Ginsenoside F2-Mediated Intestinal Microbiota and Its Metabolite Propionic Acid Positively Impact the Gut-Skin Axis in Atopic Dermatitis Mice.

Atopic dermatitis (AD) is a complex inflammatory skin disease induced by multiple factors. AD can also cause intestinal inflammation and disorders of the gut microbiota. Ginseng is a kind of edible and medicinal plant; its main active components are ginsenosides. Ginsenosides have a variety of anti-inflammatory effects and regulate the gut microbiota; however, their role in AD and the underlying mechanisms are unclear. In this study, we found that intragastric administration of ginsenoside F2 improved AD-like skin symptoms and reduced inflammatory cell infiltration, serum immunoglobulin E levels, and mRNA expression of inflammatory cytokines in AD mice. 16s rRNA sequencing analysis showed that ginsenoside F2 altered the intestinal microbiota structure and enriched the short-chain fatty acid-producing microbiota in AD mice. Metabolomic analysis revealed that ginsenoside F2 significantly increased the propionic acid (Pa) content of feces and serum in AD mice, which was positively correlated with significant enrichment of Parabacteroides goldsteinii and Lactobacillus plantarum in the intestines. Pa inhibits inflammatory responses in the gut and skin of AD mice through the G-protein-coupled receptor43/NF-κB pathway, thereby improving skin AD symptoms. These results revealed, for the first time, the mechanism by which ginsenoside F2 improves AD through the Pa (a metabolite of intestinal microbiota)-gut-skin axis.

Brown adipose tissue activation with ginsenoside compound K ameliorates polycystic ovary syndrome.

BACKGROUND AND PURPOSE: Polycystic ovary syndrome (PCOS) is a common metabolic and endocrine disease affecting women of reproductive age. Due to its complex aetiology, there is no currently effective cure for PCOS. Brown adipose tissue (BAT) activity is significantly decreased in PCOS patients, and BAT activation has beneficial effects in animal models of PCOS. Here, we investigated the effect of ginsenoside compound K (CK) in an animal model of PCOS and its mechanism of BAT activation. EXPERIMENTAL APPROACH: Primary brown adipocytes, Db/Db mice and dehydroepiandrosterone (DHEA)-induced PCOS rats were used. The core body temperature, oxygen consumption, energy metabolism related gene and protein expression were assessed to identify the effect of CK on overall energy metabolism. Oestrous cycle, serum sex hormone, ovarian steroidogenic enzyme gene expression and ovarian morphology were also evaluated following CK treatment. KEY RESULTS: Our results indicated that CK treatment could significantly protect against body weight gain in Db/Db mice via BAT activation. Furthermore, we found that CK treatment could normalize hyperandrogenism, oestrous cyclicity, normalize steroidogenic enzyme expression and decrease the number of cystic follicles in PCOS rats. Interestingly, as a potential endocrine intermediate, C-X-C motif chemokine ligand-14 protein (CXCL14) was significantly up-regulated following CK administration. In addition, exogenous CXC14 supplementation was found to reverse DHEA-induced PCOS in a phenotypically similar manner to CK treatment. CONCLUSION AND IMPLICATIONS: In summary, CK treatment significantly activates BAT, increases CXCL14 expression and ameliorates PCOS. These findings suggest that CK might be a potential drug candidate for PCOS treatment.

Ginsenoside metabolite 20(S)-protopanaxatriol from Panax ginseng attenuates inflammation-mediated NLRP3 inflammasome activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C.A. Meyer (Araliaceae), has been used in traditional medicine for preventive and therapeutic purposes in Asian countries. One of the active ginsenoside metabolites, 20(S)-Protopanaxatriol (PPT), has been associated with diverse pharmacological effects, including anti-inflammatory properties. AIM OF THE STUDY: Although the capacity of PPT as an anti-inflammatory agent has been studied, this study aimed to explore the intrinsic mechanism of PPT in regulating inflammasome activation-mediated inflammatory responses in experimental models. MATERIALS AND METHODS: Lipopolysaccharide (LPS)-primed peritoneal macrophages in vitro was used to study the role of PPT on inflammasome activation. LPS-induced septic shock and monosodium urate (MSU)-induced murine peritonitis models were employed for in vivo evaluations. RESULTS: PPT attenuated NLRP3 inflammasome activation and also reduced ASC oligomerization, leading to attenuation of interleukin (IL)-1ß secretion. Further, PPT inhibited IL-1ß secretion in both LPS-induced septic shock and MSU-induced mouse peritonitis models. CONCLUSIONS: This study revealed that ginsenoside metabolite PPT, inhibits inflammation-mediated inflammasome activation and supported the traditional use of ginseng in treating various inflammatory disorders.

