Enzymatic preparation of genuine prosapogenin, 20(S)-ginsenoside Rh1, from ginsenosides Re and Rg1

It was found that a lactase preparation from Penicillium sp. nearly quantitatively hydrolyzed ginsenosides Re and Rg1, which are major saponins in roots of Panax ginseng, to a minor saponin, 20(S)-ginsenoside Rh1 [6-O-beta-D-glucopyranosyl-20(S)-protopanaxatriol]. This is the first report on the enzymatic preparation of ginsenoside Rh1 with a high efficiency. This enzyme also readily hydrolyzed ginsenoside Rg2 to ginsenoside Rh1.     

Conversion of major ginsenoside Rb1 to 20(S)-ginsenoside Rg3 by Microbacterium sp. GS514

Ginseng saponin, the most important secondary metabolite in ginseng, has various pharmacological activities. Many studies have been directed towards converting major ginsenosides to the more active minor ginsenoside, Rg3. Due to the difficulty in preparing ginsenoside Rg3 enzymatically, the compound has been mainly produced by either acid treatment or heating. A microbial strain GS514 was isolated from soil around ginseng roots in a field and used for enzymatic preparation of the ginsenoside Rg3. Blast results of the 16S rRNA gene sequence of the strain GS514 established that the strain GS514 belonged to the genus Microbacterium. Its 16S rRNA gene sequence showed 98.7%, 98.4% and 96.1% identity with those of M. esteraromaticum, M. arabinogalactanolyticum and M. lacticum. Strain GS514 showed a strong ability to convert ginsenoside Rb1 or Rd into Rg3. Enzymatic production of Rg3 occurred by consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C-20 carbon of ginsenoside Rb1 showing the biotransformation pathway: Rb1-->Rd-->Rg3.     

Generation of ginsenosides Rg3 and Rh2 from North American ginseng

Rg3 and Rh2 ginsenosides are primarily found in Korean red ginseng root (Panax ginseng C.A. Meyer) and valued for their bioactive properties. We quantified both Rh2 and Rg3 ginseng leaf and Rg3 from root extracts derived from North American ginseng (Panax quinquefolius). Quantification was obtained by application of HPLC with ion fragments detected using ESI-MS. Ginseng leaf contained 11.3+/-0.5 mg/g Rh2 and 7.5+/-0.9 mg/g Rg3 in concentrated extracts compared to 10.6+/-0.4 mg/g Rg3 in ginseng root. No detectable Rh2 was found in root extracts by HPLC, although it was detectable by ESI-MS analysis. Ginsenosides Rg3 and Rh2 were detected following hot water reflux extraction, but not from tissues extracted with 80% aqueous ethanol at room temperature. Therefore ginsenosides Rg3 and Rh2 are not naturally present in North American ginseng, but are products of a thermal process. Using ESI-MS analysis, it was found that formation of Rg3 and Rh2, among other compounds, were a function of heating time and were breakdown products of the more abundant ginsenosides Rb1 and Rc. Our findings that heat processed North American ginseng leaf is an excellent source of Rh2 ginsenoside is an important discovery considering that ginseng leaf material is obtainable throughout the entire plant cycle for recovery of valuable ginsenosides for pharmaceutical use.     

(20S)‐Protopanaxadiol Ginsenosides Induced Cytotoxicity via Blockade of Autophagic Flux in HGC‐27 Cells

