PPD ) And Protopanaxatriol Research Articles

β-Glucosidase and Its Application in Bioconversion of Ginsenosides in Panax ginseng.

Ginsenosides are a group of bioactive compounds isolated from Panax ginseng. Conventional major ginsenosides have a long history of use in traditional medicine for both illness prevention and therapy. Bioconversion processes have the potential to create new and valuable products in pharmaceutical and biological activities, making them both critical for research and highly economic to implement. This has led to an increase in the number of studies that use major ginsenosides as a precursor to generate minor ones using β-glucosidase. Minor ginsenosides may also have useful properties but are difficult to isolate from raw ginseng because of their scarcity. Bioconversion processes have the potential to create novel minor ginsenosides from the more abundant major ginsenoside precursors in a cost-effective manner. While numerous bioconversion techniques have been developed, an increasing number of studies have reported that β-glucosidase can effectively and specifically generate minor ginsenosides. This paper summarizes the probable bioconversion mechanisms of two protopanaxadiol (PPD) and protopanaxatriol (PPT) types. Other high-efficiency and high-value bioconversion processes using complete proteins isolated from bacterial biomass or recombinant enzymes are also discussed in this article. This paper also discusses the various conversion and analysis methods and their potential applications. Overall, this paper offers theoretical and technical foundations for future studies that will be both scientifically and economically significant.

Pharmacological bioactivity of enzymatically bio-transformed ginsenosides

Many ginsenosides have shown positive effects, including anti-cancer potential and anti-inflammatory effects. Of note, protopanaxadiol (PPD) and protopanaxatriol (PPT) are not easily absorbed by the body through the digestive tract due to their hydrophilicity. From this point of view, the cytotoxic potencies of the hydrolysates of PPD and PPT on CRC are much stronger than their source compounds. Moreover, several minor ginsenosides that are absent naturally but have high disease ameliorative efficacy can be obtained from major ginsenoside by enzymatic hydrolysis. Therefore, the first aim of this study was to determine the effectiveness of the biotransformation of ginsenosides via enzymatic hydrolysis to improve their bioactivity. Second, the anti-inflammatory and anti-cancer effects of the raw and bio-transformed ginseng metabolites were determined in vitro. The results suggest that enzymes can effectively biotransform major ginsenosides (i.e., PPD and PPT) into minor ginsenosides (i.e., compound K) by hydrolyzing the β-glucosidic linkage. Moreover, the bio-transformed ginsenosides were effective in inhibiting the proliferation of HCT-116 cells and suppressing lipopolysaccharide-induced nitric oxide production in RAW 264.7 murine macrophages. Therefore, the enzymatic hydrolysis of ginsenosides can be employed to functionally produce hydrolysates with increased bioactivity.

Concomitant Administration of Red Ginseng Extract with Lactic Acid Bacteria Increases the Plasma Concentration of Deglycosylated Ginsenosides in Healthy Human Subjects.

With the increased frequency of red ginseng extract (RGE) and lactic acid bacteria (LAB) co-administration, we aimed to investigate the interactions between RGE and LAB with regard to in vitro and in vivo deglycosylation metabolism and the pharmacokinetics of ginsenosides. As a proof-of-concept study, five healthy humans were administered RGE (104.1 mg of total ginsenosides/day) with or without co-administration of LAB (2 g, 1 billion CFU/day) for 2 weeks, and the plasma concentrations of ginsenosides in human plasma were monitored. The plasma exposure to compound K (CK), ginsenoside Rh2 (GRh2), protopanaxadiol (PPD), and protopanaxatriol (PPT) in the concomitant administration RGE and LAB groups increased by 2.7-, 2.1-, 1.6-, and 3.5-fold, respectively, compared to those in the RGE administration group, without a significant change in Tmax. The plasma concentrations of GRb1, GRb2, and GRc remained unchanged, whereas the AUC values of GRd and GRg3 significantly decreased in the concomitant administration RGE and LAB groups. To understand the underlying mechanism, the in vitro metabolic activity of ginsenosides was measured during the fermentation of RGE or individual ginsenosides in the presence of LAB for 1 week. Consistent with the in vivo results, co-incubation with RGE and LAB significantly increased the formation rate of GRh2, CK, PPD, and PPT. These results may be attributed to the facilitated deglycosylation of GRd and GRg3 and the increased production of GRh2, CK, PPD, and PPT by the co-administration of LAB and RGE. In conclusion, LAB supplementation increased the plasma concentrations of deglycosylated ginsenosides, such as GRh2, CK, PPD, and PPT, through facilitated deglycosylation metabolism of ginsenosides in the intestine.

