Biotransformation of major ginsenosides to rare ginsenosides, C-K, C-Mc, and C-Y by honey mushroom mycelia

Abstract

Ginseng, root of Panax ginsengC. A. Meyer, has been used as a traditional folk medicine in East Asia such as Korea, Japan, and China for thousands of years, and has been to some extent popularized in many western countries during recent decades.Ginseng products are usually used as tonic and help the body to resist the adverse effectsof a wide range of physical, chemical and biological factors and to restore homeostasis. Ginseng saponins, namely ginsenosides are the pharmacologically active principlein ginseng.Most ginsenosides are composed of a dammarane skeleton (17 carbons in a four-ring structure) with various sugar moieties (e.g. glucose, rhamnose, xylose,and arabinose) attached to the C-3, C-6,and C-20 positionsof aglycones.The ginsenosides are classified into three major groups, protopanxadiol (PPD)-type, protopanaxatriol (PPT)-type,andoleanane-typesaponin.Ginsenoside (G)-Rb1, G-Rb2 and G-Rcare PPD-typemajor ginsenosides and find 80-90% of total ginsenosides in the root of Panax ginseng. In contracts ginsenosides such as G-Rg3, G-F2, G-Rh2,compound (C)-Mc1, C-Mc, C-O, C-Y,and C-K are minor ginsenosides, either do not exist or scarcely exist in fresh ginseng root. The naturally occurring major ginsenosides G-Rb1,G-Rb2, and G-Rc are poorly absorbed by the human intestinal tract because of their large molecular size, low solubility, and poor permeability across the cell membrane.However,minor ginsenosidesor human intestinal bacterial metabolites such as G-Rg3, G-F2, G-Rh2, C-Mc1, C-Mc, C-O, C-Y, and C-K are hydrolyzing products of major ginsenosides and pharmacologically active constituents than major ginsenosidessuch as G-Rb1, G-Rb2, and G-Rc. Moreover, minor ginsenosides are more readily absorbed into the blood stream and function as activecompounds.Therefore, numerous studies have been carried out to produce minor ginsenosides from major ginsenosides by chemical methods, microbial fermentation, and enzymatic hydrolysis. This studydeals with the formation of minor ginsenosides, especially C-K,from PPD-type ginsenosides using hydrolytic activitiesof the enzyme preparationsisolatedfrom cultured mushroom mycelia.We assayedthe hydrolytic activity of PPD-type major ginsenosides by using enzyme preparation isolated from the cultured mycelia of edible or medicinal mushroom namely; A. mellea, G. lucidum, Ph.linteus, El.applanata, and Pl.ostreatusandfoundthat A.mellea has highhydrolytic β-glucosidase activity to convert major ginsenosides such as G-Rb1, G-Rb2,and G-Rc into minor ginsenosides, especially C-K with high conversion ratio, while other mushroom myceliasuch asG. lucidum, Ph. linteus, El. applanata,and Pl. ostreatus produced remarkable amounts of G-Rd,respectively. Therefore, we selected A. melleamycelia for biotransformationof major ginsenosides.A. melleais also known as honey mushroom. The fruiting body and mycelia of this mushroom have been used in Chinese folk medicine as a health-promoting food in various forms and for dietary supplementation. Part I of the third chapter highlights enzymatic hydrolysis of G-Rb1 to minor ginsenoside,C-Kby enzyme preparation from the cultured mycelia of honey mushroom (HMEP). HMEPshowed a high hydrolyticactivity toward G-Rb1 with G-Rb1→G-Rd → G-F2 → C-Kas enzymatic transformationpathway. The optimum reaction conditions for C-Kformation from G-Rb1 were:reaction time 72-96 h, pH 4.0-4.5, and temperature 45-55°C. Part II of the third chapter deals with the formation of pharmacologicallyactive minor ginsenosides C-Mc and C-K from G-Rcby using HMEP. The optimum reaction conditions for C-Mcand C-K formation from G-Rcwere: reaction time 72-96 h, pH 4.0-4.5, and temperature 45-55°C. HMEPhydrolyzed the G-Rc along the following pathways: Rc→Rd→ F2→ C-K, and Rc → C-Mc1 → C-Mc, respectively In addition, the part III of the third chapter deals with the hydrolysis of major G-Rb2 to rare ginsenosides, C-Y and C-K by using HMEP. The optimum conditions for the transformation of G-Rb2 by HMEP showed its maximum potential activities at reaction time 72-96 h, pH 4.0-4.5, and temperature 45-55°C and the transformation pathways were G-Rb2 → Rd → C-K, and G-Rb2 → C-O → C-Y, respectively. These results suggest that HMEP can be used to produce pharmacologically active rare ginsenosides from major ginsenosides without food safety concern. To the best of our knowledge, these are the first reports on the formation of rare ginsenoside, C-K, C-Mc and C-K from PPD-type major saponins, G-Rb1, G-Rc, and G-Rb2 by using enzymes isolated from of mushroom mycelia.