(20S)‐Protopanaxadiol ginsenosides Rg3, Rh2 and PPD have been demonstrated for their anticancer activity. However, the underlying mechanism of their antitumor activity remains unclear. In the present study, we investigated the role of these three ginsenosides on cell proliferation and death of human gastric cancer cells (HGC‐27 cells). The sulforhodamine B (SRB) assay, Western blot analysis, fluorescence microscopy, confocal microscopy, high performance liquid chromatography (HPLC) analysis, flow cytometry, and transmission electron microscopy (TEM) were used to evaluate cell proliferation, apoptosis, and autophagy. The results showed that both Rh2 and PPD were more effective than Rg3 in inhibiting HGC‐27 cell proliferation and inducing cytoplasmic vacuolation, while no significant changes in apoptosis were observed. Interestingly, cytoplasmic vacuolation and blockade of autophagy flux were observed after treatment with Rh2 and PPD. Rh2 obviously up‐regulated the expression of the LC3II and p62. Furthermore, the increase in lysosomal pH and membrane rupture was observed in Rh2‐treated and PPD‐treated cells. When HGC‐27 cells were pretreated with bafilomycin A1, a specific inhibitor of endosomal acidification, cellular vacuolization was increased, and the cell viability was significantly decreased, which indicated that Rh2‐induced lysosome‐damage accelerated cell death. Furthermore, data derived from mitochondrial analysis showed that excessive mitochondrial reactive oxygen species (ROS) and dysregulation of mitochondrial energy metabolism were caused by Rh2 and PPD treatment in HGC‐27 cells. Taken together, these phenomena indicated that Rh2 and PPD inhibited HCG‐27 cells proliferation by inducing mitochondria damage, dysfunction of lysosomes, and blockade of autophagy flux. The number of glycosyl groups at C‐3 position could have an important effect on the cytotoxicity of Rg3, Rh2 and PPD.     

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.     

High performance liquid chromatographic-mass spectrometric determination of ginsenoside Rg3 and its metabolites in rat plasma using solid-phase extraction for pharmacokinetic studies

To support pharmacokinetic studies of ginsenosides, a novel method to quantitatively analyze ginsenoside Rg3 (Rg3), its prosapogenin ginsenoside Rh2 (Rh2) and aglycone 20(S)-protopanaxadiol (ppd) in rat plasma was developed and validated. The method was based on gradient separation of ginsenosides present in rat plasma using high performance liquid chromatography (HPLC), followed by detection with electrospray ionization(ESI) mass spectrometry (MS) in negative ion mode with the mobile phase additive, ammonium chloride (500 microM). Differentiation of ginsenosides was achieved through simultaneous detection of the [M(+)Cl(-)] adduct of ginsenoside Rg3 and [M(+)Cl(-)] adducts of its deglycosylated metabolites Rh2 and ppd, and other ions after solid phase extraction (SPE). The /specific ions monitored were m/z 819.50 for Rg3, m/z 657.35 for Rh2, m/z 495.40 for ppd and m/z 799.55 for the internal standard (digitoxin). The mean recoveries for Rg3, Rh2 and ppd were 77.85, 82.65 and 98.33%, respectively using 0.1 ml plasma for extraction. The lower limits of quantification were 10.0, 2.0 and 8.0 ng/ml (equivalent to 0.1, 0.02 and 0.08 ng in each 10 microl injection onto the HPLC column) for Rg3, Rh2 and ppd, respectively. The method has been demonstrated to be highly sensitive and accurate for the determination of Rg3 and its metabolites in rat plasma.     

Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts

Ginsenosides Rh2 and Rg3 represent promising candidates for cancer prevention and therapy and have low toxicity. However, the concentrations of Rh2 and Rg3 are extremely low in the bioactive constituents (triterpene saponins) of ginseng. Despite the available heterologous biosynthesis of their aglycone (protopanaxadiol, PPD) in yeast, production of Rh2 and Rg3 by a synthetic biology approach was hindered by the absence of bioparts to glucosylate the C3 hydroxyl of PPD. In this study, two UDP-glycosyltransferases (UGTs) were cloned and identified from Panax ginseng. UGTPg45 selectively transfers a glucose moiety to the C3 hydroxyl of PPD and its ginsenosides. UGTPg29 selectively transfers a glucose moiety to the C3 glucose of Rh2 to form a 1–2-glycosidic bond. Based on the two UGTs and a yeast chassis to produce PPD, yeast cell factories were built to produce Rh2 and/or Rg3 from glucose. The turnover number (k_cat) of UGTPg29 was more than 2500-fold that of UGTPg45, which might explain the higher Rg3 yield than that of Rh2 in the yeast cell factories. Building yeast cell factories to produce Rh2 or Rg3 from simple sugars by microbial fermentation provides an alternative approach to replace the traditional method of extracting ginsenosides from Panax plants.     

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.     

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.     