Pharmacokinetic Comparison of Ginsenosides between Fermented and Non-Fermented Red Ginseng in Healthy Volunteers.

Fermentation of red ginseng (RG) produces fermented red ginseng (FRG), thereby increasing the relative amount of downstream ginsenosides, including compound Y (CY), F2, Rh2, compound K (CK), compound O, protopanaxadiol (PPD), and protopanaxatriol (PPT). These downstream ginsenosides have beneficial pharmacological effects, and are easily absorbed by the human body. Based on these expectations, a randomized, single-dose, two-period, crossover clinical trial was planned to compare the pharmacokinetic characteristics of seven types (Rb1, CY, F2, CK, Rh2, PPD, and PPT) of ginsenoside components after FRG and RG administration. The safety and tolerability profiles were assessed in this clinical trial. Sixteen healthy Korean male subjects were administered 6 g of FRG or RG. All ginsenosides except Rb1 showed higher systemic exposure after FRG administration than after RG administration, based on comparisons of ginsenoside Cmax and area under the concentration-time curve (AUC) between FRG and RG. CK, the main ginsenoside component produced during the fermentation process, had 69.23/74.53-fold higher Cmax/AUClast after administration of FRG than RG, and Rh2 had 20.27/18.47-fold higher Cmax/AUClast after administration of FRG than RG. In addition, CY and F2 were detected in FRG; however, all plasma concentrations of CY and F2, except in one subject, were below the lower limit of quantification in RG. There were no clinically significant findings with respect to clinical laboratory tests, blood pressures, or adverse events. Therefore, regular administration of FRG may exert better pharmacological effects than RG.

Discovery and confirmation of protein action site AK1 of ginsenosides in brain based on DARTS technology

This study aims to explore the targets of ginsenosides in brain based on drug affinity responsive target stability(DARTS) technology. Specifically, DARTS technology was combined with label-free liquid chromatography tandem mass spectrometry(LC-MS) to screen out the proteins in the brain that might interact with ginsenosides. Based on the screening results, adenylate kinase 1(AK1) was selected for further confirmation. First, the His-AK1 fusion protein was yielded successively through the construction of recombinant prokaryotic expression vector, expression of target protein, and purification of the fusion protein. Biolayer interferometry(BLI) was employed to detect the direct interaction of Rg_1, Re, Rb_1, Rd, Rh_2, F1, Rh_1, compound K(CK), 25-OH-PPD, protopanaxa-diol(PPD), and protopanaxatriol(PPT) with AK1, thereby screening the ginsenoside monomer or sapogenin that had strong direct interaction with the suspected target protein AK1. Then, the BLI was used to further determine the kinetic parameters for the binding of PPD(strongest interaction with AK1) to His-AK1 fusion protein. Finally, molecular docking technology was applied to analyze the binding properties between the two. With DARTS and LC-MS, multiple differential proteins were screened out, and AK1 was selected based on previous research for target verification. Fusion protein His-AK1 was obtained by prokaryotic expression, and the response(nm) of Re, Rg_1, Rd, Rb_1, Rh_1, Rh_2, F1, PPT, PPD, 25-OH-PPD, and CK with His-AK1 was respectively 0.003 1, 0.001 9, 0.042 8, 0.022 2, 0.013 4, 0.037 3, 0.013 9, 0.030 7, 0.140 2, 0.016 0, and 0.040 8. The K_(on), K_(off), and K_D values of PPD and His-AK1 were determined by the BLI as 1.22×10~2 mol~(-1)·L·s~(-1), 1.04×10~(-2) s~(-1), 8.52×10~(-5) mol·L~(-1). According to the molecular docking result, PPD bound to AK1 with the absolute value of the docking score of 3.438, and hydrogen bonds mainly formed between the two. Thus, AK1 is one of the protein action sites of ginsenosides in the brain. The direct interaction between ginsenoside metabolite PPD and AK1 is the strongest.

