Jin-Woo Min

Enzymatic biotransformation of ginsenoside Rb1 to compound K by recombinant β-glucosidase from Microbacterium esteraromaticum.

We cloned and characterized a β-glucosidase (bgp3) gene from Microbacterium esteraromaticum isolated from ginseng field. The bgp3 gene consists of 2,271 bp encoding 756 amino acids which have homology to the glycosyl hydrolase family 3 protein domain. The molecular mass of purified Bgp3 was 80 kDa, as determined by SDS-PAGE. The enzyme (Bgp3) catalyzed the conversion of ginsenoside Rb1 to the more pharmacologically active minor ginsenoside Rd and compound K. The Bgp3 hydrolyzed the outer glucose moiety attached to the C-20 position of ginsenoside Rb1, followed by hydrolysis of the inner glucose moiety attached to the C-3 position. Using 0.1 mg mL(-1) enzyme in 20 mM sodium phosphate buffer at 40 °C and pH 7.0, 1.0 mg mL(-1) ginsenoside Rb1 was transformed into 0.46 mg mL(-1) compound K within 60 min with a corresponding molar conversion yield of 77%. Bgp3 hydrolyzed the ginsenoside Rb1 along the following pathway: Rb1 → Rd → compound K.

Biotransformation of Ginsenoside Rb 1 to Prosapogenins, Gypenoside XVII, Ginsenoside Rd, Ginsenoside F 2 , and Compound K by Leuconostoc mesenteroides DC102

Ginsenoside Rb1is the main component in ginsenosides. It is a protopanaxadiol-type ginsenoside that has a dammarane-type triterpenoid as an aglycone. In this study, ginsenoside Rb1 was transformed into gypenoside XVII, ginsenoside Rd, ginsenoside F2 and compound K by glycosidase from Leuconostoc mesenteroides DC102. The optimum time for the conversion was about 72 h at a constant pH of 6.0 to 8.0 and the optimum temperature was about 30℃. Under optimal conditions, ginsenoside Rb1 was decomposed and converted into compound K by 72 h post-reaction (99%). The enzymatic reaction was analyzed by highperformance liquid chromatography, suggesting the transformation pathway: ginsenoside Rb1→ gypenoside XVII and ginsenoside Rd→ginsenoside F2→compound K.

Enzymatic Transformation of Ginsenoside Rb1 by Lactobacillus pentosus Strain 6105 from Kimchi

Ginsenoside (ginseng saponin), the principal component of ginseng, is responsible for the pharmacological and biological activities of ginseng. We isolated lactic acid bacteria from Kimchi using esculin agar, to produce β-glucosidase. We focused on the bio-transformation of ginsenoside. Phylogenetic analysis was performed by comparing the 16S rRNA sequences. We identified the strain as Lactobacillus (strain 6105). In order to determine the optimal conditions for enzyme activity, the crude enzyme was incubated with 1 mM ginsenoside Rb1 to catalyse the reaction. A carbon substrate, such as cellobiose, lactose, and sucrose, resulted in the highest yields of β-glucosidase activity. Biotransformations of ginsenoside Rb1 were analyzed using TLC and HPLC. Our results confirmed that the microbial enzyme of strain 6105 significantly transformed ginsenoside as follows: Rb1→gypenoside XVII, Rd→F2 into compound K. Our results indicate that this is the best possible way to obtain specific ginsenosides using microbial enzymes from 6105 culture.

Bioconversion of ginsenoside Rb1 into compound K by Leuconostoc citreum LH1 isolated from kimchi

About 40 different types of ginsenoside (ginseng saponin), a major pharmacological component of ginseng, have been identified along with their physiological activities. Among these, compound K has been reported to prevent the development of and the metastasis of cancer by blocking the formation of tumors and suppressing the invasion of cancerous cells. In this study, ginsenoside Rb1 was converted into compound K via interaction with the enzyme secreted by β-glucosidase active bacteria, Leuconostoc citreum LH1, extracted from kimchi. The optimum time for the conversion of Rb1 to compound K was about 72 hrs at a constant pH of 6.0 and an optimum temperature of about 30oC. Under optimal conditions, ginsenoside Rb1 was decomposed and converted into compound K by 72 hrs post-reaction (99%). Both TLC and HPLC were used to analyze the enzymatic reaction. Ginsenoside Rb1 was consecutively converted to ginsenoside Rd, F2, and compound K via the hydrolyses of 20-C β-(1 → 6)-glucoside, 3-C β-(1 → 2)glucoside, and 3-C β-glucose of ginsenoside Rb1.

Pedobacter ginsengiterrae sp. nov., isolated from soil of a ginseng field

A Gram-stain-negative, oxidase- and catalase-positive bacterial strain that was motile by gliding and produced a pink pigment, designated DCY49(T), was isolated from soil of a ginseng field in a mountainous region of Chungbuk province, South Korea. 16S rRNA gene sequence analysis revealed that strain DCY49(T) belonged to the genus Pedobacter (93.0-96.3 % similarity). Strain DCY49(T) contained MK-7 as the predominant menaquinone. The major fatty acids were summed feature 3 (containing C16 : 1ω7c, C16 : 1ω6c and/or iso-C15 : 0 2-OH), iso-C15 : 0, iso-C17 : 0 3-OH and C16 : 0, and the main polar lipid was phosphatidylethanolamine. The G+C content of the genomic DNA of strain DCY49(T) was 40.5 mol%. Strain DCY49(T) differed from related Pedobacter species by a number of phenotypic characteristics. On the basis of data from the present polyphasic study, strain DCY49(T) is described as representing a novel species of the genus Pedobacter, for which the name Pedobacter ginsengiterrae sp. nov. is proposed. The type strain is DCY49(T) ( = KCTC 23317(T) = JCM 17338(T)).

