ginsenoside-rg3 and protopanaxadiol

The rare ginsenoside Rg3 is attracting more attention because of its good physiological activity and urgent need. There are many pathways to obtain ginsenoside Rg3, including chemical and biological methods. Among these, the conversion of the protopanaxadiol-type ginsenosides by microbial hydrolysis is a trend due to its high efficiency and mild conditions. For effectively extracting from the other panaxadiol saponins, the conversion process for ginsenoside Rg3 was investigated using β-glycosidase-producing endophytic fungus in Panax ginseng in this study. The metabolic pathways are as follows: ginsenoside Rb1 → Gyp-XVII and ginsenoside Rb1 → ginsenoside Rd → ginsenoside Rg3. Phylogenetic analysis of 16S rDNA gene sequence, showed that GE 32 strain belonged to Flavobacterium species. These results suggest that the process of rare ginsenoside Rg3 production by endophytic bacteria GE 32 is efficient for the industrial production and application. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on cultivable β-glycosidase-producing endophytic bacteria from Panax ginseng. Flavobacterium sp. GE32 could convert major ginsenoside Rb1 into Gyp-XVII and minor ginsenoside Rg3. Strain GE 32 has potential to be applied on the preparation for minor ginsenoside Rg3 in pharmaceutical industry.

Topics: Biotransformation; DNA, Ribosomal; Flavobacterium; Ginsenosides; Glycoside Hydrolases; Hydrolysis; Panax; Phylogeny; Sapogenins; Saponins

Ginsenosides have previously been demonstrated to effectively inhibit cancer cell growth and survival in both animal models and cell lines. However, the specific ginsenoside component that is the active ingredient for cancer treatment through interaction with a target protein remains unknown. By an integrated quantitative proteomics approach via affinity mass spectrum (MS) technology, we deciphered the core structure of the ginsenoside active ingredient derived from crude extracts of ginsenosides and progressed toward identifying the target protein that mediates its anticancer activity. The Tandem Mass Tag (TMT) labeling quantitative proteomics technique acquired 55620 MS/MS spectra that identified 5499 proteins and 3045 modified proteins. Of these identified proteins, 224 differentially expressed proteins and modified proteins were significantly altered in nonsmall cell lung cancer cell lines. Bioinformatics tools for comprehensive analysis revealed that the Ras protein played a general regulatory role in many functional pathways and was probably the direct target protein of a compound in ginsenosides. Then, affinity MS screening based on the Ras protein identified 20(s)-protopanaxadiol, 20(s)-Ginsenoside Rh2, and 20(s)-Ginsenoside Rg3 had affinity with Ras protein under different conditions. In particular, 20(s)-protopanaxadiol, whose derivatives are the reported antitumor compounds 20(s)-Ginsenoside Rh2 and 20(s)-Ginsenoside Rg3 that have a higher affinity for Ras via a low KD of 1.22 μM and the mutation sites of G12 and G60, was demonstrated to play a core role in those interactions. Moreover, the molecular mechanism and bioactivity assessment results confirmed the identity of the chemical ligand that was directly acting on the GTP binding pocket of Ras and shown to be effective in cancer cell bioactivity profiles.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Survival; Ginsenosides; Guanosine Triphosphate; Humans; Lung Neoplasms; Molecular Docking Simulation; Neoplasm Proteins; Protein Binding; Protein Conformation; Proteomics; ras Proteins; Sapogenins

A series of oxidized products have been systematically semisynthesized from 20(S)-ginsenoside Rg3, Rh2, 20(S)-protopanaxadiol (PPD) and their 20(R)-epimers and the majority of these products were evaluated for their cytotoxic activity against HeLa cells and HepG2 cells by MTT assay for the first time. Twenty-two products were obtained and elucidated based on comprehensive (1)H NMR, (13)C NMR, two-dimensional (2D) NMR, and mass spectral data and the results reported in previous literature. All the four ocotillol type saponins (20S,24R(δ86, δ85); 20S,24S(δ87, δ88); 20R,24R(δ86, δ86); 20R,24S(δ86, δ87) were obtained. In addition, eight compounds (3, 8, 9, 10, 15, 16, 19 and 22) with the cyclized side chain were firstly identified. Most of the tested compounds possessed cytotoxicity to a certain degree against the two types of cells which implied these oxidized products could play a certain role on anti-cancer functions of the raw materials in vivo. Meanwhile, the results proved that the configurations at C-20 or C-24 and the number of glycosyl at C-3 have important influence on the cytotoxicity. The products 1, 2, 11-17, 20 and 22 should possess great activities and deserved further investigation as potential cytotoxic agents.

