Evaluation Of The Antibacterial And Antiviral Activity Of Tibetan Medicinal And Investigation Of Molecular Mechanism Against HSV-2.

Objective: In order to obtain the active extracts of antibacterial and antiviral effects of Tibetan medicine. Tibetan medicinal plants: Dracocephalum heterophyllu, Aconitum tanguticum, Swertia mussotii, Gentiana urnula, Meconopsis quintuplinervia, Aconitum flavum, Tribulus terrester, Lagotis brevituba were collected and that of antibacterial activity were studied. The antiviral effects of Dracocephalum heterophyllu, Aconitum tanguticum and Swertia mussotii were studied in vitro and in vivo. The antiviral mechanism of Dracocephalum heterophyllum, Aconitum tanguticum and Swertia mussotii was also investigated and discussed.Method :1. The antibacterial activity of Tibetan medicinal plants in vitro: to evaluate the antibacterial activities of eight Tibetan medicinal plants, the agar disc diffusion method and a broth microdilution method were employed for the determination of antimicrobial activity. The inhibition zone and the minimal inhibitory concentration (MIC) values were used to estimate index of antibacterial activity. And D. heterophyllum essential oil and Aconitum tanguticum alkaloid were also studied.2. The essential oil of D. heterophyllum was subjected to gas chromatography-mass spectra (GC-MS) analysis.3. The antiviral activity of D. heterophyllum, A. tanguticum and S. mussotii in vitro: The cytotoxicity of extracts to Vero was assayed by MTT colorimetry. Acyclovir was used as positive reference drug, by using cytopathic effect (CPE), plaque reduction assay and transmission electron microscopy (TEM) , the antiviral effective rate of D. heterophyllum,A. tanguticum and S. mussotii was investigated.4. The anti-viral effect of D. heterophyllum, A. tanguticum and S. mussotii in vivo: Mice encephalitis and genital herpes model were established by infected with HSV-2 respectively to evaluate the antiviral efficacy of D. heterophyllum, A. tanguticum and S. mussotii in vivo.5. Possible mode of action and molecular mechanism of antiviral activities of D. heterophyllum, A. tanguticum and S. mussotii was investigated by using plaque reduction assay method, a real-time PCR assay and scanning electron microscope (SEM).Results:1. The antibacterial activity of Tibetan medicinal plants in vitro: a The eight extracts of D. heterophyllum, A. tanguticum, S.mussotii, G.urnula, M. quintuplinervia, A. flavum, T. terr ester and L. brevituba posses moderate antibacterial activity against tested bacterial stains. The inhibition zones and MIC values were in the range of 8-22mm, and 1.56-50.00mg/ml, respectively. D. heterophyllum and A. tanguticum showed strong antibacterial effect, while M. quintuplinervia had no effect.b The essential oil of D. heterophyllum had great potential of antimicrobial activities against all 8 bacteria and a yeast species tested. The inhibition zones and MIC values were in the range of 18-25 mm, and 0.039-0.156mg/ml respectively.c The MIC values of lipid-soluble alkaloid of A. tanguticum were 1.25mg/ml except against Candida albican whereas the MIC values of water-soluble alkaloid were 5.0mg/ml except against Enterococccus faecalis.2. GC-MS analysis resulted in the identification of eighty-three compounds representing 89.83% of the essential oil D. heterophyllum, which consisted mainly of monoterpenes(46.17%) and sesquiterpenes(28.82%).3. The antiviral effect of the extracts in vitro: The CC₅₀ value of D. heterophyllum, A. tanguticum, S. mussotii extracts and ACV was 8.43mg/mLN 5.54mg/ml,3.23mg/ml,11.02mg/ml, respectively. The EC₅₀ value of D. heterophyllum, A. tanguticum, S. mussotii extracts and ACV was 1.13mg/ml,0.53 mg/ml,1.51 mg/ml,1.64 mg/ml respectively by using cytopathic effect method. While using plaque reduction assay, the EC₅₀ value was 0.99 mg/ml,1.43 mg/ml,3.60 mg/ml and 0.29mg/ml, respectively.The therapeutic index (TI =CC₅₀/EC₅₀) of D. heterophyllum, A. tanguticum, S. mussotii and ACV was 8.91,6.36,1.05,37.4, respectively.4. The antiviral effect of the extracts in vivo: The LD50 of D. heterophyllum, A. tanguticum and S. mussotii was 9.87, 5.62, 0.85, 2.23, 1.31g/ kg respectively. D. heterophyllum and S. chirayita extracts prolonged mean survival times and the reduced protected rate of mice treated with D. heterophyllum was 20%,10% at the dose of 0.5, 1.0g/kg, while that of S. chirayita was 40%,20% at the dose of 1.0, 2.0g/kg, respectively. Whereas A. tanguticum only show minor effect to prolong mean survival times.5. Possible mode of action and molecular mechanism of antiviral activities:a The results of plaque reduction assay have shown that the extracts of D. heterophyllum , A. tanguticum and S. mussotii diminished virus infectivity significantly; D. heterophyllum inhibited the attachment greatly and S. chirayita slight inhibited while A. tanguticum had no effect; all three of extracts inhibitedreplication of HSV-2 in cells efficiently.b Scanning electron microscope further proved D. heterophyllum significantlyinhibited HSV-2 attachment.c D. heterophyllum, A. tanguticum and S. mussotii extracts produce 10⁶,10²,10³ foldsreduction in virus titre in comparison to the the virus control respectively, accordingto real-time PCR assay results.Conclusion:1. The results showed the antibacterial activity of water extracts is D. heterophyllum> A. tanguticum>S.mussotii>A. flavum>L. brevituba>T.terrester>G. urnula>M. quintuplinervia; while that of ethanol extracts is D. heterophyllum> A. tanguticum>S. mussotii>A. flavum>L. brevituba>T. terrester>G. urnula>M. quintuplinervia. The antibacterial nature of the essential oil studied is apparently related to their high monoterpenes and sesquiterpenes contents.2. The extracts of D. heterophyllum, A. tanguticum and S. mussotii showed antiviral activity in vitro. The results of CPF are accorded with that of plaque reduction assay.3. The extracts of D. heterophyllum, A. tanguticum and S. mussotii were found to be effective in encephalitis mice induced by HSV-2 infection though prolonging the mean survive time and reducing the mortality of infected mice.4. Tibetan medicine possesses strong anti-HSV-2 activity in vitro and in vivo. The antiviral mechanisms are related to different stage of virus replication.5. Our findings on antiviral activity could justify some ethno pharmacological usage of these medicinal plants as therapy for virus caused-hepatisis and other infectious diseases.