Lepidopterous Sex Attractants

Fusobacterium K-60, a ginsenoside R_b1-metabolizing bacterium, was isolated from human intestinal feces. From this Fusodobacterium K-60, a ginsenoside R_b1-metabolizing enzyme, β-glucosidase, has been purified. The enzyme was purified to apparent homogeneity by a combination of butyl-Toyopearl, hydroxyapatite ultragel, Q-Sepharose, and Sephacryl S-300 HR column chromatographies with a final specific activity of 1.52 μmol/min/mg. It had optimal activity at pH 7.0 and 40°C. The molecular mass of this purified enzyme was 320 kDa, with 4 identical subunits (80 kDa). The purified enzyme activity was inhibited by Ba⁺⁺, Fe⁺⁺, and some agents that modify cysteine residues. This enzyme strongly hydrolyzed sophorose, followed by p-nitrophenyl β-D-glucopyranoside, esculin, and ginsenoside R_b1. However, this enzyme did not change 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (IH-901) to 20(S)-protopanaxadiol, while it weakly changed ginsenoside R_b1 to IH-901. These findings suggest that the Fusobacterial β-glucosidase is a novel enzyme transforming ginsenoside R_b1.     

Anti-proliferation and apoptosis induced by a novel intestinal metabolite of ginseng saponin in human hepatocellular carcinoma cells

20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH-901), a novel intestinal bacterial metabolite of ginseng protopanaxadiol saponins, is reported to induce apoptosis in a variety of cancer cells. We purified the compound and measured its in vitro anti-tumor activity. IH-901 inhibited cell growth of human hepatocellular carcinoma SMMC7721 cells in a dose- and time-dependent manner. We also found that IH-901 induced apoptotic cell death concurrent with cell cycle arrest in G0-G1 phase in SMMC7721 cells. At the molecular level, we show that IH-901 upregulates cytochrome c, p53, and Bax expression, and downregulates pro-caspase-3 and pro-caspase-9 expressions in a dose-dependent manner, while the levels of Bcl-2 and Bcl-X(L) were unchanged in IH-901-treated SMMC7721 cells. These results provide significant insight into the anticarcinogenic action of IH-901.     

Purification and properties of a novel beta-glucosidase, hydrolyzing ginsenoside Rb1 to CK, from Paecilomyces Bainier

A novel ginsenoside-hydrolyzing beta-glucosidase was purified from Paecilomyces Bainier sp. 229 by a combination of QSepharose FF, phenyl-Sepharose CL-4B, and CHT ceramic hydroxyapatite column chromatographies. The purified enzyme was a monomeric protein with a molecular mass estimated to be 115 kDa. The optimal enzyme activity was observed at pH 3.5 and 60oC. It was highly stable within pH 3-9 and at temperatures lower than 55oC. The enzyme was specific to beta-glucoside. The order of enzyme activities against different types of beta-glucosidic linkages was beta-(1- 6)>beta-(1-2)>beta-(1-4). The enzyme converted ginsenoside Rb1 to CK specifically and efficiently. An 84.3% amount of ginsenoside Rb1, with an initial concentration of 2 mM, was converted into CK in 24 h by the enzyme at 45 degrees and pH 3.5. The hydrolysis pathway of ginsenoside Rb1 by the enzyme was Rb1-->Rd-->F2-->CK. Five tryptic peptide fragments of the enzyme were identified by a newly developed de novo sequencing method of post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. By comparing the five identified peptide sequences with the NCBI database, this purified beta-glucosidase proves to be a new protein that has not been reported before.     

Identification and characterization of the Rhizobium sp. strain GIN611 glycoside oxidoreductase resulting in the deglycosylation of ginsenosides

Using enrichment culture, Rhizobium sp. strain GIN611 was isolated as having activity for deglycosylation of a ginsenoside, compound K (CK). The purified heterodimeric protein complex from Rhizobium sp. GIN611 consisted of two subunits with molecular masses of 63.5 kDa and 17.5 kDa. In the genome, the coding sequence for the small subunit was located right after the sequence for the large subunit, with one nucleotide overlapping. The large subunit showed CK oxidation activity, and the deglycosylation of compound K was performed via oxidation of ginsenoside glucose by glycoside oxidoreductase. Coexpression of the small subunit helped soluble expression of the large subunit in recombinant Escherichia coli. The purified large subunit also showed oxidation activity against other ginsenoside compounds, such as Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, F1, and the isoflavone daidzin, but at a much lower rate. When oxidized CK was extracted and incubated in phosphate buffer with or without enzyme, (S)-protopanaxadiol [PPD(S)] was detected in both cases, which suggests that deglycosylation of oxidized glucose is spontaneous.     