Solid-State Fermentation With Aspergillus cristatus Enhances the Protopanaxadiol- and Protopanaxatriol-Associated Skin Anti-aging Activity of Panax notoginseng.

A metabolomics approach was used to profile metabolites of Panax notoginseng fermented with Aspergillus cristatus in two ways, liquid-state fermentation (LF-P) and solid-state fermentation (SSF-P) and examine metabolite markers representing antioxidant activity and skin anti-aging. Protopanaxadiol (PPD) and protopanaxatriol (PPT) contents were higher in SSF-P than in LF-P and showed a multiplicative increase over the fermentation period of four days. PPD and PPT levels also correlated with antioxidant and anti-aging effects in skin, based on the mRNA expression of dermal extracellular matrix components. In the bioactivity validation assays, PPD and PPT significantly improved the expression of type-I collagen, fibrillin-1, and elastin in human dermal fibroblasts from both young and old subjects; these were comparable with the effects of the SSF-P extracts. Overall, our results suggest that changes in the metabolites of P. notoginseng fermented with A. cristatus enhance the quality and availability of bioactive compounds associated with skin anti-aging.

Identification of three groups of ginsenoside biosynthetic UDP-glycosyltransferases from Gynostemma pentaphyllum

Ginsenosides are glycosylated dammarene-type triterpenes that have been identified in distantly related Panax ginseng and Gynostemma pentaphyllum. The phylogenetic relatedness of the ginsenoside biosynthetic genes in the two species was previously unknown. The final steps of ginsenoside biosynthesis are the glycosylations of hydroxylated triterpenes, protopanaxadiol (PPD) and protopanaxatriol (PPT), and their glycosylated forms by UDP-glycosyltransferases (UGTs). Ginsenoside biosynthetic UGTs have been identified in Panax but not in Gynostemma. Through a biochemical screening of Gynostemma UGTs (GpUGTs), we herein identified three groups of ginsenoside biosynthetic GpUGTs. These groups comprise: two GpUGTs that belong to the UGT71 family and glucosylate the C20-OH positions of PPD- and PPT-type ginsenosides; one GpUGT that belongs to the UGT74 family and glucosylates the C3-OH position of PPD-type ginsenosides; and two GpUGTs that belong to the UGT94 family and add a glucose to the C3-O-glucosides of PPD-type ginsenosides. These GpUGTs belong to the same UGT families as the ginsenoside biosynthetic Panax UGTs (PgUGTs). However, GpUGTs and PgUGTs belong to different subfamilies. Furthermore, cucumber UGTs orthologous to GpUGTs do not glucosylate ginsenosides. These results collectively suggest that, during evolution, P. ginseng and G. pentaphyllum independently opted to use the same UGT families to synthesize ginsenosides.

Efficient Production of Various Minor Ginsenosides from PPD- and PPT-type Major Ginsenosides Using a Single Recombinant BglFc Isolated from Flavobacterium chilense

Background: Rare ginsenosides (F2, Rg3, Gyp-XVII, and C-K) are pharmaceutically active components of Panax ginseng, which are derived from the conversion of major ginsenosides through various transformation methods. To date, most studies have failed to identify a competent bacterial strain and recombinant enzyme for converting protopanaxadiol (PPD)- and protopanaxatriol (PPT)-type ginsenosides to target minor ginsenosides. Method: Our study identified and employed nine sets of clones from different glycoside hydrolase bacterial strains for major ginsenoside bioconversion. Among these nine clones, only BglFc was selected based on its strong biotransformation ability and capacity to generate complete minor ginsenosides. bglFc was cloned and expressed in Escherichia coli using the pGEX-4T-1 vector system, and the recombinant enzyme was used for efficiently producing minor ginsenosides. Results: Recombinant BglFc is 2,394 bp and 798 amino acid residues long, with a predicted molecular mass of 78.8 kDa. BglFc belongs to the glycoside hydrolase family 3, and demonstrates a promising ability to convert major ginsenosides into minor ones. The Km and Vmax values of pNPG were 0.81 ± 0.06 and 4.0 ± 0.2 mM·min−1·mg−1 of protein, respectively. Under optimal conditions (37°C, pH 7.0), the ginsenoside transformation pathways for BglFc were as follows: Rb1→Rd→Rg3(S)→Rh2(S); GypXVII→GypLXXV→C-K; GypLXXV→C-K; F2→C-K; Rb2→C-O→C-Y; Rb3→C-Mx1→C-Mx; Rc→C-Mcl→C-Mc; Re→Rg2(S); and Rgl→Rhl(S), respectively. Conclusion: These results suggest that recombinant BglFc demonstrates a strong transformation activity for both PPD- and PPT-type major ginsenosides. Therefore, we conclude that BglFc can be used for gram unit production of various minor ginsenosides. Significance and Impact of Study: Previously, researchers have used a combination of enzymes for the production of minor ginsenosides. However, in this study, we found a favorable enzyme that can be used alone for the production of a different type of minor ginsenoside using the proposed conversion pathway.

