Biotransformation Of Ginsenosides From Stems And Leaves By Plant Pathogenic Fungi

Ginseng is a traditional Chinese herbal medicine, with a variety of pharmacological effects. Many studies indicated that ginseng contains ginsenosides, carbohydrates, proteins, peptides, amino acids, vitamins and other substances. Among them ginsenosides are the main active ingredients of ginseng which accounts for about 3%-4% in the dry roots, while 5%-7% in stems and leaves. At present, More than 40 ginsenosides have been isolated. Their contents in ginseng are different. Some minor ginsenosides (e.g. Rd, Rg3, CK, Rh2), whose amount in ginseng is much lower than the major ones, possess unique pharmacological activity. It is impossible to prepare the minor ginsenosides by extraction from ginseng directly while a possible pathway for preparation of the minor ginsenosides is biotransformation from structurally related major ginsenosides. Ginsenosides from stems and leaves were higher than that from ginseng roots, therefore, biotransformation of ginsenosides from stems and leaves by pathogenic fungi was studied in order to fully use ginseng resources.The main work and the results are as follows:(1) The isolation and purification of ginsenosides from stems and leaves:①28.55 g ginsenosides was obtained from 500.00 g dried ginseng stems and leaves by macroporous resin, and the yield was 5.71%.②Silica gel column chromatography method has been applied to separate 10.00 g ginsenosides from stems and leaves into fraction-Ⅰ(3.22 g, 32.2% of total), fraction-ⅠI (4.02 g, 40.2% of total), fraction-Ⅲ(1.53 g, 15.3% of total) and fraction-Ⅳ(0.28 g, 2.8% of total).③These fractions were detected by TLC and HPLC, the results indicated that fraction-Ⅰmainly contained protopanaxatriol saponins Rg1 and Rh1, etc; Fraction-ⅠI which was a mixture of protopanaxatriol saponins and protopanaxadiol saponins mainly contained Rd and Re; fraction-Ⅲmainly contained ginsenosides Rb2, Rc and a little Rb1; fraction-Ⅳmainly contained ginsenosides Rb1.(2) Pathogenic fungi which can transform ginsenosides were screened:①Strain 3.26 which can convert ginsenosides fraction-Ⅲto Rd, F2 and CK, was selected from ten pathogenic fungi. And its spores also can convert Rb1, Rb2 and Rc to Rd, F2 and CK.②Morphological and molecular biological techniques were used to identify strain 3.26 as Epicoccum LK.ex Wallr. by comparing the ITS sequences of strain 3.26 with 18S rRNA of Epicoccum sp. SP3, and the similarity is of 99%.(3) The enzyme secreted by strain 3.26 was isolated and purified by DEAE-cellulose column chromatography, yielding a parcially purified fraction E-Ⅰ. Fraction E-Ⅰhas a wide range of pH and temperature stability, whose optimum pH is 5.0 and temperature is 40℃. Fraction E-Ⅰwas used to convert ginsenosides fraction-Ⅰ, fraction-Ⅲand fraction-Ⅳ, the results indicated that the substrate protopanaxatriol saponins could not be transformed, while protopanaxadiol saponins Rb1, Rb2 and Rc could be converted to ginsenoside Rd quickly, and completely to minor ginsenoside F2 and CK if the reaction time were extended. In summary,E-Ⅰcan not only convert Rb1, Rb2 and Rc whose content is high in ginseng stems and leaves to Rd, F2 and CK, but also have a high conversion rate. If the conversion reaction time was controlled, Rd, F2 and CK could be selectively prepared.