In vitro metabolism of ginsenosides by the ginseng root pathogen Pythium irregulare

The role of ginseng saponins (ginsenosides) as modulators or inhibitors of disease is vague, but our earlier work supports the existence of an allelopathic relationship between ginsenosides and soilborne microbes. Interestingly, this allelopathy appears to significantly promote the growth of the important ginseng pathogen, Pythium irregulare while inhibiting that of an antagonistic non-pathogenic fungus, Trichoderma hamatum. Herein we report on the apparent selective metabolism of 20(S)-protopanaxadiol ginsenosides by an extracellular glycosidase from P. irregulare. Thus, when P. irregulare was cultured in the presence of a purified (> 90%) ginsenoside mixture, nearly all of the 20(S)-protopanaxadiol ginsenosides (Rb1, Rb2, Rc, Rd, and to a limited extent G-XVII) were metabolized into the minor ginsenoside F2, at least half of which appears to be internalized by the organism. No metabolism of the 20(S)-protopanaxatriol ginsenosides (Rg1 and Re) was evident. By contrast, none of the ginsenosides added to the culture medium of the non-pathogenic fungus T. hamatum were metabolized. The metabolism of 20(S)-protopanaxadiol ginsenosides by P. irregulare appears to occur through the hydrolysis of terminal monosaccharide units from disaccharides present at C-3 and/or C-20 of ginsenosides Rb1, Rc, Rb2, Rd and G-XVII to yield one major product, ginsenoside F2 and one minor product (possibly G-III). A similar transformation of ginsenosides was observed using a crude protein preparation isolated from the spent medium of P. irregulare cultures.     

Production of ginsenoside aglycons and Rb1 deglycosylation pathway profiling by HPLC and ESI-MS/MS using Sphingobacterium multivorum GIN723

Using enrichment culture, Sphingobacterium multivorum GIN723 (KCCM80060) was isolated as having activity for deglycosylation of compound K and ginsenoside F1 to produce ginsenoside aglycons such as S-protopanaxadiol (PPD(S)) and S-protopanaxatriol (PPT(S)). Through BLAST search, purified enzyme from S. multivorum GIN723 was revealed to be the outer membrane protein. The purified enzyme from S. multivorum GIN723 has unique specificity for the glucose moiety. However, it has activity with PPD and PPT group ginsenosides such as ginsenosides Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, and F1. From these results, it was predicted that the enzyme has activity on several ginsenosides. Therefore, the biotransformation pathway from Rb1, which is a major, highly glycosylated compound of ginseng, was analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry/mass spectrometry. The dominant biotransformation pathway from Rb1 to PPD(S) was determined to be Rb1 → Gp-XVII → Gp-LXXV → CK → PPD(S). S. multivorum GIN723 can be used as a whole cell biocatalyst because its activity as whole cells is nine times higher than its activity as cell extracts. The specific activity of whole cells is 2.89 nmol/mg/min in the production of PPD(S). On the other hand, the specific activity of cell extracts is 0.32 nmol/mg/min. The productivity of this enzyme in whole cell form is 500 mg/1 l of cultured cell. Its optimum reaction condition is 10 mM of calcium ions added to a phosphate buffer with a pH of 8.5.     

Purification method improvement and characterization of a novel ginsenoside-hydrolyzing beta-glucosidase from Paecilomyces Bainier sp. 229

The purification method for a novel ginsenoside-hydrolyzing beta-glucosidase from Paecilomyces Bainier sp. 229 was successfully simplified by the application of microcrystalline cellulose (MCC) as a novel chromatographic matrix. Only two chromatographic steps, Q-Sepharose FF and MCC column in sequence, were required to purify the enzyme to apparent homogeneity. The purified enzyme, with a native molecular weight estimated to be 305 KDa, was composed of three identical subunits of approximately 102 KDa each. The optimal enzyme activity was observed at pH 3.5 at 55 degrees C. It was stable within pH 3-7 and at temperatures lower than 50 degrees C. The optimal substrate for the enzyme was p-nitrophenyl-beta-D-glucoside, followed by ginsenoside Rd, gentiobiose, and ginsenoside Rb1. It converted ginsenoside Rb1 to ginsenoside Rg3 specifically and efficiently. The hydrolyzing pathway of ginsenoside Rb1 by the enzyme was Rb1-->Rd-->Rg3. The specific activities against ginsenoside Rb1 and Rd were 56.7 micromol/min/mg and 129.4 micromol/min/mg respectively.     