Inhibition of human carboxylesterases by ginsenosides: structure\u2013activity relationships and inhibitory mechanism

BackgroundHuman carboxylesterases (hCES) are key serine hydrolases responsible for the hydrolysis of a wide range of endogenous and xenobiotic esters. Although it has been reported that some ginsenosides can modulate the activities of various enzymes, the inhibitory effects of ginsenosides on hCES have not been well-investigated.MethodsIn this study, more than 20 ginsenosides were collected and their inhibitory effects on hCES1A and hCES2A were assayed using the highly specific fluorescent probe substrates for each isoenzyme. Molecular docking simulations were also performed to investigate the interactions between ginsenosides and hCES.ResultsAmong all tested ginsenosides, Dammarenediol II (DM) and 20S-O-β-(d-glucosyl)-dammarenediol II (DMG) displayed potent inhibition against both hCES1A and hCES2A, while protopanaxadiol (PPD) and protopanaxatriol (PPT) exhibited strong inhibition on hCES2A and high selectivity over hCES1A. Introduction of O-glycosyl groups at the core skeleton decreased hCES inhibition activity, while the hydroxyl groups at different sites might also effect hCES inhibition. Inhibition kinetic analyses demonstrated that DM and DMG functioned as competitive inhibitors against hCES1A-mediated d-luciferin methyl ester (DME) hydrolysis. In contrast, DM, DMG, PPD and PPT inhibit hCES2A-mediated fluorescein diacetate (FD) hydrolysis via a mixed manner.ConclusionThe structure–inhibition relationships of ginsenosides as hCES inhibitors was investigated for the first time. Our results revealed that DM and DMG were potent inhibitors against both hCES1A and hCES2A, while PPD and PPT were selective and strong inhibitors against hCES2A.

Silicon confers protective effect against ginseng root rot by regulating sugar efflux into apoplast

Root rot caused by Ilyonectria mors-panacis is a devastating fungal disease leading to defect in root quality and causes reduced yield during the perennial life cycle of Panax ginseng Meyer. This indicates the imperative need to understand the molecular basis of disease development and also to enhance tolerance against the fungus. With this idea, the protective effect of silicon (supplied as silica nanoparticles) in P. ginseng root rot pathosystem and its molecular mechanism was investigated in the current study. We have tested different concentrations of silicon (Si) to disease-infected ginseng and found that long term analysis (30 dpi) displayed a striking 50% reduction in disease severity index upon the treatment of Si. Expectedly, Si had no direct degradative effect against the pathogen. Instead, in infected roots it resulted in reduced expression of PgSWEET leading to regulated sugar efflux into apoplast and enhanced tolerance against I. mors-panacis. In addition, under diseased condition, both protopanaxadiol (PPD) and protopanaxatriol (PPT) type ginsenoside profile in roots were higher in Si treated plants. This is the first report indicating the protective role of Si in ginseng-root rot pathosystem, thereby uncovering novel features of ginseng mineral physiology and at the same time, enabling its usage to overcome root rot.

Detection of 13 Ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh2, F1, Compound K, 20(S)-Protopanaxadiol, and 20(S)-Protopanaxatriol) in Human Plasma and Application of the Analytical Method to Human Pharmacokinetic Studies Following Two Week-Repeated Administration of Red Ginseng Extract.