Mechanistic studies on protopanaxadiol, Rh2, and ginseng (Panax quinquefolius) extract induced cytotoxicity in intestinal Caco-2 cells

Certain ginsenosides, also known as triterpene glycosides, have been recently reported to have a characteristic effect on cultured intestinal and leukemia cell growth. Ginsenoside aglycones 20(S)-protopanaxadiol (PD), 20(S)-protopanaxatriol (PT), and ginsenoside Rh2 have been identified as having a strong effect on reducing cell viability. Furthermore, ginsenoside Rh2 is thought to be a rare ginsenoside not found in all ginseng products. Rather, Rh2 has been recently reported to be a breakdown product of thermal processing of North American ginseng. In this study, pure ginsenosides PD, PT, Rh2 standards and an enriched Rh2 fraction derived from ginseng leaf were tested in cultured Caco-2 cells for relative cytotoxic potency. PD and Rh2 LC50 were similar after 24 to 72 h, whereas a drop in PT LC50 occurred later at 48 and 72 h. Furthermore, PD and Rh2 affected membrane integrity as indicated by LDH secretion earlier than PT and the enriched Rh2 fraction (P < or = 0.05). Ginsenoside Rh2 showed the greatest (P < or = 0.05) build up of necrotic cells (18.3 +/- 0.1%) at the respective LC50 after 24 h and PD (21.3 +/- 0.3%) showed the largest effect after 44 h of exposure. The effect on apoptotic cells at 44 h of treatment were significantly different (P < or = 0.05) for Rh2 (21 +/- 0.4%), PD (14.6 +/- 0.1%), enriched Rh2 leaf fraction (9.9 +/- 0.6%), and PT (2.3 +/- 0.1%) treatments. Caco-2 caspase-3 activity was different between ginsenoside exposure; Rh2 (10.6 +/- 0.3 nM pNA) had the greatest (P < or = 0.05) activity followed by the enriched Rh2 leaf fraction (8.3 +/- 0.2 nM pNA), PT (7.3 +/- 0.3 nM pNA). The PD (4.8 +/- 0.04 nM pNA) treatment was similar to untreated cells (4.3 +/- 0.05 nM pNA) in caspase-3 activity. These results show variable bioactive response in cultured intestinal cell to specific ginsenosides and an enriched Rh2 North American ginseng extract which may be explained on basis of hydrophobic/hydrophilic balance.     

Utilization study of stems and leaves of Tienchi Ginseng. I. Anti-hypertensive effect of stems and leaves of Tienchi Ginseng on stroke-prone spontaneously hypertensive rat (SHRSP)

Tienchi ginseng tea was prepared from stems and leaves of Tienchi ginseng which is a special product in China. The increase in systolic blood pressure (SBP) was significantly inhibited by the consumption of a 4% this tea solution as drinking water from the prehypertensive stage (6 weeks of age) in male stroke-prone spontaneously hypertensive rats (SHRSP). A similar intake from the hypertensive stage also showed an anti-hypertensive effect. In contrast, a similar intake had no effect on SBP of normotensive Wistar Kyoto rats. We found that the rhizome of Tienchi ginseng contained two types of saponin: 20(s)-protopanaxadiol (PPD) such as ginsenosides Rb(1) and Rd having a hypotensive effect, and 20(s)-protopanaxatriol (PPT) such as ginsenosides Rg(1) and Re having a hypertensive effect. In contrast, the tea sample contained PPD and gamma-aminobutyric acid (GABA), but no PPT. These results suggest that drinking this tea infusion would be useful for controlling hypertension.     

Effects of ginsenosides and their metabolites on voltage-dependent Ca(2+) channel subtypes