Keywords: Honey mushroom, ginseng, mycelia, PPD-type ginsenosides, biotransformation, rare ginsenosides. glucose, rhamnose, xylose,and arabinose) attached to the C-3, C-6,and C-20 positionsof aglycones.The ginsenosides are classified into three major groups, protopanxadiol (PPD)-type, protopanaxatriol (PPT)-type,andoleanane-typesaponin.Ginsenoside (G)-Rb1, G-Rb2 and G-Rcare PPD-typemajor ginsenosides and find 80-90% of total ginsenosides in the root of Panax ginseng. In contracts ginsenosides such as G-Rg3, G-F2, G-Rh2,compound (C)-Mc1, C-Mc, C-O, C-Y,and C-K are minor ginsenosides, either do not exist or scarcely exist in fresh ginseng root. The naturally occurring major ginsenosides G-Rb1,G-Rb2, and G-Rc are poorly absorbed by the human intestinal tract because of their large molecular size, low solubility, and poor permeability across the cell membrane.However,minor ginsenosidesor human intestinal bacterial metabolites such as G-Rg3, G-F2, G-Rh2, C-Mc1, C-Mc, C-O, C-Y, and C-K are hydrolyzing products of major ginsenosides and pharmacologically active constituents than major ginsenosidessuch as G-Rb1, G-Rb2, and G-Rc. Moreover, minor ginsenosides are more readily absorbed into the blood stream and function as activecompounds.Therefore, numerous studies have been carried out to produce minor ginsenosides from major ginsenosides by chemical methods, microbial fermentation, and enzymatic hydrolysis. This studydeals with the formation of minor ginsenosides, especially C-K,from PPD-type ginsenosides using hydrolytic activitiesof the enzyme preparationsisolatedfrom cultured mushroom mycelia.We assayedthe hydrolytic activity of PPD-type major ginsenosides by using enzyme preparation isolated from the cultured mycelia of edible or medicinal mushroom namely; A. mellea, G. lucidum, Ph.linteus, El.applanata, and Pl.ostreatusandfoundthat A.mellea has highhydrolytic β-glucosidase activity to convert major ginsenosides such as G-Rb1, G-Rb2,and G-Rc into minor ginsenosides, especially C-K with high conversion ratio, while other mushroom myceliasuch asG. lucidum, Ph. linteus, El. applanata,and Pl. ostreatus produced remarkable amounts of G-Rd,respectively. Therefore, we selected A. melleamycelia for biotransformationof major ginsenosides.A. melleais also known as honey mushroom. The fruiting body and mycelia of this mushroom have been used in Chinese folk medicine as a health-promoting food in various forms and for dietary supplementation. Part I of the third chapter highlights enzymatic hydrolysis of G-Rb1 to minor ginsenoside,C-Kby enzyme preparation from the cultured mycelia of honey mushroom (HMEP). HMEPshowed a high hydrolyticactivity toward G-Rb1 with G-Rb1→G-Rd → G-F2 → C-Kas enzymatic transformationpathway. The optimum reaction conditions for C-Kformation from G-Rb1 were:reaction time 72-96 h, pH 4.0-4.5, and temperature 45-55°C. Part II of the third chapter deals with the formation of pharmacologicallyactive minor ginsenosides C-Mc and C-K from G-Rcby using HMEP. The optimum reaction conditions for C-Mcand C-K formation from G-Rcwere: reaction time 72-96 h, pH 4.0-4.5, and temperature 45-55°C. HMEPhydrolyzed the G-Rc along the following pathways: Rc→Rd→ F2→ C-K, and Rc → C-Mc1 → C-Mc, respectively In addition, the part III of the third chapter deals with the hydrolysis of major G-Rb2 to rare ginsenosides, C-Y and C-K by using HMEP. The optimum conditions for the transformation of G-Rb2 by HMEP showed its maximum potential activities at reaction time 72-96 h, pH 4.0-4.5, and temperature 45-55°C and the transformation pathways were G-Rb2 → Rd → C-K, and G-Rb2 → C-O → C-Y, respectively. These results suggest that HMEP can be used to produce pharmacologically active rare ginsenosides from major ginsenosides without food safety concern. To the best of our knowledge, these are the first reports on the formation of rare ginsenoside, C-K, C-Mc and C-K from PPD-type major saponins, G-Rb1, G-Rc, and G-Rb2 by using enzymes isolated from of mushroom mycelia.