Inhibitory Mechanism Against TMV And Structure-Activity Relationship Of Active Substance From Brucea Javanica

14 compounds were isolated by DCC,TLC and HPLC instrumentations from Brucea javanica seeds,guided by anti-Tobacco mosaic virus(TMV) replication activity,5 were quassinoids according to the chemical structures identification. Half-leaf and leaf-disc methods were employed to inspect the activities against TMV infection and replication of 5 quassinoids from B.javanica,together with the other 3 quassinoids from Anthus altissima.For compounds showed higher activity,the EC₅₀ of which were tested,and also the activities against Cucumber mosaic virus(CMV) and Potato virus Y(PVY) infection were examined.In addition,the structure-activity relationship(SAR) of quassinoids against phytovirus was analyzed.The results established that compounds 1�4 of 5 quassinoids from B.javanica showed much higher anti-TMV activity,which were all dose-dependent,their EC₅₀ against TMV infection were 13.7?g/mL,22.8?g/mL,30.2?g/mL and 13.7?g/mL respectively, which against TMV replication were 2.1?g/mL,24.3?g/mL,28.4?g/mL and 19.7?g/mL respectively,all tested quassinoids showed similar activity against both CMV and PVY infection to that of TMV.The yield of compounds 1 and 2 were much higher than any other compound isolated from material,which were 210 mg and 2.2 g from 50 g ethyl acetate extract of alcoholic extract matter of B.javanica seeds. According to the yield and antiviral activity of all components,it was affirmative that compounds 1 and 2 were primary antiviral components in seeds of B.javanica.The SAR analysis indicated that it was a new bio-activity of quassinoids against phytovirus,and the methylene oxygen bridge in D-ring affected dramatically decline of antiviral activity of quassinoids came from B.javanica and A.altissima;the change of C-15 easter chain influenced the antiviral activity of quassinoids came from B.javanica;the number of hydroxide was a possible factor that affected the antiviral activity of quassinoids.Action mechanism research in vitro indicated that quassinoids had ability to inhibit the foundation of TMV particles or TMV-RNA infection courses on host plants surface,via a reversible combination with them,but not destroyed the integration and configuration of the two.Besides,quassinoids influenced the polymerization of TMV-CP subunits in vitro,and accordingly interfered with the assembly of integrated TMV particles that composed by RNA with CP.The TMV-MP gene was obtained by RT-PCR from the total RNA of TMV infected leaves and cloned to pMD18-T vector,which length was 807 bp,compared with the TMV-U1 strain in GenBank,the identities of neucleotides and amino acids were both 100%.The target fragment was subcloned to an expression vector pET-29a,and the fusion protein was expressed in the E.coli BL21(DE3) strain,the highest amount of target protein was gained 4 h after IPTG inducement,which was insoluble.The antibody against the movement protein was prepared by immunized rabbit with target protein cut from SDS-PAGE,a high titer 25,600 was determined by ELISA procedure. Western-blot analysis indicated that TMV-MP was expressed in infected leaves at an early stage,and the antibody showed a good speciality also.In order to make clear the anti-TMV multiplication mechanism of quassinoids in vivo at plant and cellular level,Bruceine D was used as a representative,real time-PCR?semi-quantative RT-PCR?indirect-ELISA and western-blot procedures were employed.The results showed that quassinoids had ability to inhibit the accumulation of both TMV genomic RNA and subgenomic RNAs,accordingly hijacked the expression of TMV-CP and MP in host,and that blocked the cell to cell and long distance transport of TMV,delayed the emergence of mosaic symptom caused by TMV infection;in addition,it was found that the activity of quassinoids against TMV replication declined with the interval of TMV infection and agent application,revealed that the action of compounds against TMV replication took place at an early stage of virus infection,namely,the earlier antiviraI agents were used,the higher of antiviral activity was.The systemic resistance and protection against TMV infection on tobacco induced by Bruceine D were studied.The compound induced systemically activity enhancement of a series defense enzymes,like POD,PPO,PAL,SOD and?-1,3-glucanase,as well as the generation of isoezyme spectras of POD and PPO, which did not exit in healthy and diseased leaves;the compound also inhibited the decline of chlorophyll content and increase of MDA content caused by TMV infection; prevented the decline of soluble sugar content and soluble protein content caused by TMV infection;two kinds of pathogenesis related proteins(PRP) also generated after Bruceine D treatment,which different from that induced by TMV infection.Real time-PCR and indirect-ELISA techniques were used to research the effect of Bruceine D on multiplication of CMV genomic and subgenomic RNAs.The results illustrated that although the compound had ability to inactive CMV to infect host plants in vitro,it could not inhibit the virus replication in host after the infection.It was reasonable to believe that there were different action mechanisms that quassinoids against infection and replication of positive single strand RNA virus.To investigate the antiphytoviral mechanism of triterpenoids,67 triterpenoids and triterpenoid glycosides were screened the anti-TMV replication activity at the concentration of 200?g/mL,the SAR based on structures and activity of all compounds were analyzed too.Most of tested compounds demonstrated definite anti-TMV activity in vivo,10 compounds were over 70%,which were all dose-dependent,and their EC₅₀ values were calculated.According to the SAR analyse, it was obvious that the activities of triterpenoids and triterpenoid glycosides lied on not only in their self-structure but also in the substituents,which sometimes affected the activity dramatically,as for the triterpenes of ursane type,lactone at the position C-20 and C-28,arabinosyl at the position C-3,and glucosyl at the position C-28 are all favorable to anti-TMV;while the ones of oleanane type,?-hydroxyl at C-19, xylosyl at C-3,and glucosyl at C-28 all contribute to the inhibition.