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.     

Preventive effect of 20(S)-ginsenoside Rg3 against lipopolysaccharide-induced hepatic and renal injury in rats

The preventive effect of 20(S)-ginsenoside Rg₃ (20(S)-Rg₃) on lipopolysaccharide (LPS)-induced oxidative tissue injury in rats was investigated in this study. The elevated serum nitrite/nitrate, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and creatinine levels in LPS-treated control rats were significantly decreased following 15 consecutive days of 20(S)-Rg₃ administration. In addition, thiobarbituric acid-reactive substance levels in the serum, liver and kidney were dose-dependently lower in 20(S)-Rg₃-treated groups than in the LPS-treated control group. The nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) protein expressions in the liver and kidney were significantly increased by LPS treatment. However, the 20(S)-Rg₃ administrations significantly decreased these protein expressions except for HO-1 in the liver. On the other hand, in the kidney, oral administration of 20(S)-Rg₃ showed a tendency to reduce NF-κB and iNOS protein expressions and also significantly reduced the elevated COX-2 and HO-1 protein expressions at a dose of 10 mg/kg body weight/day. All these results suggest the preventive effect of 20(S)-Rg₃ against LPS-induced acute oxidative damage in the liver and kidney and the preventive effect of 20(S)-Rg₃ administration against LPS toxicity was thought to be more predominant in the liver than kidney.     

Preventive effect of 20(S)-ginsenoside Rg3 against lipopolysaccharide-induced hepatic and renal injury in rats

The preventive effect of 20(S)-ginsenoside Rg₃ (20(S)-Rg₃) on lipopolysaccharide (LPS)-induced oxidative tissue injury in rats was investigated in this study. The elevated serum nitrite/nitrate, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and creatinine levels in LPS-treated control rats were significantly decreased following 15 consecutive days of 20(S)-Rg₃ administration. In addition, thiobarbituric acid-reactive substance levels in the serum, liver and kidney were dose-dependently lower in 20(S)-Rg₃-treated groups than in the LPS-treated control group. The nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) protein expressions in the liver and kidney were significantly increased by LPS treatment. However, the 20(S)-Rg₃ administrations significantly decreased these protein expressions except for HO-1 in the liver. On the other hand, in the kidney, oral administration of 20(S)-Rg₃ showed a tendency to reduce NF-κB and iNOS protein expressions and also significantly reduced the elevated COX-2 and HO-1 protein expressions at a dose of 10 mg/kg body weight/day. All these results suggest the preventive effect of 20(S)-Rg₃ against LPS-induced acute oxidative damage in the liver and kidney and the preventive effect of 20(S)-Rg₃ administration against LPS toxicity was thought to be more predominant in the liver than kidney.     

Enzymatic preparation of 20(S, R)-protopanaxadiol by transformation of 20(S, R)-Rg3 from black ginseng

20(S)-protopanaxadiol (PPD(S)) and 20(R)-protopanaxadiol (PPD(R)), the main metabolites of ginsenosides Rg3(S) and Rg3(R) in black ginseng, are potential candidates for anti-cancer therapy due to their pharmacological activities such as anti-tumor properties. In the present study, we report the preparation of PPD(S, R) by a combination of steaming and biotransformation treatments from ginseng. Aspergillus niger was isolated from soil and showed a strong ability to transform Rg3(S, R) into PPD(S, R) with 100% conversion. Furthermore, the enzymatic reactions were analyzed by reversed-phase HPLC, showing the biotransformation pathways: Rg3(S) → Rh2(S) → PPD(S) and Rg3(R) → Rh2(R) → PPD(R), respectively. In addition, 12 ginsenosides including 3 pairs of epimers, namely Rg3(S), Rg3(R), Rh2(S), Rh2(R), PPD(S) and PPD(R), were simultaneously determined by reversed-phase HPLC. Our study may be highly applicable for the preparation of PPD(S) and PPD(R) for medicinal purposes and also for commercial use.     

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.     