Myristoleic acid produced by enterococci reduces obesity through brown adipose tissue activation.

OBJECTIVE: Dietary fibre has beneficial effects on energy metabolism, and the majority of studies have focused on short-chain fatty acids produced by gut microbiota. Ginseng has been reported to aid in body weight management, however, its mechanism of action is not yet clear. In this study, we focused on the potential modulating effect of ginseng on gut microbiota, aiming to identify specific strains and their metabolites, especially long-chain fatty acids (LCFA), which mediate the anti-obesity effects of ginseng. DESIGN: Db/db mice were gavaged with ginseng extract (GE) and the effects of GE on gut microbiota were evaluated using 16S rDNA-based high throughput sequencing. To confirm the candidate fatty acids, untargeted metabolomics analyses of the serum and medium samples were performed. RESULTS: We demonstrated that GE can induce Enterococcus faecalis, which can produce an unsaturated LCFA, myristoleic acid (MA). Our results indicate that E. faecalis and its metabolite MA can reduce adiposity by brown adipose tissue (BAT) activation and beige fat formation. In addition, the gene of E. faecalis encoding Acyl-CoA thioesterases (ACOTs) exhibited the biosynthetic potential to synthesise MA, as knockdown (KD) of the ACOT gene by CRISPR-dCas9 significantly reduced MA production. Furthermore, exogenous treatment with KD E. faecalis could not reproduce the beneficial effects of wild type E. faecalis, which work by augmenting the circulating MA levels. CONCLUSIONS: Our results demonstrated that the gut microbiota-LCFA-BAT axis plays an important role in host metabolism, which may provide a strategic advantage for the next generation of anti-obesity drug development.

Anti-inflammatory action of Athyrium multidentatum extract suppresses the LPS-induced TLR4 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The aerial part of Athyrium multidentatum (Doll.) Ching (AM) is widely used in the northeastern region of China as an edible wild herb, but its medicinal value, especially its anti-inflammatory effect, has not been fully explored. AIM OF THE STUDY: To investigate the anti-inflammatory activity of AM and clarify the anti-inflammatory mechanism involving the TLR4 signaling pathway using a lipopolysaccharide (LPS)-induced inflammatory model. MATERIALS AND METHODS: AM ethanol extract was used as the experimental material to investigate the effect that the extract has on the production of pro-inflammatory mediators (NO, PGE2, TNF-α, IL-1ß and IL-6); changes in LPS-induced peritoneal macrophages (PMs); and TLR4-mediated intracellular events, including MAPKs (ERK, JNK, and p38) and IκB-α in the MyD88-dependant pathway and IRF3, STAT1, and STAT3 in the TRIF-dependent pathway. In in vivo experiments, we established an LPS-induced acute lung injury (ALI) model and investigated the cell count and cytokine (TNF-α, IL-1ß and IL-6) levels in bronchoalvelar lavage fluid (BALF) of C57BL6 mice. Histological changes in the lung tissues were observed with H&E staining. RESULTS: AM extract inhibited NO and PGE2 by suppressing their synthetase (iNOS and COX-2) gene expression in LPS-induced PMs; the secretion of IL-6, IL-1ß, and TNF-α also deceased via the down-regulation of mRNA levels. Furthermore, the TLR4-mediated intracellular events involved the phosphorylated forms of MAPKs (ERK, JNK) and IκB-α in the MyD88-dependent pathway and the TRIF-dependent pathway (IRF3, STAT1, STAT3), and the relevant proteins were expressed at low levels in the AM extract groups. In in vivo experiments, the cell count and cytokine (TNF-α, IL-1ß and IL-6) levels in BALF decreased significantly in a dose-dependent manner in the AM extract groups. The lung tissue structure exhibited dramatic damage in the LPS group, and the damaged area decreased in the AM extract groups; in particular, the effect of 10 mg/kg extract was similar to that of the positive control dexamethasone (DEX). CONCLUSION: The findings demonstrate that AM protects against LPS-induced acute lung injury by suppressing TLR4 signaling, provide scientific evidence to support further study of the safety of anti-inflammatory drugs and indicate that AM can be used as an anti-inflammatory and anti-injury agent to prevent pneumonia caused by microbial infection.

