Most herbal medicines are orally administered and their components inevitably come into contact with intestinal microflora in the alimentary tract. These components may be transformed before they are absorbed from the gastrointestinal tract. Studies on the metabolism of herbal medicine components by human intestinal microflora are therefore of great importance in understanding their biological effects. 1,2) Among herbal medicines, ginseng (the root of Panax ginseng C.A. MEYER, Araliaceae) is frequently used in Asian countries as a traditional medicine. The major components of ginseng are ginsenosides.3,4) These ginsenosides have been reported to show various biological activities including antiinflammatory activity 5) and antitumor effects. 6,7) The pharmacological actions of these ginsenosides has been explained by the biotransformation of ginsenosides by human intestinal bacteria. [8][9][10][11][12] Transformed 20-O-b-D-glucopyranosyl-20(S)-protopanaxadiol (IH-901, compound K) from ginsenosides R b1 , R b2 and R c induces an antimetastatic or anticarcinogenic effect. [13][14][15] In addition, ginsenosides R b1 , R b2 , and R c are transformed to ginsenoside R g3 by treatment with mild acid such as stomach acid.16) Furthermore, ginsenoside R g3 is a main component of Red Ginseng.17) However, studies on the metabolism of ginsenoside R g3 by human intestinal bacteria are not complete.Therefore we investigated the human intestinal bacteria capable of metabolizing ginsenoside R g3 and its related biological activities, such as in vitro cytotoxicity and anti-Helicobacter pylori (HP) activity. MATERIALS AND METHODS Materials and Bacterial StrainsSodium thioglycolate and ascorbic acid were purchased from Sigma Chemical Co. (U.S.A.). General anaerobic medium (GAM) was purchased from Nissui Pharmaceutical Co., Ltd., (Japan). Tryptic soy broth was purchased from Difco Co. (U.S.A.). p-Nitrophenyl b-D-glucopyranoside (PNG) was purchased from Sigma (U.S.A.). The other chemicals were of analytical reagent grade.Tumor cell lines were purchased from the Korean Cell Bank. HP ATCC43504 was purchased from ATCC, HP NCTC11638 was purchased from NCTC. HP82516 and HP4 clinically isolated from Korean gastroscopic samples were used. They were inoculated onto brucella agar plates supplemented with 7% horse serum and cultured for 3 days at 37°C under microaerophilic conditions (AnaeroPak Campylo: 85% N 2 , 10% CO 2 , and 5% O 2 ). Isolation of Transformants of Ginsenoside R b1 under Mild Acidic Conditions Ginsenoside R b1 (2 g) was treated in mild acidic conditions 16) at 37°C for 2 h, as previously reported, concentrated at 60°C and extracted with n-BuOH. From this BuOH fraction, 20(S)-and 20(R)-ginsenoside R g3 were isolated according to the previous method.18) The BuOH fraction was chromatographed on a silica gel column using CHCl 3 -MeOH-H 2 O (10 : 3 : 1, lower layer) to produce D 20 -ginsenoside R g3 (0.05 g), and isomeric 20(S)-and 20(R)-ginsenoside R g3 (0.6 g). The isomeric mixture was dissolved in pyridine, and acetic anhydride...
Background: Ginseng (the root of Panax ginseng C.A. Meyer, Araliaceae) has been reported to possess various biological activities, including anti-inflammatory and antitumor actions. In this study, we investigated the antiallergic activity of ginsenosides isolated from ginseng. Method: We isolated ginsenosides by silica gel column chromatograghy and examined their in vitro and in vivo antiallergic effect on rat peritoneal mast cells and on IgE-induced passive cutaneous anaphylaxis (PCA) in mice. The in vitroanti-inflammatory activity of ginsenoside Rh1 (Rh1) in RAW264.7 cells was investigated. Results: Rh1 potently inhibited histamine release from rat peritoneal mast cells and the IgE-mediated PCA reaction in mice. The inhibitory activity of Rh1 (87% inhibition at 25 mg/kg) on the PCA reaction was found to be more potent than that of disodium cromoglycate (31% inhibition at 25 mg/kg); Rh1 was also found to have a membrane-stabilizing action as revealed by differential scanning calorimetry. It also inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in RAW 264.7 cells, and the activation of the transcription factor, NF-ĸB, in nuclear fractions. Conclusion: The antiallergic action of Rh1 may originate from its cell membrane-stabilizing and anti-inflammatory activities, and can improve the inflammation caused by allergies.
