Ginsenoside Rh2 Ameliorates Atopic Dermatitis in NC/Nga Mice by Suppressing NF-kappaB-Mediated Thymic Stromal Lymphopoietin Expression and T Helper Type 2 Differentiation.

Ginsenosides are known to have various highly pharmacological activities, such as anti-cancer and anti-inflammatory effects. However, the search for the most effective ginsenosides against the pathogenesis of atopic dermatitis (AD) and the study of the effects of ginsenosides on specific cytokines involved in AD remain unclear. In this study, ginsenoside Rh2 was shown to exert the most effective anti-inflammatory action on thymic stromal lymphopoietin (TSLP) and interleukin 8 in tumor necrosis factor-alpha and polyinosinic: polycytidylic acid induced normal human keratinocytes by inhibiting proinflammatory cytokines at both protein and transcriptional levels. Concomitantly, Rh2 also efficiently alleviated 2,4-dinitrochlorobenzene-induced AD-like skin symptoms when applied topically, including suppression of immune cell infiltration, cytokine expression, and serum immunoglobulin E levels in NC/Nga mice. In line with the in vitro results, Rh2 inhibited TSLP levels in AD mice via regulation of an underlying mechanism involving the nuclear factor κB pathways. In addition, in regard to immune cells, we showed that Rh2 suppressed not only the expression of TSLP but the differentiation of naïve CD4+ T-cells into T helper type 2 cells and their effector function in vitro. Collectively, our results indicated that Rh2 might be considered as a good therapeutic candidate for the alternative treatment of AD.

Gypenoside LXXV Promotes Cutaneous Wound Healing In Vivo by Enhancing Connective Tissue Growth Factor Levels Via the Glucocorticoid Receptor Pathway.

Cutaneous wound healing is a well-orchestrated event in which many types of cells and growth factors are involved in restoring the barrier function of skin. In order to identify whether ginsenosides, the main active components of Panax ginseng, promote wound healing, the proliferation and migration activities of 15 different ginsenosides were tested by MTT assay and scratched wound closure assay. Among ginsenosides, gypenoside LXXV (G75) showed the most potent wound healing effects. Thus, this study aimed to investigate the effects of G75 on wound healing in vivo and characterize associated molecular changes. G75 significantly increased proliferation and migration of keratinocytes and fibroblasts, and promoted wound closure in an excision wound mouse model compared with madecassoside (MA), which has been used to treat wounds. Additionally, RNA sequencing data revealed G75-mediated significant upregulation of connective tissue growth factor (CTGF), which is known to be produced via the glucocorticoid receptor (GR) pathway. Consistently, the increase in production of CTGF was confirmed by western blot and ELISA. In addition, GR-competitive binding assay and GR translocation assay results demonstrated that G75 can be bound to GR and translocated into the nucleus. These results demonstrated that G75 is a newly identified effective component in wound healing.

Enhanced Production of Gypenoside LXXV Using a Novel Ginsenoside-Transforming ß-Glucosidase from Ginseng-Cultivating Soil Bacteria and Its Anti-Cancer Property.

Minor ginsenosides, such as compound K, Rg3(S), which can be produced by deglycosylation of ginsenosides Rb1, showed strong anti-cancer effects. However, the anticancer effects of gypenoside LXXV, which is one of the deglycosylated shapes of ginsenoside Rb1, is still unknown due to the rarity of its content in plants. Here, we cloned and characterized a novel ginsenoside-transforming ß-glucosidase (BglG167b) derived from Microbacterium sp. Gsoil 167 which can efficiently hydrolyze gypenoside XVII into gypenoside LXXV, and applied it to the production of gypenoside LXXV at the gram-scale with high specificity. In addition, the anti-cancer activity of gypenoside LXXV was investigated against three cancer cell lines (HeLa, B16, and MDA-MB231) in vitro. Gypenoside LXXV significantly reduced cell viability, displaying an enhanced anti-cancer effect compared to gypenoside XVII and Rb1. Taken together, this enzymatic method would be useful in the preparation of gypenoside LXXV for the functional food and pharmaceutical industries.

Comparative analysis of the expression level of recombinant ginsenoside-transforming ß-glucosidase in GRAS hosts and mass production of the ginsenoside Rh2-Mix.