Purification and characterization of an extracellular beta-glucosidase produced by Phoma sp. KCTC11825BP isolated from rotten mandarin peel

A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.     

Purification and characterization of alpha-L-arabinopyranosidase and alpha-L-arabinofuranosidase from Bifidobacterium breve K-110, a human intestinal anaerobic bacterium metabolizing ginsenoside Rb2 and Rc

Two arabinosidases, alpha-L-arabinopyranosidase (no EC number) and alpha-L-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. alpha-L-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 micro mol/min/mg. alpha-L-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 micro mol/min/mg. The molecular mass of alpha-L-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that of alpha-L-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. alpha-L-Arabinopyranosidase and alpha-L-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40 degrees C and pH 4.5 and 45 degrees C, respectively. Both purified enzymes were potently inhibited by Cu(2+) and p-chlormercuryphenylsulfonic acid. alpha-L-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-alpha-L-arabinopyranoside, followed by ginsenoside Rb2. alpha-L-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-alpha-L-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-beta-galactopyranoside or p-nitrophenyl-beta-D-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified alpha-L-arabinosidases. This is the first reported purification of alpha-L-arabinopyranosidase from an anaerobic Bifidobacterium sp.     

Enzymatic biotransformation of ginsenoside Rb1 to 20(<Emphasis Type="Italic">S</Emphasis>)-Rg3 by recombinant β-glucosidase from <Emphasis Type="Italic">Microbacterium esteraromaticum</Emphasis>

Microbacterium esteraromaticum was isolated from ginseng field. The β-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 β-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 β-glucosidase.     

Screening and characterization of an enzyme with beta-glucosidase activity from environmental DNA

A novel beta-glucosidase gene, bglA, was isolated from uncultured soil bacteria and characterized. Using genomic libraries constructed from soil DNA, a gene encoding a protein that hydrolyzes a fluorogenic analog of cellulose, 4-methylumbelliferyl beta-D-cellobioside (MUC), was isolated using a microtiter plate assay. The gene, bglA, was sequenced using a shotgun approach, and expressed in E. coli. The deduced 55-kDa amino acid sequence for bglA showed a 56% identity with the family 1 glycosyl hydrolase Chloroflexus aurantiacus. Bg1A included two conserved family 1 glycosyl hydrolase regions. When using p-nitrophenyl-beta-D-glucoside (pNPG) as the substrate, the maximum activity of the purified beta-glucosidase exhibited at pH 6.5 and 55 degrees C, and was enhanced in the presence of Mn2+. The Km and Vmax values for the purified enzyme with pNPG were 0.16 mM and 19.10 micromol/min, respectively. The purified BglA enzyme hydrolyzed both pNPG and p-nitrophenyl-beta-D-fucoside. The enzyme also exhibited substantial glycosyl hydrolase activities with natural glycosyl substrates, such as sophorose, cellobiose, cellotriose, cellotetraose, and cellopentaose, yet low hydrolytic activities with gentiobiose, salicin, and arbutin. Moreover, Bg1A was able to convert the major ginsenoside Rb1 into the pharmaceutically active minor ginsenoside Rd within 24 h.     

