| In order to obtain antiviral and anticancer gisenoside-like derivatives with stronger biologic activity and lower toxicity, we modified ginsenoside Rh2 and got two new derivatives of ginsenoside Rh2 which were named as derivative B1 and derivative B2. Derivative B1 was proved to have better water-solubility.Marek's Disease (MD), a highly infectious lymphoproliferative disease in chickens, is caused by an alphaherpesvirus-Marek's disease virus (MDV), resulting in lymphoplasia. Further more, MD, served as an important animal model of viral oncogenesis and the first tumor being prevented by vaccine, has offered important contributions to veterinary medicine, basic science and comparative oncology.Due to these reasons, in the present study we investigated the antiviral effect against MDV and the inhibitive effect on the growth of MSB-1 cell of Rh2 and its derivative(B1) in vitro.At first, the model of chick embryo fibroblast cell infected by MDV was established in vitro. The viral titer was determined by tissue culture infectious dose 50 (TCID50) assay and the plaque forming methods of CEF infected with MDV was established. The TCID50 of MDV in CEF cells is 10-3.5/0.1mL. The safe concentration of Rh2 and B1 on CEF was determined by cytopathic effect (CPE) and neutral red assay. Their maximal safe concentrations to CEF were judged according to OD value and morphological variety of the cells.The results showed that the safe concentration of Rh2 and B1 were 3.125μg/mL and 50μg/mL respectively. The cytotoxic concentration 50%(CC50) of Rh2 and B1 were 4.5μg/mL and 70.5μg/mL respectively.The Rh2 and B1 in various concentrations were applied to CEF cells infected by MDV. Then the in vitro antiviral activity against MDV of Rh2 and B1 was observed from 3 aspects: (1) inhibiting the virus directly; (2) obstructing absorption; (3) inhibiting replication. The CPE inhibition data were expressed as the 50% effective (viral CPE-inhibitory) concentration (IC50), and selectivity index (SI), equal to the value of CC50/IC50. The results showed that Rh2 and B1 could inhibit multiplication on MDV in vitro. When adding medicine and virus together to the single cell layer, Rh2 and B1 all could significantly suppress the multiplication of MDV. The 50% effective (viral CPE-inhibitory) concentration (IC50) of Rh2 and B1 were 1.31μg/mL and 44.92μg/mL respectively, and selectivity index (SI) were 3.43 and 1.57 respectively. When the virus was added after the interaction of the medicine and cells, Rh2 and B1 could not suppress multiplication of MDV. When drugs were added after the attachment of the virus, B1 has significant antiviral effects, but the Rh2 has not significant antiviral effects. The 50% effective (viral CPE-inhibitory) concentration (IC50) of B1 was 32.37μg/mL, and selectivity index (SI) was 2.18. So we can conclude that the antiviral acitivity of B1 is better than that of Rh2 in the way of multiplication inhibition.We investigated the inhibitive effect of Rh2 and B1 on MSB-1 cell by MTT assay, and explored the mechanism through flow cytometry, agarose gel electrophoresis, fluorescence detection and morphological observation by transmission electron microscope. The results showed that Rh2 and B1 inhibited the growth of MSB-1 cell in dose-dependent manners. After the MSB-1 cell was treated with Rh2 for 72 hours, the IC50 was 0.65μg/mL. After the MSB-1 cell was treated with B1 (50μg/mL) for 72 hours, the inhibitory rate was 44.66%. Flow cytometry analysis indicated that the proportion of cells at S phase increased, and that of cells at G2/M phase decreased obviously. Moreover, there were apoptotic peaks in control group and drug group which may be caused by normal apoptosis, but it is higher in the drug group. After MSB-1 cell was treated with Rh2 and B1 for 72 hours, and then being stained with Annexin-V-EGFP and PI, we observed typical apototic cells with confocal microscopy. And it indicated that Rh2 and B1 could induce apoptosis of MSB-1 cell. MSB-1 cell, treated with Rh2 and B1 for 72 hours at 2μg/mL and 50μg/mL respectively, became inhomogeneous, the number of microvilli decreased, chromatin shrunk, and vacuoles increased. These results suggested that Rh2 and B1 inhibited the growth of MSB-1 cells in dose-dependent manners, and the mechanism was relative to blocking the cell cycle at S phase and inducing apoptosis of some MSB-1 cells.Based on all the information above, we concluded that Rh2 and B1 have the antiviral activity against MDV and inhibited the growth of MSB-1 cell. And the mechanism of the inhibition was relative to blocking the the cell cycle at S phase and inducing apoptosis of some MSB-1 cells. This present study offered data for developing the antitumor and antivirus resources.
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