The ginsenoside Rh2, a pharmaceutically active component of ginseng, is known to have anticancer and antitumor effects. However, white ginseng and red ginseng have extremely low concentrations of Rh2 or Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3]. To enhance the production of food-grade ginsenoside Rh2, an edible enzymatic bioconversion technique was developed adopting GRAS host strains. A ß-glucosidase (BglPm), which has ginsenoside conversion ability, was expressed in three GRAS host strains (Corynebacterium glutamicum, Saccharomyces cerevisiae and Lactococus lactis) by using a different vector system. Enzyme activity in these three GRAS hosts were 75.4%, 11.5%, and 9.3%, respectively, compared to that in the E. coli pGEX 4T-1 expression system. The highly expressed BglPm_C in C. glutamicum can effectively transform the ginsenoside Rg3-Mix [20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5] to Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, Rh3] using a scaled-up biotransformation reaction, which was performed in a 10-L jar fermenter at pH 6.5/7.0 and 37°C for 24 h. To our knowledge, this is the first report in which 50 g of PPD-Mix (Rb1, Rb2, Rb3, Rc, and Rd) as a starting substrate was converted to ginsenoside Rg3-Mix by acid heat treatment and then 24.5-g Rh2-Mix was obtained by enzymatic transformation of Rg3-Mix through by BglPm_C. Utilization of this enzymatic method adopting a GRAS host could be usefully exploited in the preparation of ginsenoside Rh2-Mix in cosmetics, functional food, and pharmaceutical industries, thereby replacing the E. coli expression system.

Mass production of the ginsenoside Rg3(S) through the combinative use of two glycoside hydrolases.

The ginsenoside Rg3(S), which is one of the exceptional components of Korean red ginseng extract, has been known to have anti-cancer, anti-metastatic, and anti-obesity effects. An enzymatic bioconversion method was developed to obtain the ginsenoside Rg3(S) with a high specificity, yield, and purity. Two glycoside hydrolases (BglBX10 and Abf22-3) were employed to produce Rg3(S) as a 100g unit. The conversion reaction transformed ginsenoside Rc to Rd using Abf22-3, followed by Rb1 and Rd to Rg3(S), using BglBX10. It was performed in a 10L jar fermenter at pH 6.0 and 37°C for 24h, with a high concentration of 50mg/ml of purified ginsenoside mixture obtained from ginseng roots. Finally, 144g of Rg3(S) was produced from 250g of root extract with 78±1.2% chromatographic purity. These results suggest that this enzymatic method would be useful in the preparation of ginsenoside Rg3(S) for the functional food and pharmaceutical industries.

Tumebacillus ginsengisoli sp. nov., isolated from soil of a ginseng field.

A gram-reaction-positive, rod-shaped, spore-forming bacterium, designated Gsoil 1105(T), was isolated from soil of a ginseng field in Pocheon Province in South Korea and characterized in order to determine its taxonomic position. Comparative analysis of the 16S rRNA gene sequence showed that the isolate belongs to the order Bacillales, showing the highest level of sequence similarity with respect to Tumebacillus permanentifrigoris Eur1 9.5(T) (94.6 %). The phylogenetic distances from other described species with validly published names within the order Bacillales were greater than 9.0 %. Strain Gsoil 1105(T) had a genomic DNA G+C content of 55.6 mol% and menaquinone 7 (MK-7) as the major respiratory quinone. The major fatty acids were iso-C(15 : 0) and anteiso-C(15 : 0). On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 1105(T) represents a novel species of the genus Tumebacillus, for which the name Tumebacillus ginsengisoli sp. nov. is proposed. The type strain is Gsoil 1105(T) ( = KCTC 13942(T)  = DSM 18389(T)).

Identification and characterization of a novel Terrabacter ginsenosidimutans sp. nov. beta-glucosidase that transforms ginsenoside Rb1 into the rare gypenosides XVII and LXXV.

A new beta-glucosidase from a novel strain of Terrabacter ginsenosidimutans (Gsoil 3082(T)) obtained from the soil of a ginseng farm was characterized, and the gene, bgpA (1,947 bp), was cloned in Escherichia coli. The enzyme catalyzed the conversion of ginsenoside Rb1 {3-O-[beta-D-glucopyranosyl-(1-2)-beta-D-glucopyranosyl]-20-O-[beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol} to the more pharmacologically active rare ginsenosides gypenoside XVII {3-O-beta-D-glucopyranosyl-20-O-[beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, gypenoside LXXV {20-O-[beta-v-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K [20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol]. A BLAST search of the bgpA sequence revealed significant homology to family 3 glycoside hydrolases. Expressed in E. coli, beta-glucosidase had apparent K(m) values of 4.2 +/- 0.8 and 0.14 +/- 0.05 mM and V(max) values of 100.6 +/- 17.1 and 329 +/- 31 micromol x min(-1) x mg of protein(-1) against p-nitrophenyl-beta-D-glucopyranoside and Rb1, respectively. The enzyme catalyzed the hydrolysis of the two glucose moieties attached to the C-3 position of ginsenoside Rb1, and the outer glucose attached to the C-20 position at pH 7.0 and 37 degrees C. These cleavages occurred in a defined order, with the outer glucose of C-3 cleaved first, followed by the inner glucose of C-3, and finally the outer glucose of C-20. These results indicated that BgpA selectively and sequentially converts ginsenoside Rb1 to the rare ginsenosides gypenoside XVII, gypenoside LXXV, and then C-K. Herein is the first report of the cloning and characterization of a novel ginsenoside-transforming beta-glucosidase of the glycoside hydrolase family 3.