In previous reports we demonstrated that ginsenosides, active ingredients of Panax ginseng, affect some subsets of voltage-dependent Ca(2+) channels in neuronal cells expressed in Xenopus laevis oocytes. However, the major component(s) of ginseng that affect cloned Ca(2+) channel subtypes such as alpha(1C) (L)-, alpha(1B) (N)-, alpha(1A) (P/Q)-, a1E (R)- and a1G (T) have not been identified. Here, we used the two-microelectrode volt-age clamp technique to characterize the effects of ginsenosides and ginsenoside metabolites on Ba(2+) currents (IBa) in Xenopus oocytes expressing five different Ca(2+) channel subtypes. Exposure to ginseng total saponins (GTS) induced voltage-dependent, dose-dependent and reversible inhibition of the five channel subtypes, with particularly strong inhibition of the a1G-type. Of the various ginsenosides, Rb(1), Rc, Re, Rf, Rg(1), Rg(3), and Rh(2), ginsenoside Rg(3) also inhibited all five channel subtypes and ginsenoside Rh(2) had most effect on the a1C- and a1E-type Ca(2+) channels. Compound K (CK), a protopanaxadiol ginsenoside metabolite, strongly inhibited only the a(1G)-type of Ca(2+) channel, whereas M4, a protopanaxatriol ginsenoside metabolite, had almost no effect on any of the channels. Rg(3), Rh(2), and CK shifted the steady-state activation curves but not the inactivation curves in the depolarizing direction in the alpha(1B)- and alpha(1A)-types. These results reveal that Rg(3), Rh(2) and CK are the major inhibitors of Ca(2+) channels in Panax ginseng, and that they show some Ca(2+) channel selectivity.     

一种真菌对人参皂苷Rg3的转化

[目的]筛选长白山人参土壤中的活性微生物,转化人参总皂苷及单体人参皂苷产生稀有抗肿瘤成份.[方法]从长白山人参根际土壤中分离各类菌株,对人参总皂苷及单体人参皂苷进行微生物转化,并通过硅胶柱层析等方法对转化产物进行分离纯化,采用波谱解析及理化常数对其进行结构鉴定;结合菌落形态、产孢结构、孢子形态特征以及菌株ITS rDNA核酸序列分析,对活性菌株进行鉴定.[结果]从长白山人参根际土壤中分离各类真菌菌株68株,有12株菌株对人参总皂苷有转化活性,其中菌株SYP2353对二醇组人参皂苷Rg3具有较强的转化活性.[结论]阳性菌株SYP2353被鉴定为疣孢漆斑菌(Myrothecium verrucaria),能将人参皂苷Rg3转化为稀有人参皂苷Rh2及二醇组人参皂苷苷元PPD,为稀有人参皂苷Rh2的制备提供了新的方法.     

Enzyme kinetics of ginsenosidase type IV hydrolyzing 6-O-multi-glycosides of protopanaxatriol type ginsenosides

In this work, the kinetics of ginsenosidase type IV hydrolyzing the 6-O-multi-glycosides of protopanaxatriol type ginsenosides (PPT) from Aspergillus sp.39g strain were investigated. The enzyme molecular weight was about 56 kDa. The enzyme hydrolyzes the 6-O-α-l-(1 → 2)-rhamnoside of ginsenoside Re and 6-O-β-d-(1 → 2)-xyloside of R1 into Rg1, and subsequently hydrolyzes 6-O-β-d-glucoside of Rg1 into F1. The enzyme hydrolyzes 6-O-α-l-(1 → 2)-rhamnoside of Rg2 and 6-O-β-d-(1 → 2)-glucoside of Rf into Rh1, and subsequently hydrolyzes 6-O-β-d-glucoside of Rh1 into its aglycone. The enzyme K_m and V_max for Re were 22.2 mM, and 7.94 mM/h; the K_m and V_max for R1 were 7.06 mM and 1.61 mM/h; the enzyme transformation velocity (V₀) at 5 mM substrate was 1.46 mM/h for Re, and 0.67 mM/h for R1. Therefore, the enzyme hydrolysis on the Re rhamnoside was faster than that on R1 xyloside. The enzyme V₀ on Rg1 was 0.05 mM/h that indicated the enzyme hardly hydrolyzed the 6-O-β-d-glucoside of Rg1. The enzyme kinetic parameters of Rg2 and Rf were 5.74 and 9.43 mM for K_m; 2.70 and 2.84 mM/h for V_max; 1.26 and 0.98 mM/h for V₀ at 5 mM substrate, respectively. Thus the enzyme hydrolysis on Rg2 rhamnoside was faster than that on the glucoside of Rf.     

