Antiviral Activity Of Selaginella Moellendorfii Hieron Extract Against CVB3 & HSV-I

Selaginella moellendorfii Hieron is the whole herbs from SelaginellaSpring, Selaginellaceae, which is commonly used as Chinese Herbs & EthnicMedicines. It can clear heat & expel miasma, cure cancer & stop bleeding.It isused for cancer, pneumonia, acute tonsillitis, pink eye & mastitis treatment ect.It is applied to icteric hepatitis, hemoptysis of pulmonary tuberculosis &general dropsy in Yizu Yunnan, while, cancer, diarrhoea, gonorrhea, infantileconvulsion, bloodspitting, hematochezia, menostasis, hemorrhoids, burn andscald, traumatic bleeding in Tujia ethnic minority. Moreover, tablet made ofwhich can cure idiopathic thrombocytopenic purpura, susceptible purpura,secondary purpura & various haemorrhagic diseases.Recently, phytochemistry researchers have been separated all kinds ofchemical composition, such as flavonoid, alkaloid, phenol, organic acid,amino acid & polysaccharide ect. The study focused mainly on Ethyl acetateas well as n-Butanol extracts, from which separate dozens of flavonoids,which are biflavonoids besides apigenin. In which, Amentoflavone is in themajority, that extract from Selaginella tamariscina by Okigawa et al in 1971,at the earliest.Our research team separate 19 bis-flavonoids,1 flavonoids & glycosidesfrom Selaginella, from which 5 are neoflavonoid, 15 are found for the firsttime, which can restrain XOD, LOX&COX-2 activation. We discovered thatamentoflavone, robustaflavone & Robustaflavone 4'-Me are commoncharacteristic components of Selaginella, which can anti oxidation, virus ortumor. They are active indicators. We separated 6 bis-flavonoids from Selaginella moellendorfii Hieron,which identified as7,4',7"-tri-O-methylrobustaflavone(Ⅰ),7,7"-di-O-methylrobustaflavone(Ⅱ),4'-O-methytrobustaflavone(Ⅲ),robustaflavone(Ⅳ), hinokiflavone(Ⅴ) &amentoflavone(Ⅵ). CompoundsⅠ-Vare found first from Selaginellamoellendorfii Hieron,Ⅰ&Ⅱare robustaflavonoids, which are discovered onlyfrom Podocarpus imbricatus, Podocarpus, Podocarpaceae, Gymnosperm, thatalso separated from Selaginella & other plants initially.Pre-study also manifested that Selaginella Spring has anti-CVB3 action invitro, e.g. S moellendorfii, S labordei & S delicatula etc. Ethyl acetateextraction of Selaginella moellendorfii Hieron can inactivate CVB3 directly &inhibit biological synthesis, of which active ingredient are flavonoids.Therefore, we'll extract and separate total flavonoids, research on and developpreparation technology & quality standards. All the antiviral tests in vitro &vivo showed the effect is confirmed and credible.Firstly, about preparation technology of Selaginella moellendorfii Hieronextract, ethanol reflux extraction is taken as the crude extraction method bysingle-factor experiments. The optimum condition is refluxing twice, 1h eachtime by nine times 85% alcohol, that method is orthogonal trial, index is totalflavonoids quantity & yield. The top condition of MSO degreasing is refluxingtwice, 1h each time by six times, that method is also orthogonal trial, index isamentoflavone quantity & yield. Then dissolve it into low concentrationethanol, which is filtered to be purified by macroporous resin. After that, weget final product: powder of total flavonoids. The best technology is eluted at2BV/h by 8BV 70% alcohol through D101 type, weight of resin: herbs is 3:1,which could be used for 3 times.On quality standards of Selaginella moellendorfii Hieron, content of totalflavonoids & amentoflavone is assayed by UV spectrophotometry & HPLC.We have done method validation and stipulated that content of total flavonoids is not less than 50.0%, while, amentoflavone is 35.0%.Meanwhile, we also draft QS of herb, determine its name, source,character, identification, assay, processing, natures&flavors, meridialdistribution, function&indication, usage&administration, storage. On assaying,amentoflavone is measured by HPLC, reference substance is amentoflavone,which has been proved by method validation. So the content of amentoflavoneis no less than 0.30% of dry crude medicine.Subsequently, antiviruses of Selaginella moellendorfii Hieron extract werestudied, that we selected CVB3 & HSV-I. According to replication cycle, 3treatment groups were taken, which are anti-adhesion, directly inactivate &inhibit biological synthesis. Cytotoxicity test showed that TC50 is 60μg/ml ofthe Selaginella moellendorfii Hieron extract. So we choose the drug levelbelow:5,10,20,30,40,50μg/ml. The study on anti-CVB3 showed that IC50 is36μg/ml, the maximum inhibition ratio is 60%, TI is 1.7 about themechanism of directly inactivating; while, IC50 is 16μg/ml, the maximuminhibition ratio is 70%, TI is 3.8 about the mechanism of inhibiting biologicalsynthesis. Meanwhile, the research on anti HSV-I manifested that IC50 is 21μg/ml, the maximum inhibition ratio is 80%, TI is 2.9 about the mechanismof directly inactivating; while, IC50 is 12μg/ml, the maximum inhibitionratio is 90%, TI-is 5.1 about the mechanism of inhibiting biological synthesis.The result showed that Selaginella moellendorfii Hieron extract showed littlecytotoxicity as well as function of inactivating virus directly & inbitingbiological Synthesis, which has high biological value.Finally, we have researched priority on anti-CVB3 action in vivo ofSelaginella moellendorfii Hieron extract.Acute toxicity test indicated that mortality is 10%, 30%, 60%, 80%, 100%as the drug level is 4.0, 5.0, 6.3, 7.9, 10mg/g i.g, which showed that LD50 is6.2 mg/g of Selaginella moellendorfii Hieron extract. Therefore, we take 0.1, 0.3, 0.9 mg/g for LMH drug concentration for antiviral therapy.CVB3 0.2ml(TCID50) was inoculated i.p in male km mouse(10±2g)toestablish experimental model of viral myocarditis. Mice were divided into 4groups at random as normal control group, model control group, ACV groupand administering group, 0.2ml/10g i.g once a day, 15 days. It demonstratedthat recovery rate is 30% & mortality is 50% when 0.1mg/g(low concentration)i.g; recovery rate is 50% & mortality is 20% when 0.3mg/g(mid concentration)i.g; recovery rate is 70% & mortality is 10% when 0.9mg/g(high concentration)i.g. So ED50 is 0.4mg/g, the curative ratio can be 70% & minimum mortalityis 10% of Selaginella moellendorfii Hieron extract. Analysis all the indexcomprehensively, it displays that total flavonoids extract can suppressproliferation of CVB3 actively, protect viscera, lessen symptoms distinctively,reduce mortality, which is dose-dependency distinctively.The research manifested little cytotoxicity(TC50:60μg/ml) & lowtoxicity(LD50:6.2mg/g) of Selaginella moellendorfii Hieron extract, whichcan inhibit CVB3 & HSV-I apparently in vitro as well as cure ViralMyocarditis induced by CVB3 effectively in vivo. It is a new potential validanti CVB3&HSV-I drug, which provides a new option for antiviral medicinedevelopment.