Screening And Antitumor Activity Of Adenosine Deaminase Inhibitor From Endophytic

Endophytes is a natural and huge resource,and the richness and diversity of theirs metabolites would provide new way and new methods for drug discovery.Adenosine deaminase（ADA）is an purine nucleosides enzymes which can irreversible catalytic adenosine metabolism to inosine and hypoxanthineand,and plays an important role at maintaining the normal function of organism.Studies have shown that much human disease is associated with abnormal adenosine,especially cancer,virosis and immune system diseases.However,there are some defects in the present ADA inhibitors,such as irreversibility,side-effect and higher toxicity,etc.These limited its further application in clinical.In view of this,20 species of medicinal plantseeds and 6 kinds of medicinal plants were chosen to investigate their endophytes and ADA inhibitory activity of their endophytes metabolite.The results were obtained as follows:Isolation and identication of endophyte with ADA inhibitory activity.In the present study,the methods of tissue homogenate and streak were used,and 182 endophytes were isolated from 20 species of medicinal plant seeds and 6 species of medicinal plants.Subsequently,those endophytes were fermented in PDA medium with shaking flask,and182 kinds of sample were extracted by ethyl acetate from culture broth and further used to determinate their ADA inhibitory activity by spectrophotometric method.The resultd showed that there are 56 kinds of extracts inhibit the rate of more than 50%,22 extracts more than 70%and 4 extracts more than 90%,accounting for 30.77%,12.08%and 2.19%of the total,respectively.The extracts from WT-9 strains and QJ-4 strains in the concentration of 1mg/ml had remarkable ADA inhibitory activity with an rate of 97.05%and95.67%,respectively.The results of phylogenetic analysis based on 18S rDNA sequences and microscopic morphology observation showed that WT-9 strain and QJ-4 strain belonged to Cochliobolus sativus and Aspergillus niger,respectively.Isolation and purification of active ingredients of ADA inhibition.Using chromatographic methods,including silica gel column chromatography,preparative TLC,Sephadex LH-20 and high HPLC,etc.,finally,11 compounds were isolated from ethyl acetate extractes of WT-9 strains and QJ-4 strains.Further,their structures were elucidated by e x t e n si v e s p e c t r os c o p i c an a l ys i s an d w e r e i d e n t i f i ed a s D e ox yc h o-lic Acid（H1）;13β,14β-androstan-17-one（H2）;methyl 11-hydroxyhexadec-ano-ate（H3）;3β-O-cis-p-Coumaroyl-2α-hydroxy-urs-12-en-28-oic acid（H4）;Diglucosyldirhamnoside（H5）;1,3-diacyl glycerol（H6）;methyl 11-hydroxyhexadecanoate（H 7）;l u p i n i n e（H 8）;3-（4-n i t r o p h e n y l）-5-p h e n y l i s o x a z o l e（H 9）;k a e m p f e r o l-3-O-β-D-g l u c o p yr a n o s i d e（H 1 0）;m yr i c e t i n（H11）.Subsequently,the ADA inhibitory activity of thses compounds were evaluated.The results showed that compounds 3 and 9 had better ADA inhibitory activity and,their IC50were 31μg/ml and 908μg/ml,respectively.By using Lineweaver Burk double bottom curve method,the inhibition type on ADA of Compounds 3 and 9 were identified as mixed inhibition and anticompetitive inhibition,respectively.Evaluation compound antitumor activity with MTT assay.As a classical cytotoxic analysis,the MTT assay were used in the present study.The results showed that compounds H3 and H9 exhibited remarkable dose-time-effect relationships in 72 and 48 hours respectively with growth inhibitory property at the concentration(IC₅₀)of 0.385μg/ml and0.165μg/ml in human hepatoma（HepG2）cells.Our results suggested that endophytic would be a better resources for screening ADA inhibitor of antitumor drugs.At the same time,compound H3 would be exploited as a new potential ADA inhibitor.