Screening And Characterization Of Calcineurin Inhibitors From Extrat Of Medicinal Plants With Yeast Model

Calcineurin(CN)is the only known phosphatase at present which is regulated by cellar second messenger Ca+2 and Calmodulin(CaM).It distributes widely in different tissues of mammals.Calcineurin not only participates in the regulation of many important physiological functions like the activation of T cell,the expression of IL-2,study and memory and the cell apoptosis,but also has close relationships with some serious diseases such as Alzheimers’ disease and self-immunity disease.The studies on biological roles of calcineurin have progressed to the important discovery that it is the common target of the immunosuppressant drugs cyclosporin A(CsA)and FK506 and that discovery also promotes the researches of immunosuppressant drugs using CN as the target enzyme.However,the use of these CN inhibitors clinically has significant side effects,such as nephrotoxicity,neurotoxicity,hyperlipidemia and hyperpotassemia.Traditional medicinal plants has many advantages,It is of significance to screen CN inhibitors from the medicinal plants compounds with high efficiency and low toxicity.In this study,using the novel yeast Ca2+-signal pathway positive screen model and yeast report gene assaywith CN as the target,hundreds of Chinese medicinal herbs had been screened.Data of yeast positive sreening model showed that 15 kinds of extracts of medicinal plants rescued yeast growth from the inhibition.Among them,polygonum multiflorum thunb and sculellaria barbata showed strong growth rescue effect.Based on the test with yeast synthetic lethal model,we found that the traditional medicinal herbs such as polygonum multiflorum thunb,sculellaria barbata,snow-in-june and corn stigma show inhibition on S.cerevisiae △mpk1 and can be presumed potential calcineurin inhibitor.Among them,polygonum multiflorum thunb showed the highest inhibition.The monomeric compounds such as 2,3,5,4,-Tetrahydroxystilbene-2-O-beta-D-glucopyran-Side(THSG),naringenin,baicalein and isorhamnetin from Polygonum multiflorum Thunb were further studied.Experiments using yeast positive sreening model and yeast report gene assay indicated that THSG inhibited CN activity effectively.The enzyme activity assay showed that THSG has obvious inhibition on CN with RⅡ peptide as substrate.At 500 μmol/L,the highest inhibition was 25.4%.The THSG could directly inhibit CN and it is independent of matchmaker protein.The inhibitory manner was competitive.Also,the CN inhibition could be enhanced with the presence of CaM.In summary,THSG had significant inhibition on CN and it is a good candidate drug of calcineurin inhibitor.