We aimed to develop a sensitive method for detecting 13 ginsenosides using liquid chromatography–tandem mass spectrometry and to apply this method to pharmacokinetic studies in human following repeated oral administration of red ginseng extract. The chromatograms of Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh2, F1, compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT) in human plasma were well separated. The calibration curve range for 13 ginsenosides was 0.5–200 ng/mL and the lower limit of quantitation was 0.5 ng/mL for all ginsenosides. The inter- and intra-day accuracy, precision, and stability were less than 15%. Among the 13 ginsenosides tested, nine ginsenosides (Rb1, Rb2, Rc, Rd, Rg3, CK, Rh2, PPD, and PPT) were detected in the human plasma samples. The plasma concentrations of Rb1, Rb2, Rc, Rd, and Rg3 were correlated with the content in red ginseng extract; however, CK, Rh2, PPD, and PPT were detected although they are not present in red ginseng extract, suggesting the formation of these ginsenosides through the human metabolism. In conclusion, our analytical method could be effectively used to evaluate pharmacokinetic properties of ginsenosides, which would be useful for establishing the pharmacokinetic–pharmacodymic relationship of ginsenosides as well as ginsenoside metabolism in humans.

A review of Ginseng species in different regions as a multipurpose herb in traditional Chinese medicine, modern herbology and pharmacological science

Ginseng is the most famous of the Chinese herbs throughout the world, and has been one of the most valued herb in China. Traditional Chinese medicine as an essential element of alternative and complementary medicine, advanced over thousands of years with its own distinctive arrangement of therapies, diagnostics theories and in Asian countries, particularly China. In most parts of the world, especially western countries, Ginseng has been largely employed in recent decades and has become renowned for its important function in treating and preventing so many diseases. Panax ginseng consisted of a number of active constituents, like ginsenosides, nitrogenous substances, carbohydrates, phytosterol, organic acids, essential oils, amino acids, peptidoglycans, it's repeated, nitrogen-containing compounds, fatty acids, vitamins, minerals and other phenolic compounds. Ginsenosides are classified into two main groups known as protopanaxadiol (PPD) and protopanaxatriol (PPT). Pharmacological activities of ginseng extracts are effects on the central nervous system, antipsychotic action, tranquilizing effects, protection from stress ulcers, increase of gastrointestinal motility, anti-fatigue action, endocrinological effects, enhancement of sexual behaviour, acceleration of metabolism, or synthesis of carbohydrates, lipids, RNA, and proteins. More clinical studies are necessary to uncover the numerous substances and their effects in ginseng that contribute to public health. Key words: Ginseng, traditional Chinese medicine, herbology, pharmacological science.

The ginsenoside PPD exerts anti-endometriosis effects by suppressing estrogen receptor-mediated inhibition of endometrial stromal cell autophagy and NK cell cytotoxicity

Endometriosis (EMS) is an estrogen-dependent gynecological disease with a low autophagy level of ectopic endometrial stromal cells (eESCs). Impaired NK cell cytotoxic activity is involved in the clearance obstruction of the ectopic endometrial tissue in the abdominopelvic cavity. Protopanaxadiol (PPD) and protopanaxatriol (PPT) are two metabolites of ginsenosides, which have profound biological functions, such as anti-cancer activities. However, the role and mechanism of ginsenosides and metabolites in endometriosis are completely unknown. Here, we found that the compounds PPD, PPT, ginsenoside-Rg3 (G-Rg3), ginsenoside-Rh2 (G-Rh2), and esculentoside A (EsA) led to significant decreases in the viability of eESCs, particularly PPD (IC50 = 30.64 µM). In vitro and in vivo experiments showed that PPD promoted the expression of progesterone receptor (PR) and downregulated the expression of estrogen receptor α (ERα) in eESCs. Treatment with PPD obviously induced the autophagy of eESCs and reversed the inhibitory effect of estrogen on eESC autophagy. In addition, eESCs pretreated with PPD enhanced the cytotoxic activity of NK cells in response to eESCs. PPD decreased the numbers and suppressed the growth of ectopic lesions in a mouse EMS model. These results suggest that PPD plays a role in anti-EMS activation, possibly by restricting estrogen-mediated autophagy regulation and enhancing the cytotoxicity of NK cells. This result provides a scientific basis for potential therapeutic strategies to treat EMS by PPD or further structural modification.

Therapeutic Potential of Ginsenosides as an Adjuvant Treatment for Diabetes.

