Sequential Separation Of Fermentative Product Of An Endophytic Fungus Isolated From Wheat And Effects Of Each Sequential Separation Product On Rabbit Chondrocytes

Kaschin-beck is a endemic Disease which does great harm to human body of steochondral.The etiology and mechanism of Kaschin-beck are still hidden. This research intends to testify the etiological hypothesis that Kaschin-Beck Disease may caused by entophytic fungus taken from the wheat in Qinghai province,and provide evidence for the etiology of KBD.This study research on petroleum ether extracts of fermentation products with strain 5-5-1 that was isolated from the wheat in the Kaschin-Beck Disease district of Qinghai province. To separate and identify fermentation products of petroleum ether partial step by step by silica gel column chromatography, TLC, HPLC, GC/MSmethod. In order to step by step screen out petroleum ether which extracts could damage chodrocytes, the survival rate of the chodrocytes was examined by methylthiazolyldiphenyl-tetrazoliumbromide (MTT), although the expressions of collagen ? and aggrecan were detected by toluidine blue (TB) and immunological histological chemistry (IHC). RT-PCR detected the mRNA expressions of collagen ? and aggrecan.Petroleum ether crude separated A, B, C, D, E, F, G, H eight groups samples by silica gel column chromatography and thin layer chromatography (TLC). Results show that E, F, G samples could inhibit the survival rate of chondrocytes, synthesis of collagen II and aggrecan and its mRNA expression, and have been concentration and time effect. F sample was the strongest damage to rabbit chondrocytes.Thus F sample was separated F1, F2, F3 three portions by HPLC, result shows that F1, F2 could inhibit the survival rate of chondrocytes, synthesis of collagen ?and aggrecan and its mRNA expression. Fl may exist in the fusarium toxin of diacetoxyscirpenol by GC/MS detection analysis. DAS standard substance compared with the sample of F1 by HPLC, result display that F1 certain include DAS, and different concertration DAS could inhibit the survival rate of chondrocytes,synthesis of collagen ? and aggrecan and its mRNA expression, and have been concentration and time effect. The sample of F2 included complicated materials that were detected by GC/Ms and needed further separation and identification. There was no influence on chondrocyte sample of F3.It was DAS and other substances that could damage chondrocytes in petroleum ether partial of strain 5-5-1 fermentation products. What's more we found there were various substances that could injure chondrocytes in entophytic fungus from the Kaschin-Beck Disease district and provided new theoretical basis to Kaschin-beck Disease etiology and pathogenesis research.