An application of β-glycosidase to transformation of ginsenosides for the effective production of specific ginsenosides with biological efficacy

Over the past several decades, the pharmacological effects of ginsenosides in Panax ginseng roots have been extensively investigated. Here, we developed a method for producing specific ginsenosides (F1 and F2) with good yields (F1:162 mg/g, F2:305 mg/g) using ??-glycosidase purified from Aspergillus niger. In addition, each ginsenoside (at least 25 species) was separated and purified by high performance liquid chromatography (HPLC) using five different types of solvents and different purification steps. In addition, the Rg3:Rh2 mixture (1:1, w/w) was shown to inhibit a specific lung cancer cell line (NCI-H232) in vivo, displaying an anticancer effect at a dose lower than achieved using treatments with single Rg3 or Rh2. This finding suggests that the combination of ginsenosides for targeting anticancer is more effective than the use of a single ginsenoside from ginseng or red ginseng.     

Simultaneous enrichment of deglycosylated ginsenosides and monacolin K in red ginseng by fermentation with Monascus pilosus

To improve its bioavailability and pharmacological effects in humans, red ginseng was fermented with a newly isolated fungus, Monascus pilosus KMU103. Most of the ginsenosides were converted to deglycosylated ginsenocides, such as Rh(1), Rh(2), and Rg(3). The total amount of ginsenosides Rh(1), Rh(2), and Rg(3) was 838.7 mg/kg in the red ginseng, and increased to 4,117 mg/kg after 50 L fermentation in 13% red ginseng and 2% glucose. In addition, the Monascus-fermented red ginseng contained 3,089 mg/kg of monacolin K, one of the metabolites produced by Monascus known to reduce cholesterol in the blood. This newly developed Monascus-fermented red ginseng should result in improved health effects, not only by biotransforming gisenosides to deglycosylated ones but also by creating additional bioactive compounds.     

Can ginsenosides protect human erythrocytes against free-radical-induced hemolysis?

Many studies have focused on the free-radical-initiated peroxidation of membrane lipid, which is associated with a variety of pathological events. Panax ginseng is used in traditional Chinese medicine to enhance stamina and capacity to deal with fatigue and physical stress. Many reports have been devoted to the effects of ginsenosides, the major active components in P. ginseng, on the lipid metabolism, immune function and cardiovascular system. The results, however, are usually contradictory since the usage of mixture of ginsenosides cannot identify the function of every individual ginsenosides on the experimental system. On the other hand, every individual ginsenosides is not compared under the same experimental condition. These facts motivate us to evaluate the antioxidant effect of various individual ginsenosides on the experimental system of free-radical-initiated peroxidation: the hemolysis of human erythrocyte induced thermally by water-soluble initiator, 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). The inhibitory concentration of 50% inhibition (IC(50)) of AAPH-induced hemolysis of the erythrocyte has been studied firstly and found that the order of IC(50) is Rb3 - Rb1Rc>Re>Rh1>R1>Rg2>Rb3. Rg3, Rd and Rh2, however, act as synergistic prooxidants in the above experimental system. Rg1 does not show any synergistic antioxidative property. Although the antioxidative and prooxidative mechanism of various ginsenosides with or without TOH in AAPH-induced hemolysis of human erythrocytes will be further studied in detail, this information may be useful in the clinical usage of ginsenosides.     

Ginsenosides 20(S)-protopanaxadiol and Rh2 reduce cell proliferation and increase sub-G1 cells in two cultured intestinal cell lines, Int-407 and Caco-2

Ginsenosides derived from 20(S)-protopanaxatriol (PT) and 20(S)-protopanaxadiol (PD) groups had similar characteristic cytotoxic effects on the growth of two intestinal cells lines, Int-407 and Caco-2. Pure Rh2, a ginsenoside structurally related to PD, inhibited intestinal cell growth at greater than twice the concentration of PD, while Rh1, a ginsenoside structurally related to aglycone PT, had no cytotoxic effect. Concentrations causing growth inhibition of 50% of cells (LC50) for the compounds PD, PT, and Rh2 were 23, 26, and 53 microg/mL, respectively, for Int-407 cells. In comparison, the LC50 for PD and PT was determined to be 24 microg/mL, and that for Rh2 was 55 microg/mL in Caco-2 cells. A standardized North American ginseng extract with a known ginsenosides composition did not induce cytotoxicity in either of the intestinal cell lines. Cell cycle analysis showed characteristically different (P = 0.05) effects of ginsenosides PD, Rh2, and PT in both cell lines. Rh2 treatment of Int-407 caused a significantly (P = 0.05) higher production of sub-G1 (apoptotic) cells (35% +/- 1%) compared with untreated cells (14% +/- 0.3%) after 24 h. PD and Rh2 treatments were both significantly (P < 0.05) higher in apoptotic cells than in untreated cells after 48 and 72 h. Similar results were obtained for treatment of Caco-2 cells. Lactate dehydrogenase (LDH) activity in both cell lines was similar for PD and Rh2 and higher (P = 0.05) than for PT treatment at most time periods. These results show a specific structure-function relationship for bioactive ginsenosides in two contrasting intestinal cell types.     

