In vitro anti-coxsackievirus B3 effect of ethyl acetate extract of Tian-hua-fen

Table 1 Cytotoxity of ethyl acetate extract to HeLa cells shown in the value of A₆₀₀

Dilution	1:2	1:4	1:8	1:16	1:32	1:64	1:128	1:256	1:512	1:1024
Value of A600	0.20 ± 0.029	0.22 ± 0.030	0.32 ± 0.022	0.29 ± 0.069	0.38 ± 0.043	0.59 ± 0.035	0.69 ± 0.075	1.30 ± 0.069	1.23 ± 0.061	1.29 ± 0.056

The A₆₀₀ value of the cells co-cultured with the extract at concentration from 1:2 to 1: 128 mg/mL was lower than that of normal HeLa cells (1.32 ± 0.034), which showed that the extract had some toxicity to HeLa cells at these concentrations. From 1:256 mg/mL the A₆₀₀ value became close to that of normal HeLa cells, which indicated that from 1:256 mg/mL the extract had no toxicity to HeLa cells again.

Table 2 Protective effect of ethyl acetate extract on HeLa cells shown in the value of A₆₀₀

Dilution	1:256	1:512	1:1024	1:2048	1:4096	1:8192
Value of A600	1.09 ± 0.017	1.02 ± 0.122	0.89 ± 0.060	0.91 ± 0.039	0.83 ± 0.048	0.77 ± 0.028

The A₆₀₀ value of the cells co-cultured only with RPMI1640 was 1.32±0.02; the A₆₀₀ value of the cells co-cultured only with 10^-5 CVB_3m was 0.22 ± 0.026; and the A₆₀₀ value of the cells co-cultured only with extract at concentration of 1:256 mg/mL was 1.21 ± 0.042. As shown in the table the value of A₆₀₀ of the cells treated with extract is higher than that of the cells infected with CVB_3m while not treated with the extract (^bP < 0.01), indicating the protective effect of the extract on’ HeLa cells from CVB_3m infection.

Table 3 Protective effect of ethyl acetate extract on HeLa cells shown in percentage (A₆₀₀ value of cells protected with the extract/ A₆₀₀ value of normal HeLa cells

Dilution	1:256	1:512	1:1024	1:2048	1:4096	1:8192	10-5 CVB3m
The ratio of A600	88.56 ± 0.032	79.46 ± 0.008	73.07 ± 0.057	65.57 ± 0.023	61.83 ± 0.038	56.1 ± 0.015	16.7 ± 0.027

The percentage of vital cells protected with the extract accounted for compared with normal HeLa cells decreased with the in-creased extract dilution, and it was preferable from concentration 1 : 256 mg/mL to 1:1 024 mg/mL. ^bP < 0.01 vs group of 10^-5 CVB_3m.

Table 4 The value of A₆₀₀ obtained from various infective phase

Dilution/A600	1:256	1:512	1:1024	1:2048	1:4096	1:8192
First group	0.45 ± 0.041	0.56 ± 0.011	0.63 ± 0.036	0.65 ± 0.043	0.37 ± 0.076	0.38 ± 0.029
Second group	0.72 ± 0.022	0.96 ± 0.013	0.69 ± 0.009	65.00 ± 0.025	0.78 ± 0.056	0.73 ± 0.054
Third group	0.53 ± 0.030	0.48 ± 0.036	0.40 ± 0.049	0.39 ± 0.066	0.39 ± 0.077	0.32 ± 0.015

The A₆₀₀ value of the cells co-cultured only with RPMI1640, cells co-cultured only with 10 ^-5 CVB_3m and cells co-cultured only with extract (1:256 mg/mL) was 1.29 ± 0.011, 0.24 ± 0.023 and 1.20 ± 0.029, respectively. The value of A₆₀₀ of the cells treated with extract of variable concentration at different time of CVB_3m infection was higher than that of the cells infected with CVB_3m and not treated with the extract, which indicated the protective effect of the extract existed through all the processes of CVB_3m attachment, entry, biosynthesis and assemble in cells.