Isolation, Purification, Identification And Bioactivities Of Liver Protective Compounds From Submerged Culture Of Acremonium Implicatum

In order to make full use of the bioactive compounds in the mycelia of Acremonium implicatum, the liver protection substances from the mycelia derived from submerged culture was studied. The main works include bioactive components extraction, purification, structure identification and bioactivity study.The optimum extraction condition was determined as follows: degreased the mycelia by petroleum ether, immerge the degreased mycelia powder in 8 times volume water, extract reelingly at 50℃ for 90 minutes, concentrate the supernatant by centrifuging, add 95% ethanol to a final concentration of 70% to remove polysaccharide, and then concentrate the supernatant by centrifuging to small volume. The concentrated substance was crude HG.HG was purified through the following procedure: crude HG → silica gel columnchromatogramphy → Sephadex LH-20 column chromatogramphy → highperformance liquid chromatogramphy →pure HG.In silica gel column chromatogramphy, the solid phase was silica gel(200-300 mesh), and the mobile phase is trichloromethane:acetic:isopropanol:water =8:4:4:0.5 (1% aqueous ammomia). The yield of crude HG dHG was 0.25%.Sephadex LH-20 column chromatogramphy was used to purify dHG. In the experiments of mobile phase selection, 100%, 80% and 40% methanol were used, the result revealed that the effect of 80% methanol and 40% methanol were close, and 80% methanol excelled 100% methanol, but they all did not result in 100% pure component. After the purification of LH-20 column chromatography, only hypo-HG was obtained.High performance liquid chromatogramphy was used with reverse phase C18 HPLC preparative column to purify hypo-HG. Two pure components HG-1 and HG-2 wereyielded, HG-1 was the main component with a proportion at about 90%, and HG-2 is the subordinative component with a proportion at about 10%.The purity tests of HG-1 and HG-2 by TLC, analytical HPLC and chemical methods proved that they were both single.The structure identification of HG-1 was performed with element analysis, MS and NMR. The element analysis of HG-1 indicated that the ratio of C, N, H was 1:1:1 in HG-1, and there was no oxygen element; Low resolution MS showed that its molecular weight was 135, and high resolution MS revealed that its accurate molecular weight was 135.0549 and a possible molecular formula was C5N5H5. 'H-NMR spectrum showed one hydrogen signal at 8 8.38 and 8 8.33 respectively, and two active hydrogen at 8 7.08. 'JC-NMR spectrum showed one carbon atom was at 8 155, 8 153, and 8 143, respectively. In combination with molecular formula and all these data, HG-1 was attributed deductively to adenine, though some of the carbon signals did not appear.The structure identification of HG-2 was only performed with HR-MS and 'H-NMR because of its insufficient amout. HR-MS showed that its accurate molecular weight was 113.0347, and a possible molecular formula was C4H4N2O2. 1H-NMR spectrum showed that there were 2 coupling hydrogens, which conformed well with the two hydrogens on the unsaturated carbon of the uracil ring. The general analysis indicated that HG-2 was possibly uracil, but its structure was to be further proved by 13C-NMR.The pharmacological experiment showed that pretreatment with crude HG, e.g. dHG, relieved significantly liver injury induced by IR in in vivo IR model, suggesting obvious protective function to liver.The experiment of in vivo IR model also showed the level of GSH-Px in rat liver rose by dHG pretreatment, while the level of LPO declined obviously. That indicated dHG restrained the production of free radical by raising the activity of GSH-Px of liver cell, and it also lightened lipide peroxide of liver cell membrane, and protected liver.The comparative pharmacological experiment between dHG and hypo-HG indicated that they both reduced liver injury induced by CCl4 after pretreatment and had significant protective function to liver.The experiment showed the raised activities of GPT and GOT in injuried mouse liver, induced by CCl4, declined, while the l...