人参皂苷活性单体-20(R)-人参皂苷Rg3药理作用及其机制的研究进展

20(R)-人参皂苷Rg3是人参皂苷的主要活性单体之一,主要用于抗肿瘤治疗。近年来,20(R)-人参皂苷Rg3的药理作用不断取得进展,其中保护心脑血管、增强免疫功能、抗增生性瘢痕、抗疲劳的药理作用尤为值得关注。本文整理近年来国内外与20(R)-人参皂苷Rg3相关文献,论述其主要药理作用及其作用机制,为进一步开发20(R)-人参皂苷Rg3提供了科学依据。     

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.     

20(S)-Ginsenoside Rh2 as aldose reductase inhibitor from Panax ginseng

The root of Panax ginseng C. A. Meyer (Araliaceae) is a well-known herbal medicine in East Asia. The major bioactive metabolites in this root are commonly identified as ginsenosides. A series of ginsenosides were determined for in vitro human recombinant aldose reductase. This Letter aims to clarify the structural requirement for aldose reductase inhibition. We discovered that only ginsenoside 20(S)-Rh2 showed potent against aldose reductase, with an IC₅₀ of 147.3 μM. These results implied that the stereochemistry of the hydroxyl group at C-20 may play an important role in aldose reductase inhibition. An understanding of these requirements is considered necessary in order to develop a new type of aldose reductase inhibitor. Furthermore, P. ginseng might be an important herbal medicine in preventing diabetic complications.     

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.     

Bioconversion of major ginsenosides Rg(1) to minor ginsenoside F(1) using novel recombinant ginsenoside hydrolyzing glycosidase cloned from Sanguibacter keddieii and enzyme characterization

This study focuses on the cloning, expression, and characterization of recombinant ginsenoside hydrolyzing glycosidase from Sanguibacter keddieii in order to biotransform ginsenosides efficiently. The gene, termed bglSk, consists of 1857bp and revealed significant homology to that of glycoside hydrolase family 3. The enzyme was over-expressed in Escherichia coli BL21 (DE3) using a GST-fused pGEX 4T-1 vector system. The over-expressed recombinant enzymes could convert six major ginsenosides Rb(1), Rb(2), Rc, Rd, Re and Rg(1) into more pharmacologically active rare ginsenosides such as C-Y, C-Mc, C-K, Rg(2)(S), and F(1). Especially, BglSk could completely convert the Rg(1) into F(1). The GST-fused BglSk was purified with GST·bind agarose resin and then characterized. The kinetic parameters for β-glucosidase had apparent K(m) values of 0.456±0.009 and 0.167±0.003mM and V(max) values of 30.2±0.7 and 4.1±0.1μmolmin(-1)mg of protein(-1) against p-nitrophenyl-β-d-glucopyranoside and Rb(1), respectively.     

20(R)-Ginsenoside Rh2, not 20(S), is a selective osteoclastgenesis inhibitor without any cytotoxicity

Increased osteoclastic bone resorption plays a central role in the pathogenesis of many bone diseases, and osteoclast inhibitors are the most widely used treatments for these diseases. Ginsenosides, the main component of ginseng, have been known for their medicinal effects such as anti-inflammatory and anti-proliferative activities. In this study, we investigated the inhibitory effects of ginsenosides (ginsenoside 20(R)-Rh2 and ginsenoside 20(S)-Rh2) on osteoclastgenesis using RAW264 cells in vitro. Only ginsenoside 20(R)-Rh2 showed selective osteoclastgenesis inhibitory activity without any cytotoxicity up to 100 μM. These results implied that the stereochemistry of the hydroxyl group at C-20 may play an important role in selective osteoclastgenesis inhibitory activity.     

Enhanced production of ginsenoside Rh2(S) from PPD-type major ginsenosides using BglSk cloned from Saccharibacillus kuerlensis together with two glycosidase in series