Ginseng (the root of Panax ginseng C. A. MEYER, Araliaceae) is frequently used in Asian countries as a traditional medicine. The major components of ginseng are ginsenosides.1,2) These ginsenosides have been reported to show various biological activities including antiinflammatory activity 3) and antitumor effects. 4,5) The pharmacological actions of these ginsenosides have been explained by the biotransformation of ginsenosides by human intestinal bacteria. [6][7][8][9][10][11] Transformed 20-O-b-D-glucopyranosyl-20(S)-protopanaxadiol (IH-901, compound K) from ginsenosides R b1 , R b2 and R c induces an antimetastatic or anticarcinogenic effect. [12][13][14] However, studies on the metabolism of ginsenoside R c by human intestinal bacteria and its related biological activities are not complete.We therefore investigated the human intestinal bacteria capable of metabolizing ginsenoside R c and its related antiallergic activity. MATERIALS AND METHODSInstrument Melting points were determined on an electrothermal digital melting point apparatus.1 H-and 13 C-NMR spectra were recorded on a Bruker-AM 500 with tetramethylsilane (TMS) as an internal standard. The TLC chromatogram of metabolites was quantitatively analyzed with Shimadzu model CS-9301PC (Japan).Materials and Bacterial Strains Sodium thioglycolate, ascorbic acid and disodium cromoglycate (DSCG) were purchased from Sigma Co. (U.S.A.). General anaerobic medium (GAM) was purchased from Nissui Pharmaceutical Co., Ltd. (Japan). Tryptic soy broth was purchased from Difco Co. (U.S.A.). Ginsenoside R c was isolated by the previous method.15) The other chemicals were of analytical reagent grade.Isolation of Metabolites of Ginsenoside R c by Human Intestinal Microflora The reaction mixture containing 100 mg of 20(S)-ginsenoside R c and 500 mg of fresh fecal suspension (Bifidobacterium K-103 or K-506) was incubated for 24 h at 37°C. The reaction mixture was extracted with ethyl acetate, evaporated to dryness, and applied to silica gel column chromatography; solvent, CHCl 3 -MeOH-H 2 O (65 : 35 : 10 v/v, lower phase). From the reaction mixture, we isolated ginsenoside M c , compound K and 20(S)-protopanaxadiol (5 mg). Ginsenosides R d (12 mg) and F 2 (10 mg) were isolated as metabolites by Bifidobacterium K-103, and ginsenoside M b (15 mg) was isolated as a metabolite by Bifidobacterium K-506.Ten grams of fresh feces were suspended with 100 ml of anaerobic diluted media and centrifuged at 200ϫg for 5 min, and the precipitate was discarded. The supernatant was centrifuged at 10000ϫg for 30 min. The precipitate was washed twice with saline and used in the experiment.Ginsenoside Screening of Bacteria Metabolizing Ginsenoside R cThe bacteria (Bacteroides HJ-15, Bacteroides JY-6, Eubacterium A-44, Bifidobacterium K-110, and Fusobacterium K-60) previously isolated from human intestinal microflora were cultured in 50 ml of tryptic soy broth containing 0.01% sodium thioglycolate and 0.1% ascorbic acid (TSTA), and then each cultured cell was centrifuged at 3000ϫg for 10 min an...
We previously reported that the in vivo and in vitro suppression of Nuclear Factor of Activated T Cells (NFAT) signaling increases osteoblast differentiation and bone formation. To investigate the mechanism by which NFATc1 regulates osteoblast differentiation, we established an osteoblast cell line that overexpresses a constitutively active NFATc1 (ca-NFATc1). The activation of NFATc1 significantly inhibits osteoblast differentiation and function, demonstrated by inhibition of alkaline phosphatase activity and mineralization as well as a decrease in gene expression of early and late markers of osteoblast differentiation such as osterix and osteocalcin, respectively. By focusing on the specific role of NFATc1 during late differentiation, we discovered that the inhibition of osteocalcin gene expression by NFATc1 was associated with a repression of the osteocalcin promoter activity, and a decrease in TCF/LEF transactivation. Also, overexpression of NFATc1 completely blocked the decrease in total histone deacetylase (HDAC) activity during osteoblast differentiation and prevented the hyperacetylation of histones H3 and H4. Mechanistically, we show by Chromatin Immunoprecipitation (ChIP) assay that the overexpression of NFATc1 sustains the binding of HDAC3 on the proximal region of the osteocalcin promoter, resulting in complete hypoacetylation of histones H3 and H4 when compared to GFP-expressing osteoblasts. In contrast, the inhibition of NFATc1 nuclear translocation either by cyclosporin or by using primary mouse osteoblasts with deleted calcineurin b1 prevents HDAC3 from associating with the proximal regulatory site of the osteocalcin promoter. These preliminary results suggest that NFATc1 acts as a transcriptional co-repressor of osteocalcin promoter possibly in an HDAC-dependent manner.
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