Ginseng, one of the oldest traditional Chinese medicinal herbs, has been used widely in China and Asia for thousands of years. Ginsenosides extracted from ginseng, which is derived from the roots and rhizomes of Panax ginseng C. A. Meyer, have been used in China as an adjuvant in the treatment of diabetes mellitus. Owing to the technical complexity of ginsenoside production, the total ginsenosides are generally extracted. Accumulating evidence has shown that ginsenosides exert antidiabetic effects. In vivo and in vitro tests revealed the potential of ginsenoside Rg1, Rg3, Rg5, Rb1, Rb2, Rb3, compound K, Rk1, Re, ginseng total saponins, malonyl ginsenosides, Rd, Rh2, F2, protopanaxadiol (PPD) and protopanaxatriol (PPT)-type saponins to treat diabetes and its complications, including type 1 diabetes mellitus, type 2 diabetes mellitus, diabetic nephropathy, diabetic cognitive dysfunction, type 2 diabetes mellitus with fatty liver disease, diabetic cerebral infarction, diabetic cardiomyopathy, and diabetic erectile dysfunction. Many effects are attributed to ginsenosides, including gluconeogenesis reduction, improvement of insulin resistance, glucose transport, insulinotropic action, islet cell protection, hepatoprotective activity, anti-inflammatory effect, myocardial protection, lipid regulation, improvement of glucose tolerance, antioxidation, improvement of erectile dysfunction, regulation of gut flora metabolism, neuroprotection, anti-angiopathy, anti-neurotoxic effects, immunosuppression, and renoprotection effect. The molecular targets of these effects mainly contains GLUTs, SGLT1, GLP-1, FoxO1, TNF-α, IL-6, caspase-3, bcl-2, MDA, SOD, STAT5-PPAR gamma pathway, PI3K/Akt pathway, AMPK-JNK pathway, NF-κB pathway, and endoplasmic reticulum stress. Rg1, Rg3, Rb1, and compound K demonstrated the most promising therapeutic prospects as potential adjuvant medicines for the treatment of diabetes. This paper highlights the underlying pharmacological mechanisms of the anti-diabetic effects of ginsenosides.

Protopanaxadiol and Protopanaxatriol-Type Saponins Ameliorate Glucose and Lipid Metabolism in Type 2 Diabetes Mellitus in High-Fat Diet/Streptozocin-Induced Mice

Ginsenoside is a major active component of ginseng, which exhibits various pharmacological properties such as hepatoprotection, tumor suppression and diabetes resistance. In this study, the anti-diabetic effects of protopanaxadiol (PPD) and protopanaxatriol (PPT)-type saponins were explored and compared in high-fat diet/streptozocin-induced type 2 diabetes mellitus (T2DM) mice. Our results showed that low or high dose (50 mg/kg bodyweight or 150 mg/kg bodyweight) PPD and PPT significantly reduced fasting blood glucose, improved glucose tolerance and insulin resistance in T2DM mice. PPD and PPT also regulated serum lipid-related markers such as reduced total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol in T2DM mice. In addition, PPD and PPT dramatically ameliorated the inflammatory responses by suppressing the secretion of pro-inflammatory cytokines like tumor necrosis factor-alpha and interleukin-6 in serum level and gene expression in liver level, and improved the antioxidant capacity by increasing the superoxide dismutase and decreasing malondialdehyde levels in the serum of T2DM mice. Moreover, the anti-diabetic effect of PPD and PPT appeared to be partially mediated by the suppression of hepatic metabolism genes expression such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase, as well as facilitating lipid metabolism genes expression such as microsomal TG transfer protein in the liver tissues of T2DM mice. Taken together, our results indicated that PPD and PPT might potentially act as natural anti-diabetic compounds to be used for preventing and treating the T2DM and its complications in the future.

Optimal Conditions for Extracting the Ginsenosides Rg3, Rg5, and Rk1 from Black Ginseng

Ginseng is known to contain numerous ginsenosides, which exhibit beneficial health effects. In particular, ginsenoside Rg3, Rg5, and Rk1, derived from high temperature-processed ginseng, have higher bioactivities compared to other types of ginsenosides. In this study, we determined the optimal conditions for extraction of the valuable ginsenosides Rg3, Rg5, and Rk1 from black ginseng. Our results revealed that black ginseng extracted with 100°C distilled water for 0.5 hours and 6 hours yields large amounts of Rg3, Rg5, and Rk1 compared with other extraction times. We also found that protopanaxadiol (PPD) and protopanaxatriol (PPT) were also efficiently extracted from black ginseng with 100 °C distilled water at 0.5 hours and 6 hours and then rapidly degraded in a time-dependent manner. Therefore, we posit that short extraction times such as 0.5 hours and 6 hours might be optimal for isolation of the ginsenosides Rg3, Rg5, and Rk1 from black ginseng as opposed to longer extraction periods.