BackgroundGinsenoside Rh2(S) is a promising compound for the prevention of various kinds of cancers, inflammation, and diabetes. However, due to its low concentration (<0.02%), researchers are still trying to find an efficient glycoside hydrolase for the scaled-up production of Rh2(S).MethodThree glycoside hydrolases (BglBX10, Abf22-3, and BglSk) were cloned in Escherichia coli BL21 (DE3) and the expressed recombinant enzyme was used for the scaled-up production of Rh2(S) through the conversion of PPD-type (protopanaxadiol) major ginsenosides (Rb1, Rc, and Rd, except Rb2) extracted from Korean red ginseng. Specific and specialized bioconversion pathways were designed that evolved the initial bioconversion of PPD-mix → Rg3(S) → Rh2(S). The reaction was started with 50 mg/mL of PPD-mix, 20 mg/mL of BglBX10, Abf22-3, and BglSk in series, respectively. The process was completed in a 10 L jar fermenter with a 5 L working volume at 37 °C for 48 hrs.ResultsThe designed bioconversion pathways show that Abf22-3 and BglBX10 were responsible for the conversion of Rb1, Rc and Rd → Rg3(S), and then Rg3(S) was completely transformed to Rh2(S) by BglSk. As a result, 15.1 g of ginsenoside Rh2(S) with 98.0 ± 0.2% purity was obtained after strict purification using the Prep-HPLC system with a 100 φ diameter column. Additionally, BglSk was also investigated for its production activity with seven different kinds of PPD-mix type ginsenosides.ConclusionOur pilot data demonstrate that BglSk is a suitable enzyme for the gram unit production of ginsenoside Rh2(S) at the industrial level.     

20(S)-Ginsenoside Rh1 inhibits cisplatin-induced hearing loss by inhibiting the MAPK signaling pathway and suppressing apoptosis in vitro

As an anticancer drug, cisplatin is widely used, but its clinical application is restricted due to its severe side effects of ototoxicity. Therefore, this study was dedicated to assessing the benefit of ginsenoside extract, 20(S)-Ginsenoside Rh1 (Rh1), on cisplatin-induced ototoxicity. HEI-OC1 cells and neonatal cochlear explants were cultured. Cleaved caspase-3, TUNEL, and MitoSOX Red were observed in vitro by immunofluorescence staining. CCK8 and LDH cytotoxicity assays were detected to measure cell viability and cytotoxicity. Our results showed that Rh1 significantly increased cell viability, reduced cytotoxicity, and alleviated cisplatin-induced apoptosis. In addition, Rh1 pretreatment decreased the excessive accumulation of intracellular reactive oxygen species. Mechanistic studies indicated that Rh1 pretreatment reversed the increase of apoptotic protein expression, accumulation of mitochondrial ROS, and activation of the MAPK signaling pathway. These results suggested that Rh1 can act as an antioxidant and anti-apoptotic agent against cisplatin-induced hearing loss by suppressing the excessive accumulation of mitochondrial ROS, activation of MAPK signaling pathway and apoptosis.     

Enzymatic preparation of an immunostimulant, the disaccharide-dipeptide, N-acetyl-beta-D-glucosaminyl-(1----4)-N-acetylmuramyl-L-alanyl-D-is ogl utamine, from a bacterial peptidoglycan

The disaccharide-dipeptide N-acetyl-beta-D-glucosaminyl-(1----4)-N-acetylmuramyl-L-alanyl-D-isog lut amine has been obtained by an enzymatic degradation of the peptidoglycan of Actinomadura R39. The peptidoglycan was hydrolyzed successively by the three following enzymes: lysozyme, DD-carboxypeptidase from Streptomyces albus G and gamma-D-glutamyl-meso-diaminopimelate endopeptidase I from Bacillus sphaericus 9602. The by-products of the last reaction were eliminated by successive ion-exchange and gel-permeation chromatographies. Both chemical analysis and mass spectrometry show that the resulting disaccharide-dipeptide is a pure compound.     

Simple and efficient preparation of ginsenoside (S)-Rg2 from ginsenoside Re by biotransformation with Cellulosimicrobium sp. 21

A novel ginsenoside-hydrolyzing strain was isolated from ginseng-cultivation soil in Changbai Mountain (China). The strain was identified as Cellulosimicrobium sp. 21 by 16S rDNA sequencing. Using the β-glucosidases secreted from Cellulosimicrobium sp. 21, protopanaxatriol-type ginsenoside Re was converted to the highly active neuroprotective molecule (S)-Rg2 by removal of the C-20-glucopyranosyl residue. The α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranose at the C-6 position of Rg2 was not further attacked by Cellulosimicrobium sp. 21, so the transformation shows high specificity. To simplify the transformation and product-preparation process, a simple and efficient transformation system was developed in a phosphate buffer system instead of organic media. The optimum conditions for transforming ginsenoside Re into Rg2 by Cellulosimicrobium sp. 21 were determined through single-factor experiments and response surface methodology. Under the optimized conditions: transformation buffer, 50 mM phosphate buffer, at pH: 7.00; temperature: 27.6°C; substrate concentration: 0.50 mg/ml; biotransformation period: 12 h; the biotransformation efficiency reached 89.8% (molar ratio) in 2-L reaction system. This simple biotransformation with high specificity and efficiency has potential for use in Rg2 preparation in the pharmaceutical industry.     