Cloning and characterization of ginsenoside Ra1-hydrolyzing beta-D-xylosidase from Bifidobacterium breve K-110

beta-D-Xylosidase (E.C. 3.2.1.37) from Bifidobacterium breve K-110, which hydrolyzes ginsenoside Ra1 to ginsenoside Rb2, was cloned and expressed in Escherichia coli. The (His6)-tagged recombinant enzyme, designated as XlyBK- 110, was efficiently purified using Ni2?-affinity chromatography (109.9-fold, 84% yield). The molecular mass of XylBK- 100 was found to be 55.7 kDa by SDS-PAGE. Its sequence revealed a 1,347 bp open reading frame (ORF) encoding a protein containing 448 amino acids, which showed 82% identity (DNA) to the previously reported glycosyl hydrolase family 30 of Bifidobacterium adolescentis ATCC 15703. The Km and Vmax values toward p-nitrophenyl-beta-D-xylopyranoside (pNPX) were 1.45mM and 10.75 micromol/min/mg, respectively. This enzyme had pH and temperature optima at 6.0 and 45 degrees C, respectively. XylBK-110 acted to the greatest extent on xyloglucosyl kakkalide, followed by pNPX and ginsenoside Ra1, but did not act on p-nitrophenyl-alpha-Larabinofuranoside, p-nitrophenyl-beta-D-glucopyranoside, or p-nitrophenyl-beta-D-fucopyranoside. In conclusion, this is the first report on the cloning and expression of beta-Dxylosidase- hydrolyzing ginsenoside Ra1 and kakkalide from human intestinal microflora.     

Microbial conversion of major ginsenoside rb(1) to pharmaceutically active minor ginsenoside rd

More than seventy strains of aerobic bacteria showing beta-glucosidase activity were isolated from a ginseng field, using a newly designed Esculin-R2A agar, and identified by their 16S rRNA gene sequences. Of these microorganisms, twelve strains could convert the major ginsenoside, Rb(1), to the pharmaceutically active minor ginsenoside Rd. Three strains, Burkholderia pyrrocinia GP16, Bacillus megaterium GP27 and Sphingomonas echinoides GP50, were phylogenetically studied, and observed to be most potent at converting ginsenoside Rb(1) almost completely within 48 h, as shown by TLC and HPLC analyses.     

Isolation and characterization of novel ginsenoside-hydrolyzing glycosidase from Microbacterium esteraromaticum that transforms ginsenoside Rb2 to rare ginsenoside 20(S)-Rg3

Ginsenoside Rb2 was transformed by recombinant glycosidase (Bgp2) into ginsenosides Rd and 20(S)-Rg3. The bgp2 gene consists of 2,430 bp that encode 809 amino acids, and this gene has homology to the glycosyl hydrolase family 2 protein domain. SDS-PAGE was used to determine that the molecular mass of purified Bgp2 was 87 kDa. Using 0.1 mg ml^?1 of enzyme in 20 mM sodium phosphate buffer at 40 °C and pH 7.0, 1.0 mg ml^?1 ginsenoside Rb2 was transformed into 0.47 mg ml^?1 ginsenoside 20(S)-Rg3 within 120 min, with a corresponding molar conversion yield of 65 %. Bgp2 hydrolyzed the ginsenoside Rb2 along the following pathway: Rb2 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb2 to ginsenoside 20(S)-Rg3 using the recombinant glycosidase.     

Purification and characterization of two novel β-glucosidases from Penicillium oxalicum and their application in bioactive ginsenoside production

Abstract

The fungus Penicillium oxalicum is able to selectively metabolize the 20(S)-protopanaxadiol ginsenosides Rb1, Rb2 and Rc to the bioactive ginsenoside compound K using extracellular glycosidases. In this study, two novel extracellular ginsenoside-hydrolyzing enzymes GH3-1 and GH3-2 were purified and characterized from P. oxalicum culture. Using ginsenosides as substrates, GH3-1 and GH3-2 synergistically catalyzed the hydrolysis of Rb1, Rb2 and Rc to yield the final product Compound K (C-K). The hydrolysis pathways were determined to be: Rb1→Rd→F2→C-K, Rb2→CO→CY→C-K and Rc→Mb→Mc→C-K for GH3-1 and GH3-2, respectively. The two enzymes differ, especially in composition, molecular weight, stability and substrate specificity, from GH1, a glycosidase previously purified from the same fungus. These enzymes could be of interest in glycoside degradation, especially in the production of minor ginsenosides.     

Purification and properties of lactate dehydrogenase from Nocardia asteroides.

