Studies On Hypericum Perforatum L. Extract Against Influenza A Virus And Immune Regulatory Mechanism

Hypericin and hyperoside are the major effective components in Hypericum perforatum L. Extract (HPE). The pharmacological experiments showed that they had the effects of anti-viral, anti-tumor, anti-depression, anti-inflammation, anti-oxidation and raise immune function. The studies on preparation of Hypericum perforatum L. extract, determine of qualitation and quantitation of hypericin and hyperoside. Anti-viral effects of HPE against influenza A virus (IAV) in vitro and in vivo were carried out respectively in this paper. Anti-viral mechanism was approached according to pathomorphism, anti-oxidation, immune function and secretion of immune-inflammation correlation factor.Thin-layer chromatography (TLC) was used to detect effective components qualitatively in HPE. High performance liquid chromatography (HPLC) was applied to detect the content of hypericin and hyperoside in HPE. The experiment results showed that the content of hypericin was 1.45 % and hyperoside was 4.72 %.Cell survival rate and inhibition rate were served as target to evaluate antiviral activity of HPE against IAV in vitro by method thiazolyl tetrazolium (MTT) and the influence of HPE against proliferation of IAV in MDCK cell. The experiment results indicated that within the scope of biggest nontoxic concentration, the effect of HPE in deactivating IAV is not obvious as compared with oseltamivir and the difference was significan(tP<0.01,P<0.05). But the effects of preventing IAV from adsorbing target cell and inhibiting its replication were obvious as compared with the virus control and differences were significant(P<0.01,P<0.05). The anti-viral effect was dose-dependent with drug concentrations (12.5μg/mL -100μg/mL). HPE could decrease maximally the TCID50 of IAV from 4.6 to 3.5.This study was performed to investigate anti-viral effect of HPE by mouse model in whole level. Mice were infected with IAV through nasal cavity to make model mice. After 4 hours, mice infected with IAV were treated with HPE 100 mg·kg-1, HPE 50 mg·kg -1 and ribavirin 10 mg·kg-1 respectively by mouth for 5 days, twice daily. The major criteria for evaluating the effects of HPE on mice infected with IAV in whole level including influence on the mortality rate, life-prolong rate of mice, lung index and the change of viral pneumonia. Pathomorphology of the lung tissue, spleen index and thymus index of mice were observed to approach further the mechanism of HPE on mice infected with IAV. The changes of SOD and MDA in lung tissues were detected by XO and TBA colorimetric analysis antigenic to observe the effect of removing free radicle and anti-oxidation of HPE. The change of T, B leukomonocyte cells multiplication were determined by MTT in order to understand the mechanism of HPE on mice infected with IAV in cellular level. The contents of cytokine IL-6, IL-10, TNF-αand IFN-γin the lung tissues and serum were observed dynamically in two phases (the third day and the fifth day) using ELISA to research on the influence of HPE. And their mRNA in the lung tissue (the fifth day) were observed by RT-PCR to probe further into mechanism of action of HPE.The research results showed that the death rate of infectious model group was 80 %, survival time was 8.8 days. But the death rate of two HPE groups were respectively 40 % and 50 %, survival time were 12.1 and 11.3 days. There were significant differences as compared with the infectious model group(P<0.01,P<0.05).Therefore HPE could decrease the mortality rate and prolong life-time of the mice infected with IAV. The lung index and virus hemagglutinin titre of the infectious model group were increaser than those of the normal group. The lung index and virus hemagglutinin titre of HPE groups decreased and the change of viral pneumonia lessened as compared with the infectious model group (P<0.05,P<0.01).The lung pathohistology results showed that the lungs were consolidation, bleed and the changes of interstitial pneumonia in the infectious model group by microscope. But the catabatic interstitial pneumonia were showed in two HPE groups, alveolar septum were thinner and the number of mononuclear cell was less in alveolar and bronchiole wall than those of the infectious model group. The content of SOD and MDA in the infectious model group decreased and increased respectively than those of the normal group (P<0.01). But the content of SOD and MDA in the HPE groups increased and decreased respectively than those of the infectious model group (P<0.01). The spleen index and thymus index of the infectious model group lowered than those of the normal group ( P<0.01). But the thymus of HPE increaser than those of the infectious model group ( P<0.01). T, B leukomonocyte cells multiplication function of the infectious model group were decreased as compared with the normal group (P<0.01). T, B leukomonocyte cell multiplication function of the HPE groups were increased as compared with the infectious model group (P<0.01). The contents of cytokines IL-6 and TNF-αof the lung tissues and serum in infectious model group in two phases were higher than those of the normal group and the differences were significant (P<0.05,P<0.01). But the contents of cytokines IFN-γand IL-10 of lung tissues in infectious model group in two phases were lower than those of the normal group and the difference were significant(P<0.05,P<0.01). The amounts of protein expression of IL-6 and TNF-αof HPE groups decreased and the differences were only significant on the fifth day (P<0.05). The content of IFN-γand IL-10 of HPE groups were decreased and the difference were significant (P<0.05,P<0.01) as compared with those of the infectious model group. As for the mRNA transcription level of IL-6, IL-10, TNF-αand IFN-γon the fifth day shared the same tendency as their protein expression.To sum up, HPE could lessen the lung damage caused by the lung tissues lipid peroxidation and strengthen anti-oxidation through raising the content of SOD and decreasing the content of MDA. It could improve the immune function of mice infected with IAV by improving the thymus index and the multiplication of lymphocytes. It could hold the inflammatory reaction under control by inhibiting the secretion of pro-inflammatory cytokines and promoting that of anti-inflammatory factors, so it could recover the immune damage caused by IAV.