Topics: Antineoplastic Agents; Chemistry Techniques, Synthetic; Ginsenosides; HeLa Cells; Hep G2 Cells; Humans; Oxidation-Reduction; Sapogenins; Stereoisomerism; Structure-Activity Relationship; Triterpenes

20(S)- and 20(R)-ginsenoside Rg3 are a pair of epimers which could be deglycosylated to ginsenoside Rh2 and protopanaxadiol (PPD) in vivo. To better understand the differences of pharmacokinetic parameters and metabolism behaviors of Rg3 epimers in rat plasma, a sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and fully validated. This chromatographic method separate 20(S)-/20(R)-Rg3, 20(S)-/20(R)-Rh2 and 20(S)-/20(R)-PPD by gradient elution of 10 mM ammonium acetate solution (pH 5.0) and acetonitrile on a C18 column with a total run time of 15 min. 20(S)-protopanaxatriol (PPT) was used as internal standard, and multiple reaction monitoring (MRM) mode with negative electrospray ionization were performed. The lower limit of quantitations (LLOQs) were between 4.2 and 4.8 ng/ml, and the accuracies were between 91.7% and 112.2% with intra- and inter-day precisions less than 11.6%. This method was successfully applied to a pharmacokinetic study of intravenous and intra-gastric administration of 20(S)-Rg3 and 20(R)-Rg3 to rats. It has been found that both epimers can be deglycosylated to their corresponding chiral metabolites, i.e., Rh2 and PPD, with different extents. However, 20(R)-Rg3 underwent single direction chiral inversion to 20(S)-Rg3 in rats. Stereoselective pharmacokinetic parameters, metabolic degrees and chiral inversion extents of Rg3 epimers in rats were also discussed for the first time.

Topics: Animals; Chromatography, Liquid; Ginsenosides; Male; Plasma; Rats; Rats, Sprague-Dawley; Sapogenins; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Tandem Mass Spectrometry

A β-glucosidase from Gordonia terrae was cloned and expressed in Escherichia coli. The recombinant enzyme with a specific activity of 16.4 U/mg for ginsenoside Rb1 was purified using His-trap chromatography. The purified enzyme specifically hydrolyzed the glucopyranosides at the C-20 position in protopanaxadiol (PPD)-type ginsenosides and hydrolyzed the glucopyranoside at the C-6 or C-20 position in protopanaxatriol (PPT)-type ginsenosides. The reaction conditions for the high-level production of Rg3 from Rb1 by the enzyme were pH 6.5, 30°C, 20 mg/ml enzyme, and 4 mg/ml Rb1. Under these conditions, G. terrae β-glucosidase completely converted Rb1 and Re to Rg3 and Rg2, respectively, after 2.5 and 8 h, respectively. Moreover, the enzyme converted Rg1 to Rh1 at 1 h with a molar conversion yield of 82%. The enzyme at 10 mg/ml produced 1.16 mg/ml Rg3, 1.47 mg/ml Rg2, and 1.17 mg/ml Rh1 from Rb1, Re, and Rg1, respectively, in 10% (w/v) ginseng root extract at pH 6.5 and 30°C after 33 h with molar conversion yields of 100%, 100%, and 77%, respectively. The combined molar conversion yield of Rg2, Rg3, and Rh1 from total ginsenosides in 10% (w/v) ginseng root extract was 68%. These above results suggest that this enzyme is useful for the production of ginsenosides Rg3, Rg2, and Rh1.

Topics: beta-Glucosidase; Escherichia coli; Ginsenosides; Gordonia Bacterium; Hydrogen-Ion Concentration; Molecular Weight; Panax; Plant Extracts; Plant Roots; Sapogenins; Substrate Specificity; Temperature

Ginseng is a medicinal herb that requires cultivation under shade conditions, typically for 4-6 years, before harvesting. The principal components of ginseng are ginsenosides, glycosylated tetracyclic terpenes. Dammarene-type ginsenosides are classified into two groups, protopanaxadiol (PPD) and protopanaxatriol (PPT), based on their hydroxylation patterns, and further diverge to diverse ginsenosides through differential glycosylation. Three early enzymes, dammarenediol-II synthase (DS) and two P450 enzymes, protopanaxadiol synthase (PPDS) and protopanaxatriol synthase (PPTS), have been reported, but glycosyltransferases that are necessary to synthesize specific ginsenosides have not yet been fully identified. To discover glycosyltransferases responsible for ginsenoside biosynthesis, we sequenced and assembled the ginseng transcriptome de novo and characterized two UDP-glycosyltransferases (PgUGTs): PgUGT74AE2 and PgUGT94Q2. PgUGT74AE2 transfers a glucose moiety from UDP-glucose (UDP-Glc) to the C3 hydroxyl groups of PPD and compound K to form Rh2 and F2, respectively, whereas PgUGT94Q2 transfers a glucose moiety from UDP-Glc to Rh2 and F2 to form Rg3 and Rd, respectively. Introduction of the two UGT genes into yeast together with PgDS and PgPPDS resulted in the de novo production of Rg3. Our results indicate that these two UGTs are key enzymes for the synthesis of ginsenosides and provide a method for producing specific ginsenosides through yeast fermentation.

