Transformation And Degradation Of Two Kinds Of Natural Active Products

The effective constituents in the medical plants are the material basis for preventing and curing disease. Medical effective constituents can be converted and metabolized when getting inside the body. In this article Cunninghamella elegans AS3.2028which had similar effect with human cytochrome P450was chosen, in order to find new activated transformation products. Besides, the derivative of Sal B has already been used as injection fluid clinically, however, its drug metabolism in vivo is still unclear. The theoretical support for clarifying its metabolism in vivo will be provided by studying its stability and degradation mechanism in vitro.First,Curcumol can be strongly transformed by Cunninghamella elegans AS3.2028. A major product was separated and purified from the biotransformation products, and it was identified as10S,14-epoxycurcumol, as epoxidation at the double bond outside the seven-memebered ring by spectroscopic analysis. The HPLC-analyzing method of10S,14-epoxycurcumol was established for the first time. Detector:differential refractometer detector. The transformation curve of Cunninghamella elegansAS3.2028resting cells was designed, and the status of the amount of substrate and product changing with time history and the dynamic process of stereoselectivity in biotransformation reaction were studied. Stereoselective epoxidation at the C10-C14double bond of curcumol can be catalyzed by C. elegans AS3.2028. After60h transformation almost all of curcumol has been converted, Cunninghamella elegans AS3.2028catalyze substrate shows the high stereoselectivity.Curcumol can be converted by Cunninghamella elegans AS3.2028, meanwhile its isomer can also be effectively converted by Cunninghamella elegans AS3.2028. Curdione was converted96h for biotransformation by Cunninghamella elegans AS3.2028, and two main products were separated and purified through silica-gel column chromatography from the biotransformation products. It was identified that the epoxidation occurred at the C-C double bond outside the ten-memebered ring by using spectroscopic analysis, generating both (1S,10S)-1,10-epoxycurdione and (1R,10R)-1,10-epoxycurdione. The HPLC-UV analysis method was established. Detector wavelength:285nm. The biotransformation curve of curdione converted by growth cells was designed. After96h biotransformation for curdione, only76%percentage of curdione was converted into new products, however, the productivity of curdione epoxidation was not very high; some other products were also generated. The maximum concentration of (1S,10S)-1,10-epoxycurdione in biotransformation was determined to appear at96h, but the products stereoselectivity were not too high.The high stereoselectivity for Cunninghamella elegans AS3.2028to catalyze substrate shows that it can be considerably utilized in chiral catalyzing. And it will have important significance to get further study. It was supposed that the cytochrome P450enzyme system in Cunninghamella elegans AS3.2028may catalyze the oxidation reaction. The cytochrome P450enzyme system in microorganism was widely used in bio-asymmetric synthesis epoxidation for a long time, so the classical inducers of cytochrome P450enzymes phenobarbital, n-tetradecane and the inhibitors piperonyl butoxide,1H-benzotriazol-1-amine were chosen and studied. It was identified that the cytochrome P450enzyme system participated the biotransformation of curcumol and curdione by Cunninghamella elegans AS3.2028.According the standard and plans of Clinical and Laboratory Standards Institute (CLSI), the antibacterial and antifungal activity of curcumol, curdione and their epoxidation biotransformation products in vitro was studied for the first time. It was demonstrated from the results that both curcumol and curdione have well antimicrobial activity to molds in the test, and also have good antimicrobial activity to the yeast like fungi Cryptococcus neoformans and Rhodotorμla, and have no antimicrobial activity to the typical Gram-positive bacteria Staphyloccocus aureusm, Bacillus subtillis, Bacillus subtillis subsp.subtillis, Sarcina lutea and Gram-negative bacteria Escherichia coli and Enterobacter aerogenes.Second,the test of influence factors of the Sal B powder shows that Sal B was reduced in the condition of60℃, while no remarkable decrease was observed at40℃. Under high temperature and high humidity conditions, Sal B powder absorbed a substantial amount of water, but no marked change was observed under the conditions of strong light. Sal B as a potential drμg material shoμld be stored at normal temperatures, and exposure to moisture shoμld be avoided. In the solid state, Sal B packaged in aluminum foil bags was stable for6months under "accelerated conditions", and Sal B powder was relatively stable during the accelerated testing. Accelerated testing resμlt of Sal B in NS solution indicates a first-order mechanism of decomposition. T90for Sal B was reached after9days’storage at room temperature, indicates that potential drμg material Sal B coμld be used in a solid formμlation, but it is not suitable for using as a liquid formμlation.The degradation products of Sal B were identified throμgh HPLC analysis and ESI-TOFMS in negative mode. Twelve components were identified in the degraded sample of Sal B. Among them, nine components were identified as degradation products of Sal B. Rosmarinic acid and isosalvianolic acid B were determined to be impurities. HPLC was used to measure the changes with time of nine major degradation products content when they were treated in thermocompression condition, and according to the results, the possible pathway of degradation of Sal B was deduced. The pathway is feasible on the basis that Sal B has two ester bonds and one benzofuran, which are susceptible to decomposition. Sal B obtains a proton to generate Sal E, while the concurrent loss of DSU and carbon dioxide resμlts in the formation of Sal A, which then undergoes ester hydrolysis to Sal F. Sal B can also be hydrolyzed to release DSU to form Lit, which is converted into Sal D and Prolit after the loss of PRO or DSU. It is possible that Cafa is generated from Prolit via the ring-opening of benzofuran and the loss of one Cafa. The resμlts of stability testing of Sal B in solution provide more evidence towards elucidating the degradation pathway of Sal B.