Study On The Inhibition Of Xantine Oxidase, Lipoxygenase, And Cyclooxygenase By Seven Kinds Of Medicinal Plants From Selaginella

Xantine Oxidase(XOD),Lipoxygenase(LOX)and Cyclooxygenase(COX)are important oxidases dealing with oxidation process in human body.The high expression of the oxidases is closely related with free radicals and inflammation, sclerosis,cardiovascular disease,aging,cancer and other pathological process.The inhibitors of the oxidases is of great significance for the removal of free radicals, reducing inflammation,promoting tissue repair,controleling the development of diseases(such as cancer,viral diseases).There are about 60～70 species in Selaginella in China,among them,the 14 medicinal species are found in Hubei Province,China.They are used for the treatment of cancer,viral hepatitis and other diseases.Pharmacological research confirmed that these medicinal plants have extensive pharmacological activity, particularly in anti-tumor,anti-virus,hypoglycemic effect,anti-bacteria and anti-inflammatory.Selaginella plants generally contain a variety ofbiflavones,mainly amentoflavone,which are probable active substances for anti-cancer.Some species contain the activity components in anti-virus.Thus,in this thesis the screening experiments were made on the inhibition of Xantine Oxidase,Lipoxygenase,and on the active substance to descend Cyclooxygenase-2mRNA by seven kinds of medicinal plants from Selaginella,and also the experiments were made on anti-tumor and anti-virus.The above studies provide the basis on search for the development of new adjuvant therapy drugs of anti-cancer,anti-virus.The ultraviolet spectrophotometer with a dynamic analysis software was used to detect Xanthine Oxidase inhibitory activities in vitro of 28 extracts,three kinds of flavonoids mixture,three kinds of compounds and 12 kinds of fluent fractions from Selaginella.The results showed that ethyl acetate extracts and 95%alcohol extracts are the effective fractions inhibiting Xanthine Oxidase of the medicinal plants from Selaginella,and that three kinds of flavonoid mixtures(flavonoid mixture from Selaginella uncinata;flavonoid mixture from S.moellendorfii;flavonoid mixture from S.labordei)may inhibit relatively strong the activity of Xanthine Oxidase,and that so are three kinds of compounds(robustaflavone;2",3"-dihydro-3',3"'-biapigenin;2",3"-dihydroochnaflavone),and among them,robustaflavone has stronger activity to inhibit XOD that is similar to positive reference substance quercetin.The experiments also showed that the Fr5 has the strongest activity to inhibit XOD among ten fluent fractions(Fr1～Fr10)from methanol extract of S. pulvinata,and the activity of Fr5 is descended after Fr5 was separated and purifyed,and the results showed that the antioxidant activity of the biflavones from S. pulvinata is reflected not only in a single component,but also reflected in their overall effect.Lipoxygenase inhibitory experiment in vitro was made,the samples being 28 extracts,12 kinds of compounds and 3 kinds of component mixture from Selaginella, in order to detecting the impact of the samples to Lipoxygenase activity.The results showed that ethyl acetate extracts and 95%alcohol extracts are mainly the effective fractions,and there are six kinds of compounds show stronger inhibitory activity than others,Their order from strong to weak being:eriodictyol;amentoflavone; robustaflavone;4'-methylether robustaflavone;2",3"-dihydroochna flavone;2",3"-dihydro-3',3"'-biapigenin.COX-2 mRNA expression level in HT-29 was detected by Reverse Transcript Polymerase Chain Reaction(RT-PCR),the samples used being five kinds of compounds extracted from S.labordei.The results showed that the brightness value of band had a negative correlation with their concentration on all samples.These five kinds of compounds being amentoflavone;4'-methylether robustaflavone;2",3"-dihydro-5,5",7,7",3"'-pentahydroxy-6,6"-dimethyl-[4'-0-3"']-biflavone;2",3",-dihy dro-5,5",7,7",3'-pentahydroxy-6,6"-dimethyl-[3'-0-4"']-biflavone and 2",3"-dihydroochnaflavone.MTT assay was used to analysis the effect of the samples on HT-29 cell viability and proliferation in vitro.The results showed that these four compounds can inhibit the growth and proliferation of colon cells in a dose-effect mode When the concentration is between 12.5μg/mL to 100μg/mL.These four compounds are amentoflavone;4'-methyletherrobustaflavone;2",3"-dihydro-5,5",7,7",3'-pentahydroxy-6,6"-dimethyl-[3'-0-4"']-biflavone and 2",3"- dihydro-5,5",7,7",3"' -pentahydroxyl-6,6"-dimethyl-[4'-0-3"']-biflavone.Hep-2 cells infected by Coxsackie Virus B₃(CVB₃)were cultured with 13 extracts from Selaginella with different concentration for 72 hours,and cytopathic effect(CPE)were observed and compared,and the drug effectiveness to inhibit CVB₃ was measured by therapeutic index(TI=TC₅₀/IC₅₀)as an evaluation indicators.The results showed that CVB₃ can be killed or inhibited by the samples these are ethyl acetate extracts and 95%alcohol extracts from S delicatuta,,ethyl acetate extracts and 95%alcohol extracts from S.remotifolia,ethyl acetate extracts and 95%alcohol extracts from S.moellendorfii,95%alcohol extracts from S.pulvinata,aqua extracts from S.labordei,and CVB₃ can not be killed and inhibited by the extracts from S. uncinata.The above study also showed that ethyl acetate extracts and 95%alcohol extracts are the effective fractions to inhibit Xanthine Oxidase and to inhibit Lipoxygenase of the medicinal plants from Selaginella.There is one compound that 2",3"-dihydroochnaflavone can inhibiting the activity of Xanthine Oxidase,Lipoxygenase and Cyclooxygenase-2.There are two compounds that robustaflavone and 2",3"-dihydro-3',3"'-biapigenin have inhibition activity both to Xanthine Oxidase and to Lipoxygenase.There are four compounds that amentoflavone;4'-methylether robustaflavone;2",3"-dihydro-5,5",7,7",3'-pentahydroxy-6,,6"-dimethyl-[3'-0-4"'] -biflavone and 2",3"-dihydro-5,5",7,7",3"'-pentahydroxy-6,6"-dimethyl-[4'-0-3"']-biflavone can inhibit Cyclooxygenase-2,and also inhibit the the growth and reproduction of HT-29 cell.Flavonoids are a group of relatively moderate components,their role in the maintenance of human health also have been gradually increasing emphasis,with profoundly understanding of their anti-oxidation and free radicals removal.The above-mentioned researches would provide certain experimental basis,in order to control the development of inflammation and to prevent the worsening of the disease from the multi-target,multi-channel of Selaginella medicinal plants,and also for the use of Selaginella plant resources scientificly and reasonably.