Biotransformation of 20(S)-protopanaxadiol by Mucor spinosus

Biotransformation of 20(S)-protopanaxadiol (1) by the fungus Mucor spinosus AS 3.3450 yielded eight metabolites (2–9). On the basis of NMR and MS analyses, the metabolites were identified as 12-oxo-15α,27-dihydroxyl-20(S)-protopanaxadiol (2), 12-oxo-7β,11α,28-trihydroxyl-20(S)-protopanaxadiol (3), 12-oxo-7β,28-dihydroxyl-20(S)-protopanaxadiol (4), 12-oxo-15α,29-dihydroxyl-20(S)-protopanaxadiol (5), 12-oxo-7β,15α-dihydroxyl-20(S)-protopanaxadiol (6), 12-oxo-7β,11β-dihydroxyl-20(S)-protopanaxadiol (7), 12-oxo-15α-hydroxyl-20(S)-protopanaxadiol (8), and 12-oxo-7β-hydroxyl-20(S)-protopanaxadiol (9), respectively. Among them, 2–5, 7, and 8 are new compounds. These results indicated that M. spinosus could catalyze the specific C-12 dehydrogenation of 20(S)-protopanaxadiol, as well hydroxylation at different positions. These biocatalytic reactions may be difficult for chemical synthesis. The biotransformed products showed weak in vitro cytotoxic activities.     

Evidence that the tertiary structure of 20(S)-ginsenoside Rg(3) with tight hydrophobic packing near the chiral center is important for Na(+) channel regulation

Ginsenosides are the active ingredients of Panax ginseng. Ginsenoside Rg(3) exists as two stereoisomers of carbon-20: 20-S-protopanaxatriol-3-[O-beta-d-glucopyranosyl (1-->2)-beta-glucopyranoside] (20(S)-Rg(3)) and 20-R-protopanaxatriol-3-[O-beta-d-glucopyranosyl (1-->2)-beta-glucopyranoside] (20(R)-Rg(3)). Recently, we reported that 20(S)-Rg(3) regulates voltage-dependent Ca(2+) channel activity and several types of ligand-gated ion channels, whereas 20(R)-Rg(3) does not have this activity. In this study, we investigated the structure-activity relationship of these two stereoisomers by NMR spectroscopy and by measurement of the current in Xenopus oocytes expressing the mouse cardiac voltage-dependent Na(+) channel (Na(v)1.5). We found that 20(S)-Rg(3) but not 20(R)-Rg(3) inhibited Na(+) channel current in a dose- and voltage-dependent manner. The difference between Rg(3) epimers in voltage-dependent ion channel regulation indicates that the structure of 20(S)-Rg(3) may be geometrically better aligned than that of 20(R)-Rg(3) for interaction with receptor regions in Na(+) channels. The (1)H and (13)C NMR chemical shifts, including all hydroxyl protons of 20(S)-Rg(3) and 20(R)-Rg(3), were completely assigned, and their tertiary structures were determined. 20(S)-Rg(3) has more tight hydrophobic packing near the chiral center than 20(R)-Rg(3). Tertiary structures and activities of 20(S)-Rg(3) and 20(R)-Rg(3) indicate that 20(S)-Rg(3) may have stronger interactions with the receptor region in ion channels than 20(R)-Rg(3). This may result in different stereoselectivity of Rg(3) stereoisomers in the regulation of voltage-dependent Na(+) channel activity. This is the first structural approach to ginsenoside action on ion channel.