Topics: Ginsenosides; Glycosyltransferases; Molecular Sequence Data; Panax; Plant Proteins; Plant Roots; Plants, Medicinal; Sapogenins

The ability of fresh lemon (Citrus limon) to convert protopanaxadiol-type saponins into ginsenoside Rg3 was investigated, and the structures of 20(S)-ginsenoside Rg3 (1) and 20(R)-ginsenoside Rg3 (2) were identified by 1H NMR and 13C NMR spectroscopy. The experiment showed that lemon possesses the strong ability to hydrolyze ginsenosides. When protopanaxadiol-type saponins (16 mg/mL) were hydrolyzed by fresh lemon juice at 80 degrees C for 3 hrs, the conversion ratios of ginsenoside Rb1, Rb2, Rc and Rd were 92.9%, 90.0%, 96.90/0 and 55.5%, respectively, and the yields of 20(S)-ginsenoside Rg3 and 20(R)-ginsenoside Rg3 were, respectively, 31.2% and 28.3%.

Topics: Citrus; Ginsenosides; Hydrolysis; Magnetic Resonance Spectroscopy; Sapogenins

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.

Topics: Antineoplastic Agents; Aspergillus niger; Biotransformation; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Ginsenosides; Panax; Sapogenins; Stereoisomerism

We recently isolated 20(S)-25-methoxyl-dammarane-3beta, 12beta, 20-triol (25-OCH3-PPD), a natural product from Panax notoginseng, and demonstrated its cytotoxicity against a variety of cancer cells. Here we report the effects of this compound in vitro and in vivo on human prostate cancer cells, LNCaP (androgen-dependent) and PC3 (androgen-independent), in comparison with three structurally related ginsenosides, ginsenoside Rh2, ginsenoside Rg3, and 20(S)-protopanaxadiol. Of the four test compounds, 25-OCH3-PPD was most potent. It decreased survival, inhibited proliferation, induced apoptosis, and led to G1 cell cycle arrest in both cell lines. It also decreased the levels of proteins associated with cell proliferation (MDM2, E2F1, cyclin D1, and cdks 2 and 4) and increased or activated pro-apoptotic proteins (cleaved PARP, cleaved caspase-3, -8, and -9). In LNCaP cells, 25-OCH3-PPD inhibited the expression of the androgen receptor and prostate-specific antigen. Moreover, 25-OCH3-PPD inhibited the growth of prostate cancer xenograft tumours. Combining 25-OCH3-PPD with conventional chemotherapeutic agents or with radiation led to potent antitumour effects; tumour regression was almost complete following administration of 25-OCH3-PPD and either taxotere or gemcitabine. 25-OCH3-PPD also demonstrated low toxicity to noncancer cells and no observable toxicity in animals. In conclusion, our preclinical data indicate that 25-OCH3-PPD is a potential therapeutic agent against both androgen-dependent and androgen-independent prostate cancer.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Cell Cycle; Cell Proliferation; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Ginsenosides; Humans; In Vitro Techniques; Male; Mice; Mice, Nude; Prostate-Specific Antigen; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sapogenins; Survival Rate; Triterpenes; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The hydrolysis of protopanaxadiol-type saponin mixture by various glycoside hydrolases was examined. Among these enzymes, crude preparations of lactase from Aspergillus oryzae, beta-galactosidase from A. oryzae, and cellulase from Trichoderma viride were found to produce ginsenoside F(2) [3-O-(beta-D-glucopyranosyl)-20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol], compound K [20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol], and ginsenoside Rd {3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl]-20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol}, respectively, from protopanaxadiol-type saponin mixture in large quantities. Moreover, the crude preparation of lactase from Penicillium sp. having a high producing activity of ginsenoside Rh(1) (6-O-beta-D-glucopyranosyl-20(S)-protopanaxatriol) from protopanaxatriol-type saponin mixture gave ginsenoside Rd as a main product, ginsenoside Rg(3) {3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, and compound K from protopanaxadiol-type saponin mixture. The hydrolytic pathways of ginsenosides Rb(1), Rb(2), and Rc to ginsenosides Rd, Rg(3), and F(2), and compound K by crude preparations of four glycoside hydrolases were also studied. This is the first report on the enzymatic preparation of an intestinal bacterial metabolite, ginsenoside F(2), in quantity, and a considerable amount of a minor saponin, ginsenoside Rg(3), from a protopanaxadiol-type saponin mixture.

