SHL markedly decreased viral amounts compared to CBF at all concentrations (< 0.001) in A549 cells and at concentrations higher than 4.6 g/mL (< 0.01) in the Acotiamide hydrochloride trihydrate suspension. g/mL on the HEp-2 cells (< 0.05) (a); chlorogenic acid, baicalin, and forsythia glycosides A UBCEP80 (CBF) showed cytotoxicity above the concentrations of 74.2 g/mL on both cells (< 0.001) (b). Data are represented as mean S.D. of nine tests. * < 0.05; ** < 0.01; *** < 0.001 were compared to the cell control. 3.2. SHL Attenuated Virus Proliferation More Significantly Than CBF SHL and CBF dose-dependently decreased virus proliferation in HEp-2 and A549 cells. The effect of SHL, however, showed better suppression than CBF in both HEp-2 cells and A549 cells (< 0.05) (Figure 3). This effect was significantly different at all concentrations except for 2.3 and 1.2 g/mL on Hep-2 cells (< 0.05). It indicated that SHL was more effective at inhibiting the proliferation of the virus than Acotiamide hydrochloride trihydrate that by CBF. Open in a separate window Figure 3 SHL and CBF were dose-dependently effective against human adenovirus III (HAdV3) in both cell types as determined by plaque reduction assay (< 0.05); SHL decreased more plaque formation than CBF at all the concentrations (< 0.05) in A549 cells (a) and at the higher concentrations than 4.6 g/mL (< 0.01) in HEp-2 cells (b). Data are represented as mean S.D. of nine tests. * < 0.05; ** < 0.01; *** < 0.001 were compared to CBF. # < 0.05 was compared to the virus Acotiamide hydrochloride trihydrate control. 3.3. SHL Decreased Plaque Formation More Than CBF When Viral Inoculation Was Given in Different Working Points To better understand the therapeutic intervention during virus invasion, time of addition assay in A549 and HEp-2 cells was employed to explore its working points. SHL and CBF time-dependently and dose-dependently decreased plaque formation in A549 and HEp-2 cells. SHL decreased plaque formation more than CBF when viral inoculation was given in different working points (< 0.05) (Figure 4). It showed that both SHL and CBF were better at inhibiting virus activity when given before viral inoculation than after in the two cells types. As the exposure duration of cells to SHL and CBF before viral inoculation increased, so did the significance of the antiviral activity. Open in a separate window Open in a separate window Figure 4 SHL and CBF were time-dependently and dose-dependently effective against HAdV3 when given viral inoculation in different administrations (< 0.05), and SHL decreased more plaque formation than CBF in both cell types (< 0.05). Data are represented as mean S.D. of nine tests. * < 0.05; ** < 0.01; *** < 0.001 were compared to CBF. # < 0.05 was compared to the virus control. 3.4. SHL Inhibited Viral Attachment Better Than CBF in A549 and HEp-2 Cells Because SHL and CBF anti-virus activity was mainly effective by supplementation before viral inoculation, we predicted that they worked by disrupting viral attachment and that the anti-viral effect of SHL was superior to that of CBF. Results from the attachment assay confirmed this hypothesis, as both SHL and CBF dose-dependently inhibited viral attachment. SHL decreased plaque formation more than CBF at concentrations higher than 4.6 g/mL in A549 cells (< 0.01) (Figure 5a), and SHL Acotiamide hydrochloride trihydrate decreased plaque formation more than CBF at all concentrations in HEp-2 cells (< 0.01) (Figure 5b). These results were consistent with those of the anti-viral effect assay (Figure 3) and the time course assay (Figure 4). It demonstrated that viral attachment was inhibited more with SHL than with CBF, and the effect was not significantly different between A549 and HEp-2 cells. Open in a separate window Figure 5 SHL and CBF.