Effects of Red Ginseng Extract on the Epididymal Sperm Motility of Mice Exposed to Ethanol

The protective effects of red ginseng extract and ginseng wine against ethanol-induced male reproductive toxicity were evaluated in male mice using computer-assisted sperm analysis. Mice were divided into 4 groups of 10 and fed plain saline, 6 g/kg per d of ethanol in saline, red ginseng extract plus ethanol, or a fermented preparation of red ginseng extract daily for 5 weeks. We found that the average seminal vesicle weight was significantly lower in the ethanol-treated group compared to the control group, while those of the ginseng-treated groups tended to be higher than the ethanol-treated group. We found a significant decrease in sperm motility and progressiveness in mice treated with ethanol for 5 weeks, while administration of ethanol plus red ginseng extract appeared to minimize the negative effects of ethanol toxicity on male fertility. Serum testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were insignificantly lower in the ethanol-treated group than in the control group.

Use of Lactobacillus rossiae DC05 for bioconversion of the major ginsenosides Rb1 and Re into the pharmacologically active ginsenosides C-K and Rg2

Rb1 and Re are the major ginsenosides in protopanaxadiol and protopanaxatriol with contents of 38.89 and 13.34%, respectively. β-Glucosidase-producing food grade Lactobacillus rossiae DC05 was isolated from kimchi using esculin-MRS agar and an enzyme of L. rossiae DC05 was used for bioconversion of the major ginsenosides Rb1 and Re. Strain DC05 showed strong activity in converting ginsenosides Rb1 and Re into the minor ginsenosides compound-K and Rg2, respectively. Within 4 days, 100% of ginsenoside Rb1 was decomposed and converted into C-K, while 85% of Re was decomposed and converted into Rg2 after 6 days of incubation. The biosynthesis rate of ginsenoside C-K was 72.88%, and the biosynthesis rate of Rg2 was 53.94%. Strain DC05 hydrolyzed ginsenosides Rb1 and Re along the pathway Rb1→Rd→F2→CK and the pathway Re→Rg2, respectively. The optimum temperature and pH of the enzyme were 30°C and 7.0, respectively.

Microbacterium panaciterrae sp. nov., isolated from the rhizosphere of ginseng.

Strain DCY56(T) was isolated from a soil sample taken from a ginseng field. The strain was Gram-reaction positive, catalase-positive, oxidase-negative, aerobic and non-motile. Phylogenetic analysis, based on 16S rRNA gene sequence analysis, indicated that strain DCY56(T) belonged to the genus Microbacterium. The closest relatives were Microbacterium azadirachtae AI-S262(T), Microbacterium aerolatum V-73(T) and Microbacterium phyllosphaerae DSM 13468(T) (98.0 %, 98.0 % and 97.5 % gene sequence similarity, respectively). The G+C content of the genomic DNA of strain DCY56(T) was 68.5 mol%. The DNA-DNA relatedness values between strain DCY56(T) and the most closely related type strains were lower than 36 %. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid. The predominant fatty acids contained iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The menaquinones were MK-12 and MK-13. The diagnostic diamino acid of strain DCY56(T) was ornithine. The dominant whole-cell sugars were glucose, rhamnose and ribose. The results of the genotypic analysis, in combination with chemotaxonomic and physiological data, demonstrate that strain DCY56(T) represents a novel species within the genus Microbacterium, for which the name Microbacterium panaciterrae sp. nov. is proposed. The type strain is DCY56(T) ( = KCTC 19884(T) = JCM 17839(T)).

Amelioration of insulin resistance by Rk 1 + Rg 5 complex under endoplasmic reticulum stress conditions

Background: Diabetes mellitus is a metabolic syndrome exaggerated by stress conditions. Endoplasmic reticulum stress (ERS) impairs the insulin signaling pathway making the diabetic conditions worsen. Pharmacological agents are supplied externally to overcome this malfunction. Ginsenosides from Panax ginseng C.A Meyer possesses many pharmacological properties and are used for the treatment of diabetes. Objective: To investigate the effects of the Rk1 +Rg5 complex on the amelioration of insulin resistance in 3T3-L1 cells under endoplasmic reticulum stress conditions. Materials and Methods: Heat-processed ginseng extracts are found to contain many pharmacologically active ginsenosides. Among them Rk1 +Rg5 is found to be present in higher concentrations than the other minor ginsenosides. The Rk1 +Rg5 complex was tested for its effect in the 3T3-L1 insulin-resistant model and subjected to the MTT assay, glucose oxidase assay and gene expression studies using RT-PCR and real-time PCR under endoplasmic reticulum stress conditions. Results: Rk1 +Rg5 treatment is found to increase the glucose uptake into the cells when compared to that of a positive control (tunicamycin treatment group, TM). Further we have analyzed the role at gene expression level. The Rk1 +Rg5 complex was found to show an effect on the IGF 2R receptor, CHOP-10, and C/EBP gene at a particular treated concentration (50 μM). Moreover, stress condition (about 50% decreases) was overcome by the ginsenoside treatments at 50 μM. Conclusion: The present results showed that under endoplasmic reticulum stress conditions Rk1 +Rg5 complex exhibits a potential protective role in insulin-resistant 3T3-L1 cells.