Metabolite profiling of ginsenosides in rat plasma, urine and feces by LC-MS/MS and its application to a pharmacokinetic study after oral administration of Panax ginseng extract.

Panax ginseng is widely consumed as a functional food in the form of tea, powder, capsules, among others, and possesses a range of pharmacological activities including adaptogenic, immune-modulatory, anti-tumor, anti-aging and anti-inflammatory effects. The aim of this study was to identify and quantify the major ginsenosides and their metabolites in rat plasma, urine and feces after administration of P. ginseng extract using LC-MS/MS. We collected rat plasma samples at 0.5, 1, 2, 4, 8, 12, 24 and 48 h, and the amounts of urine and fecal samples accumulated in 24 h. Fourteen major ginsenosides and their metabolites were observed in fecal samples at high levels; however, low levels of 11 ginsenosides were detected in urine samples. The pharmacokinetics of the major ginsenosides and their metabolites was investigated in plasma. The results indicated that the maximum plasma concentration, time to maximum concentration and area under the curve of compound K were significantly greater than those of other ginsenosides. This study thus provides valuable information for drug development and clinical application of P. ginseng.

Comparative Analysis of the Rats' Gut Microbiota Composition in Animals with Different Ginsenosides Metabolizing Activity.

Following oral intake of Panax ginseng, major ginsenosides are metabolized to deglycosylated ginsenosides by gut microbiota before absorption into the blood. As the composition of gut microbiota varies between individuals, metabolic activities are significantly different. We selected 6 rats with low efficiency metabolism (LEM) and 6 rats with high efficiency metabolism (HEM) from 60 rats following oral administration of Panax ginseng extract, and analyzed their gut microbiota composition using Illumina HiSeq sequencing of the 16S rRNA gene. The components of gut microbiota between the LEM and HEM groups were significantly different. Between the 2 groups, S24-7, Alcaligenaceae, and Erysipelotrichaceae occupied most OTUs of the HEM group, which was notably higher than the LEM group. Furthermore, we isolated Bifidobacterium animalis GM1 that could convert the ginsenoside Rb1 to Rd. The result implies that these specific intestinal bacteria may dominate the metabolism of Panax ginseng.

Phycicoccus ginsengisoli sp. nov., isolated from cultivated ginseng soil.

Ginseng-cultivated soil is an excellent habitat for soil-borne bacteria to proliferate. A novel strain, DCY87T, was isolated from ginseng-cultivated soil in Gochang County, Republic of Korea, and subsequently characterized by polyphasic approach. Cells were rod shaped, non-motile, aerobic, Gram-reaction-positive, oxidase-negative and catalase-positive. 16S rRNA gene sequence analysis showed that strain DCY87T shared the highest similarity to 'Phycicoccus ochangensis' L1b-b9 (98.7 %). Closely phylogenetic relatives of strain DCY87T were identified: Phycicoccus ginsenosidimutans BXN5-13T (97.9 %), Phycicoccus soli THG-a14T (97.8 %), Phycicoccus bigeumensis MSL-03T (97.3 %), Phycicoccus cremeus V2M29T (97.3 %), Phycicoccus aerophilus 5516T-20T (97.3 %), Phycicoccus dokdonensis DS-8T (97.3 %) and Phycicoccus jejuensis KSW2-15T (97.1 %). The major polar lipids were classified as phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acids were composed of iso-C15 : 0, anteiso-C15:0, C17 : 0 and C17 : 1ω8c. The menaquinone was resolved as MK-8(H4). Strain DCY87T contained meso-diaminopimelic acid as diamino acid in the cell-wall peptidoglycan and glucose, xylose and rhamnose in the whole-cell sugar. The genomic DNA G+C content was calculated to be 72.7 mol%. DNA-DNA hybridization value between strain DCY87T and 'P. ochangensis' L1b-b9 was estimated to be 50 %. However, DNA-DNA hybridization value obtained between strain DCY87T and P. ginsenosidimutans BXN5-13T, P. soli THG-a14T and P. bigeumensis MSL-03T was well below 17 %. In general, polyphasic taxonomy demonstrated that DCY87T strain represented a novel species within the genus Phycicoccus. Accordingly, we propose the name Phycicoccus ginsengisoli sp. nov. The type strain is DCY87T (=KCTC 39635T=JCM 31016T).

