active extract were hydrolysed
according to slightly modified method of Proestos et al. [34]. Forty millilitres
of methanol containing BHT (1 mg/ml) were added to 0.5 g of dried
subfractions. Then, 10 ml of 6 M HCl. The mixture was stirred carefully and
then sonicated for 15 min and refluxed in a water bath at 90°C for 2 hours. The
obtained mixture was filtered through a 0.45 μm membrane filter and injected
to RP-HPLC. The separation of phenolics was performed with an Agilent 1100
series HPLC system equipped with in-line degasser (G 1322A), quaternary
pump (G 1311A), a thermostatic auto sampler (G 1313A), column heater (G
1316A), and diode array detector (G 1315A).
Instrument control and data analysis was carried out using Agilent HPLC
Chemstation 10.1 edition through Windows 2000. The separation was carried
out on a reverse phase ODS C18 (4 μm, 250 × 4.6 mm, Hypersil) column used
as stationary phase at ambient temperature. The mobile phase consisted of
acetonitrile (solvent A) and water with 0.2% sulfuric acid (solvent B). The
flow rate was kept at 0.5 ml/min. The gradient program was as follows: 15
A/85 B 0–12 min, 40% A/60% B12–14 min, 60% A/40% B 14–18 min, 80%
A/20% B 18–20 min, 90% A/10% B 20–24 min, 100% A 24– 28 min. The
injection volume was 20 μl and peaks were monitored at 280 nm. Peaks were
identified by congruent retention times compared with standards.
2.9. Statistical Analysis
The results were given as the average ± SD for at least three replicates for
each sample. The 50% cytotoxic concentration (CC 50 ) was defined as the
compound’s concentration (μg/mL) required for the reduction of cell viability
by 50%, which were calculated by regression analysis [y = f(x); where y =%
viability and x = concentration of extract, μg/mL]. The 50% inhibitory
concentration (IC 50 ) was defined as the concentration that inhibited 50% of
viral replication when compared to virus controls. The data were subjected to
ANOVA, and Duncan’s multiple range test was used to compare means.
Statistical analyses were performed with the SPSS statistical software program
(SPSS v.16). P values <0.05 were regarded as significant. To evaluate the
antiviral activity in vitro, the selectivity index (SI =CC 50 /IC 50 ) was determined.
The selectivity index describes the ratio between the cytotoxic and the
antiviral activity of a tested compound.