An improved protocol for the preparation of (S)-vinylglycine from (S)-methionine

We present an optimized procedure for the synthesis of (S)-vinylglycine from (S)-methionine. The key step is a solvent free pyrolysis of an intermediate sulfoxide at high temperature. Using our optimized reaction conditions, Cbz-protected vinylglycine was obtained in high yield and with almost no side products. The protocol is scalable, fast and avoids the use of poisonous reagents.     

Enzymatic preparation of optically pure t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate by a novel alcohol dehydrogenase discovered from Klebsiella oxytoca

Alcohol dehydrogenases can catalyze the inter-conversion of aldehydes and alcohols. The t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate is a key chiral intermediate in the synthesis of statin-type drugs such as Crestor (rosuvastatin calcium) and Lipitor (atorvastatin). Herein, a novel alcohol dehydrogenase (named as KleADH) discovered from Klebsiella oxytoca by a genome mining method was cloned and characterized. The KleADH was functionally overexpressed in Escherichia coli Rosetta (DE3) and the whole cell biocatalyst was able to convert t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate to t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate with more than 99% diastereomeric excess (de) and 99% conversion in 24 h without adding any expensive cofactors. Several factors influencing the whole cell catalyst activity such as temperature, pH, the effects of metal ions and organic solvent were determined. The optimum enzyme activity was achieved at 30 °C and pH 7.0 and it was shown that 1 mM Fe³⁺ can increase the enzyme activity by 1.2 times. N-hexane/water and n-heptane/water biphasic systems can also increase the activity of KleADH. Substrate specificity studies showed that KleADH also exhibited notable activity towards several aryl ketones with high stereoselectivity. Our investigation on this novel alcohol dehydrogenase KleADH reveals a promising biocatalyst for producing chiral alcohols for preparation of valuable pharmaceuticals.     

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.     

Isolation of (S)-2,3-dichloro-1-propanol assimilating bacterium,its characterization,and its use in preparation of (R)-2,3-dichloro-1-propanol and (S)-epichlorohydrin

Summary Linear alkylbenzene sulfonate (LAS) is a widely used anionic surfactant. Although approximately 1 million metric tons of LAS are produced annually, relatively little is known about the bacteria or the genetic factors that control LAS degradation in the environment. The objectives of this research were to: i) compare bacterial populations in wastewater and pristine pond systems; ii) determine the frequency of plasmids in bacteria from these sites; and iii) compare the frequency of DNA sequences coding for aromatic catabolism in isolates from these two sites. Plate counts indicated that exposure to wastewater resulted in higher levels of both heterotrophic bacteria and bacteria capable of growing on LAS containing medium (LAS/YEPG). In addition to higher numbers, a higher proportion of heterotrophs from the wastewater system were capable of growth on LAS/YEPG medium. Thus, the high levels of LAS in the wastewater system apparently selected fro organisms that were able to tolerate and/or degrade, it. Mineralization of¹⁴C-ring labelled LAS in any habitat related to the presence of organisms that grew on LAS/YEPG. Although may of these isolates could carry out primary degradation, no isolate, could mineralize¹⁴C-ring LAS in pure culture. A higher incidence of plasmids was found in bacteria from the wastewater pond and among bacteria that grew on LAS containing medium. However, the presence of plasmid, DNA did not necessarily confer the ability to degrade LAS nor was the ability to degrade LAS dependent on the presence of a plasmid. The incidence of selected genotypes for aromatic catabolism was similar among isolates on LAS/YEPG at both sites, suggesting that LAS ring degradation may be present in other populations or encoded by alternative sequences. In conclusion, LAS mineralization is mediated by a consortium and the evidence that initial attack of LAS is plasmid mediated is inconclusive.