Topics: Aspergillus oryzae; beta-Galactosidase; Cellulase; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Ginsenosides; Glycoside Hydrolases; Lactase; Plants, Medicinal; Sapogenins; Saponins; Substrate Specificity; Trichoderma; Triterpenes

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the investigation of the pharmacokinetics of 20(R)-ginsenoside Rg3 in dog. The plasma samples were pretreated by liquid-liquid extraction and analyzed using LC/MS/MS with an electrospray ionization interface. Dioscin was used as the internal standard. The method had a lower limit of quantitation of 0.5 ng/mL for Rg3 in 200 microL of plasma or 2 ng/mL in 100 microL of plasma, which offered a satisfactory sensitivity for the determination of Rg3 in plasma. The intra- and inter-day precisions were measured to be below 8% and accuracy between -1.5 and 1.4% for all quality control samples. This quantitation method was successfully applied to pharmacokinetic studies of Rg3 after both an oral and an intravenous administration to beagle dogs. No Rh2 and protopanaxadiol were detected in plasma.

Topics: Administration, Oral; Animals; Blood Chemical Analysis; Chromatography, Liquid; Dogs; Female; Ginsenosides; Injections, Intra-Arterial; Male; Mass Spectrometry; Metabolic Clearance Rate; Reproducibility of Results; Sapogenins; Sensitivity and Specificity; Triterpenes

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.

Topics: Animals; Chromatography, High Pressure Liquid; Drug Stability; Freezing; Ginsenosides; Rats; Rats, Sprague-Dawley; Sapogenins; Spectrometry, Mass, Electrospray Ionization; Triterpenes

We showed recently that ginsenosides inhibit the activity of various types of ion channel. Here we have investigated the role of the carbohydrate component of ginsenoside Rg3 in the inhibition of Na+ channels. The channels were expressed in Xenopus oocytes by injecting cRNAs encoding rat brain Nav1.2 alpha and beta1 subunits, and analyzed by the two-electrode voltage clamp technique. Treatment with Rg3 reversibly inhibited the inward Na+ peak current (INa) with an IC50 of 32.2 +/- 4.5 microM, and the inhibition was voltage-dependent. To examine the role of the sugar moiety, we prepared a straight chain form of the second glucose and a conjugate of this glucose with 3-(4-hydroxyphenyl) propionic acid hydrazide (HPPH). Neither derivative inhibited INa. Treatment with the carbohydrate portion of ginsenoside Rg3, sophorose [beta-D-glucopyranosyl (1-->2)- beta-glucopyranoside], or the aglycone (protopanaxadiol), on their own or in combination had no effect on INa. These observations indicate that the carbohydrate portion of ginsenoside Rg3 plays an important role in its effect on the Na+ channel.

Topics: Animals; Ginsenosides; Glucans; NAV1.2 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Neurons; Oocytes; Patch-Clamp Techniques; Sapogenins; Sodium Channels; Structure-Activity Relationship; Triterpenes; Xenopus

The aim of the present study was to characterize the endothelium-dependent relaxation elicited by ginsenosides, a mixture of saponin extracted from Panax ginseng, in isolated rat aorta. Relaxations elicited by ginsenosides were mimicked by ginsenoside Rg1 and ginsenoside Rg1, two major ginsenosides of the protopanaxatriol group. Ginsenoside Rg3 was about 100-fold more potent than ginsenoside Rg1. The endothelium-dependent relaxation in response to ginsenoside Rg3 was associated with the formation of cycle GMP. These effects were abolished by N(G)-nitro-L-arginine and methylene blue. Relaxations in response to ginsenoside Rg3 were unaffected by atropine, diphenhydramine, [D-Pro2, D-Trp7,9]substance P, propranolol, nifedipine, verapamil and glibenclamide but were markedly reduced by tetraethylammonium. Tetraethylammonium modestly reduced the relaxation induced by sodium nitroprusside. These findings indicate that ginsenoside Rg3 is a major mediator of the endothelium-dependent nitric oxide-mediated relaxation in response to ginsenosides in isolated rat aorta, possibly via activation of tetraethylammonium-sensitive K+ channels.

Topics: Adrenergic beta-Antagonists; Animals; Antineoplastic Agents, Phytogenic; Aorta, Thoracic; Atropine; Calcium Channel Blockers; Chromatography, High Pressure Liquid; Diphenhydramine; Dose-Response Relationship, Drug; Endothelium, Vascular; Ginsenosides; Glyburide; Histamine H1 Antagonists; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle Relaxation; Nitroprusside; Potassium Channel Blockers; Potassium Channels; Propranolol; Rats; Rats, Sprague-Dawley; Sapogenins; Saponins; Tetraethylammonium; Triterpenes; Vasodilator Agents; Vasomotor System