Biotransformation of gypenoside XVII to compound K by a recombinant ß-glucosidase.

OBJECTIVE: To study the ß-glucosidase gene (bgy1) from Lactobacillus brevis that was cloned and expressed in Escherichia coli BL21 (DE3) and then using it for the biotransformation of gypenoside XVII. RESULTS: The bgy1 gene consists of 2283 bp encoding 761 amino acids, with homology to the glycosyl hydrolase family-3 protein domain. The enzyme (Bgy1) hydrolyzed the glucose moieties at the C-3 position and the outer glucose moieties at the C-20 position of gypenoside XVII. Using 0.1 mg enzyme ml(-1) in 20 mM sodium phosphate buffer at 30 °C and pH 6.0, 1 mg gypenoside XVII ml(-1) was transformed into 0.58 mg compound K ml(-1) within 6 h, with a corresponding molar conversion yield of 89 %. CONCLUSION: The recombinant Bgy1 is considered potentially useful for the practical preparation of compound K.

Enzymatic biotransformation of ginsenoside Rb1 to 20(S)-Rg3 by recombinant ß-glucosidase from Microbacterium esteraromaticum.

Microbacterium esteraromaticum was isolated from ginseng field. The ß-glucosidase gene (bgp1) from M. esteraromaticum was cloned and expressed in Escherichia coli BL21 (DE3). The bgp1 gene consists of 2,496 bp encoding 831 amino acids which have homology to the glycosyl hydrolase family 3 protein domain. The recombinant ß-glucosidase enzyme (Bgp1) was purified and characterized. The molecular mass of purified Bgp1 was 87.5 kDa, as determined by SDS-PAGE. Using 0.1 mg ml(-1) enzyme in 20 mM sodium phosphate buffer at 37°C and pH 7.0, 1.0 mg ml(-1) ginsenoside Rb1 was transformed into 0.444 mg ml(-1) ginsenoside Rg3 within 6 h. The Bgp1 sequentially hydrolyzed the outer and inner glucose attached to the C-20 position of ginsenosides Rb1. Bgp1 hydrolyzed the ginsenoside Rb1 along the following pathway: Rb1 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 using the recombinant ß-glucosidase.

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 ¥â-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 30¨¬C. Under optimal conditions, ginsenoside Rb1 was decomposed and converted into compound K by 72 hrs post-reaction (99 percent). 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 ¥â-(1 ¡æ 6)-glucoside, 3-C ¥â-(1 ¡æ 2)glucoside, and 3-C ¥â-glucose of ginsenoside Rb1.

Stenotrophomonas ginsengisoli sp. nov., isolated from a ginseng field.

A Gram-negative, non-spore-forming, rod-shaped bacterium, designated strain DCY01(T), was isolated from soil from a ginseng field in South Korea and was characterized in order to determine its taxonomic position. 16S rRNA gene sequence analysis revealed that strain DCY01(T) belonged to the Gammaproteobacteria and was most closely related to Stenotrophomonas koreensis KCTC 12211(T) (98.4 % similarity), Stenotrophomonas humi R-32729(T) (97.2 %), Stenotrophomonas terrae R-32768 (97.1 %), Stenotrophomonas maltophilia DSM 50170(T) (96.9 %) and Stenotrophomonas nitritireducens DSM 12575(T) (96.8 %). Chemotaxonomic analyses revealed that strain DCY01(T) possessed a quinone system with Q-8 as the predominant compound, and iso-C(15 : 0) (28.2 %), C(16 : 0) 10-methyl (13.2 %), iso-C(15 : 1) F (10.8 %) and C(15 : 0) (7.5 %) as major fatty acids, corroborating assignment of strain DCY01(T) to the genus Stenotrophomonas. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The results of DNA-DNA hybridization and physiological and biochemical tests clearly demonstrated that strain DCY01(T) represents a species distinct from recognized Stenotrophomonas species. Based on these data, DCY01(T) (=KCTC 12539(T)=NBRC 101154(T)) should be classified as the type strain of a novel species of the genus Stenotrophomonas, for which the name Stenotrophomonas ginsengisoli sp. nov. is proposed.