摘 要：
【Objective】To evaluate the antiviral activity mechanism action of phillyrin and other Chinese traditional medicine components to swine transmissible gastroenteritis virus(TGEV), to confirm the antiviral effect of the drug components in vitro, and therefore to provide scientific basis for screening drugs of antiviral activity to TGEV.【Method】Animal cell culture techniques, MTT detection and cytopathic effect(CPE) methods were used to determine the toxic effect of six Chinese traditional medicine components, including phillyrin, forsythiaside, chlorogenic acid, caffeic acid, eugenol and paeonol, on Swine testis(ST) cells. The maximum safe concentrations of the drugs on ST cells were determined by observing CPE of cells, virus TCID_(50) was determined at the same time, and 100·TCID_(50) with cell culture maintenance medium was prepared. The original solutions of the 6 drug components were diluted within the maximum safe concentration scope, and their antiviral effects for cell growth inhibition in vitro were measured with three arrangement routes: TGEV before drugs adding, TGEV after drugs adding and TGEV-and-drugs at the same time. Normal cell control and virus control were arranged for each testing group, with three replications for all treatments. The OD630 was determined by using Eliasa approach, the rate of inhibition of six Chinese traditional medicine components to TGEV in different ways was determined, and the inhibition of the best Chinese traditional medicine component on the replication of TGEV was screened, the best antiviral effect concentration of each drug component was recorded. After the six Chinese traditional medicine components reacted with the TGEV, the TCID_(50), RT-PCR was used to evaluate the viral titer in the supernatant. Furthermore, the proliferation inhibition of six Chinese traditional medicine components on TGEV in ST cell was determined.【Result】The results showed that the highest safe concentrations of Phillyrin, Forsythiaside, Chlorogenic acid, Caffeic acid, Eugenol, Paeonol were 320, 200, 80, 125, 100, 200 μmol·L~(-1), respectively; the best antiviral effect concentration were 160, 100, 20, 62.5, 25, 100 μmol·L~(-1), respectively. By using Karber method, the TCID_(50) of initial TGEV was estimated to be 10~(-6.25)/0.1 m L. All the six Chinese traditional medicine components had inhibition effects on TGEV in ST cells in vitro. In particular, caffeic acid at 62.5 μmol·L~(-1) mixed with TGEV at 100·TCID_(50) showed the best inhibition effect. With this caffeic acid treatment, 72 h after the test started, the tested cells could still keep smooth, without pycnosis, complete, and profile clear between cells, with only a few cells fell off, or death. At that moment, the measured TGEV TCID_(50) was 10~(-3.75)/0.1 m L in the supernatants. The results showed that the virus in treatment with caffeic acid was significantly different, compared to the virus control group with 10~(-6.45)/0.1 m L(P<0.01). The inhibition rate reached 84.4% according to the OD630 value in the direct inactivation experiment. For other tested components, the TGEV TCID_(50) of phillyrin, forsythiaside, chlorogenic acid, paeonol and eugenol were 10~(-4.75), 10~(-5.55), 10~(-5.55), 10~(-5.65), 10~(-5.75)/0.1 m L, respectively, with no significant difference, compared to the virus control group, with most of the rate of inhibition below 50%. In addition, all of the six Chinese traditional medicine components had proliferation inhibition on TGEV in ST cells, directly killing effect was the most affective one, followed by adsorpt blocking effect, with replicate blocking effect behind. RT-PCR was used to evaluate the viral titer in the supernatant, the result showed that caffeic acid group was dark compared with the virus control group, the virus titer was low, inhibition effect was remarkable to virus. The next were Phillyrin, Forsythiaside, Chlorogenic acid, Paeonol and Eugenol.【Conclusion】All the six Chinese traditional medicine components had proliferation inhibition on TGEV in ST cells, and caffeic acid showed the best inhibition effect. It has the potential to develop into the antiviral drugs.