The lactate dehydrogenase (LDH) from Nocardia asteroides was purified to homogeneity by ammonium sulphate precipitation, gel filtration on Sephadex G-150 and DEAE-Sepharose column chromatography. The purified enzyme showed a single band in native condition which indicated its homogeneity. SDS-PAGE of the purified enzyme showed the presence of three bands which correspond to molecular weights of 60, 66 and 74 kDa. The pH and temperature optima of the purified enzyme were 9.5 and 50 degrees C, respectively. The metal ions Mn++, Fe++, Co++, Mg++ and Ca++, increased the purified LDH activity. On the other hand, enzyme activity was completely inhibited by CuCl2. Potassium chloride, ammonium sulphate and sodium chloride did not alter the enzyme activity. The purified enzyme exhibited a Km value of 1.6 x 10(-5) M for pyruvate.     

Microbial conversion of ginsenoside Rb1 to minor ginsenoside F2 and gypenoside XVII by Intrasporangium sp. GS603 isolated from soil

A new strain, GS603, having beta-glucosidase activity was isolated from soil of a ginseng field, and its ability to convert major ginsenoside Rb(1) to minor ginsenoside or gypenoside was studied. Strain GS603 was identified as an Intrasporangium species by phylogenetic analysis and showed high ginsenoside-converting activity in LB and TSA broth but not in nutrient broth. The culture broth of the strain GS603 could convert ginsenoside Rb(1 )into two metabolites, which were analyzed by TLC and HPLC and shown to be the minor ginsenoside F(2) and gypenoside XVII by NMR.     

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.     

20(S)-protopanaxadiol and the ginsenoside Rh2 inhibit Na+ channel-activated depolarization and Na+ channel-dependent amino acid neurotransmitter release in synaptic fractions isolated from mammalian brain

The ginsenoside Rh(2) and its aglycone 20(S)-protopanaxadiol are known to inhibit the binding of [(3)H]batrachotoxinin 20alpha-benzoate to site 2 on voltage-gated sodium channels and electrophysiological investigations conducted by others have shown that ginsenosides cause voltage-dependent inhibition of reconstituted forms of the sodium channel. Here we describe the actions of Rh(2) and 20(S)-protopanaxadiol on sodium channel function and release of neurotransmitters resulting from activation of native sodium channels in synaptic preparations isolated from whole mouse brain. Rh(2) and 20(S)-protopanaxadiol inhibited veratridine-dependent (tetrodotoxin-suppressible) depolarization of synaptoneurosomes as determined using the rhodamine 6G method although 20(S)-protopanaxadiol was more potent as an inhibitor than Rh(2). Veratridine- (sodium channel-) dependent release of the neurotransmitters L-glutamate and GABA was almost fully inhibited by 20(S)-protopanaxadiol, however, less complete inhibition was observed with Rh(2). At its maximum inhibitory concentration, Rh(2) also produced release of l-glutamate and GABA from synaptosomes, in contrast to 20(S)-protopanaxadiol. We conclude that low to moderate micromolar concentrations of Rh(2) and 20(S)-protopanaxadiol inhibit sodium channel function and sodium channel-activated release of neurotransmitters. Apparently the ginsenoside Rh(2) cannot achieve complete inhibition of sodium channel-activated transmitter release because at high concentrations it also stimulates release.     

The Ginsenoside 20-O-β-D-Glucopyranosyl-20(S)-Protopanaxadiol Induces Autophagy and Apoptosis in Human Melanoma via AMPK/JNK Phosphorylation

Studies have shown that a major metabolite of the red ginseng ginsenoside Rb1, called 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (GPD), exhibits anticancer properties. However, the chemotherapeutic effects and molecular mechanisms behind GPD action in human melanoma have not been previously investigated. Here we report the anticancer activity of GPD and its mechanism of action in melanoma cells. GPD, but not its parent compound Rb1, inhibited melanoma cell proliferation in a dose-dependent manner. Further investigation revealed that GPD treatment achieved this inhibition through the induction of autophagy and apoptosis, while Rb1 failed to show significant effect at the same concentrations. The inhibitory effect of GPD appears to be mediated through the induction of AMPK and the subsequent attenuation of mTOR phosphorylation. In addition, GPD activated c-Jun by inducing JNK phosphorylation. Our findings suggest that GPD suppresses melanoma growth by inducing autophagic cell death and apoptosis via AMPK/JNK pathway activation. GPD therefore has the potential to be developed as a chemotherapeutic agent for the treatment of human melanoma.