Study On The Inhibitory Action On Coxsackie Virus B₃ And Herpesvirus-â…  Replication With Emodin Combined With Other Chinese Medicine Components

HSV and CV are common viral pathogen for human being,and can lead to human diseases,Acyclovir and its derivatives are used widespreadly for the thrapy of diseases caused by HSV,but some shortcomings,such as side-effects and relatively higher price,restrained their application.So far there are not very effective drugs developed for the therapy of the diseases caused by CVB₃.The activity of anti-CVB₃ and anti-HSV of extract from Rheum and Rhizoma was discovered previously in our group.Emodin,the main bioactive component exists within these two TCMs,therefor was investigated on its efficacy of anti-CVB3 and antiHSV.But there exist some problems when using the emodin directly:low water-solubility,high cytotoxicity,teratogenesis and mutagenesis.To resolve these problems,three methods were developed:(1)Emodin was dispersed with PEG6000 for the improvement of its water-solubility;(2)Emodin was formulated with curcumin and matrinc to eradicate its cytotoxicity,while its antiviral activity was enhanced;(3)The literature of extraction was optimized to reduce the toxicity,teratogenesis and mutagenesis of the emodin.Orthogonal design was used to optimize the formulation of emodin,curcumin and matrine according the effect of anti-CVB3 and anti-HSV-1.Hep-2(a cell-line)infected with CVB₃ were cultured with different concentration and proportion of the Emodin,Curcumin and matrine for 72 hours and their inhibitory effect on CVB₁ replication was evaluated with MTT Cell Proliferation Assay.The cytotoxicity of emodin was reduced 10 folds as before, while the anti-viral ctivity was enhanced after proportional formulation with curcumin and matrine.All of combinations could inhibit cytopathic effect(CPE) and improve the survival rates of CVB₃-infected Hep-2 cells.The ratio which kill viruses directly was 2:1:2 of emodin:curcumin:matrine,and which inhibit the replication of CVB₃ in ceils was 2:0:1 of emodin:curcumine:matrine.The results demonstrated that the toxicity of emodin was reduced and its anti-CVB₃ activity was enhanced by the combination with other active component.Vero(a cell-line)infected with HSV-1 were cultured with different concentration and proportion of the emodin,cucumin and matrine for 72 hours and their inhibitory effect on HSV-1 replication were investigated with MTT Cell Proliferation Assay and barren spot subtractor assay.The cytotoxicity of emodin was reduced 11 folds as before,while the anti-HSV effect was enhanced by the way of combination with curcumin andmatrine.In the veto cell system,some of the drug combination could inhibit cytopathic effect(CPE)of HSV- infected cells. The formulation which stop the viral adsorption most effectively was 2:1:2 and 1:2:2 of emodin:curcumin:matrine;The formulation which has the best effect of killing HSV-1 directly was matrine,and inhibiting the replication of HSV in cells was 2:0:1 of emodin:curcumine:matrine.The result demonstrated that the toxicity of emodin was reduced and its anti-HSV activity was enhanced by the optimized formulation with other components.The best formulation(emodin extract:curcumine:banlangen was 2:1:2)was chosen to protect mice from being infected by CVB₃.the mice infected with CVB₃ were administered with optimized emodin formulation for consecutly 15 days. Recovery rate and death rate were recorded.After treatment,all the mice were killed,and the heart,liver,lung and kidney were taken out to be observed the physiological change and immunofluorescence,and virus titer was calculated, and the extraction of mRNA and operation of PCR were carried out.The results showed that the symptoms of CVB₃-infected mice were alleviated with the proper emodin formulation,and that mortality was reduced;The virus titer in organs was lower after treatment;Fluorescence was not observed in the organs of treated mice and mRNA could not be extracted from treated nice.The study demonstrated that the emodin formulation can inhibit CVB₃ multiplication in heart,lung and kidney and protect these organs from infection.The anti-viral effect of proposed emodin formulation was better than that of Acyclovir.The present study also showed that CVB₃ could not proliferate in liver.Inbred Kunming mice were chosen to be investigated whether the emodin,the matrine and the proper combination have the acute toxicity on mice.Healthy adult mice were randomly divided into groups.The toxicity on mice was analyzed by observing the general status,pathological dissection.The general status. Pathological dissection that the high dose of emodin and matrine showed toxicity on mice organs,but the toxicity was reduced after proper formulation.The toxicity of banlangen and emodin extract was lower than that of pure compound; Accumulation toxicity test displayed that the emodin combination has no cumulative toxicity.The result from pregnant mice during the sensitive period showed that there was no significant difference between medication groups and normal groups,and emodin formulation showed no teratogenic effect.The procedures of the extraction of emodin and banlangen were studied. Orthogonal design was adopted to optimize the entire process of extraction. 8-folds of 80%alcohol was used to extract Rhizoma three times and for 1.5h each time.HPLC was used to determine the concentration of Emodin,and the content of emodin in extract should not be lower than 10%and the purity should not be lower than 70%.Mobile phase wasacetonitrile:0.1%phosphoric acid(80:20),and wave-length of detection wasλ₂₅₄.The ion exchange resin was used to purify the matrine,6-folds of 60%alcohol refluxed three times and for 1.5h each time.The optimized procedure is as follows: after 0.5%hydrochloric acid treatment,pH12 ammonia was added in and 3-folds BV of 80%ethanol was used toelute the total alkaloid of matrine.UPLC was used to determine the content of matrine.Mobile phase was acetonitrile:absolute alcohol:3%phosphoric acid(70:15:15),and the wave-length of detection wasλ₂₂₀.The HPLC condition for curcumin was as follows:mobile phase was methanol:0.6% acetyl acid(75:25),the wave-length of detection wasλ₄₂₈,and the content of curcumin should not be lower than 80%.The present work focus on the activities of anti-CVB and anti-HSV-1 of emodin formulation,through the way of cell biology and animal model.the optimized formulation(emodin extract:curcumine:banlangen was 2:1:2)was achieved,which showed less cytotoxicity and no acute toxicity.