Microwave Heating Rapidly Alters Ginsenoside Composition of Fresh Ginseng ( Panax ginseng CA Meyer)

Heating and drying is an important process to improve the preservation of herbs and its bioactive constituents. Ginseng is most popular medical herbs worldwide. Red ginseng is one of the products, which is prepared by heating and drying fresh ginseng. Red ginseng have ginsensides Rg3, Rg3, and Rk1 which is known for beneficial effect on inflammation, cancer, and obesity compared with other types of ginseng product. However, it is required to spend for long time to produce red ginseng with traditional preparation method. Thus, we aimed to develop a convenient ginseng process method that increases the unique form of bioactive ginsenoside in fresh ginseng. Microwave heating was used as a source of heating energy to dry and heating of fresh ginseng. Our result showed that fresh ginseng treated with microwave heating for 40 min efficiently increased the both of protopanaxadiol (PPD) and protopanaxatriol (PPT) in ginseng. In particular, red ginseng specific-detected ginsenosides Rg3, Rg5, and Rk1 were found in ginseng treated with microwave heating for 40 min compared with other periods of microwave heating condition. In addition, the appearance quality of ginseng after heating and drying affect the market value of ginseng. We observed that fresh ginseng treated with microwave heating for 40 min increased the composition of ginsenoside with proper color change of ginseng. Thus, microwave heating not only can alter the composition of ginsenosides in the short time, but also can improve the appearance of ginseng after heating and drying the processes. Moreover, our method might bring a benefit to produce high quantity of bioactive ginsenoside with a convenient process in ginseng-associated food industry.

Alteration of Panax ginseng saponin composition by overexpression and RNA interference of the protopanaxadiol 6-hydroxylase gene (CYP716A53v2)

BackgroundThe roots of Panax ginseng contain noble tetracyclic triterpenoid saponins derived from dammarenediol-II. Dammarene-type ginsenosides are classified into the protopanaxadiol (PPD) and protopanaxatriol (PPT) groups based on their triterpene aglycone structures. Two cytochrome P450 (CYP) genes (CYP716A47 and CYP716A53v2) are critical for the production of PPD and PPT aglycones, respectively. CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes PPT production from PPD in P. ginseng. MethodsWe constructed transgenic P. ginseng lines overexpressing or silencing (via RNA interference) the CYP716A53v2 gene and analyzed changes in their ginsenoside profiles. ResultOverexpression of CYP716A53v2 led to increased accumulation of CYP716A53v2 mRNA in all transgenic roots compared to nontransgenic roots. Conversely, silencing of CYP716A53v2 mRNA in RNAi transgenic roots resulted in reduced CYP716A53v2 transcription. HPLC analysis revealed that transgenic roots overexpressing CYP716A53v2 contained higher levels of PPT-group ginsenosides (Rg1, Re, and Rf) but lower levels of PPD-group ginsenosides (Rb1, Rc, Rb2, and Rd). By contrast, RNAi transgenic roots contained lower levels of PPT-group compounds and higher levels of PPD-group compounds. ConclusionThe production of PPD- and PPT-group ginsenosides can be altered by changing the expression of CYP716A53v2 in transgenic P. ginseng. The biological activities of PPD-group ginsenosides are known to differ from those of the PPT group. Thus, increasing or decreasing the levels of PPT-group ginsenosides in transgenic P. ginseng may yield new medicinal uses for transgenic P. ginseng.

Two ginseng UDP-glycosyltransferases synthesize ginsenoside Rg3 and Rd.

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.

Effects of Protopanaxatriol-Ginsenoside Metabolites on RatN-Methyl-D-Aspartic Acid Receptor-Mediated Ion Currents

Ginsenosides are low molecular weight glycosides found in ginseng that exhibit neuroprotective effects through inhibition of N-methyl-D-aspartic acid (NMDA) receptor channel activity. Ginsenosides, like other natural compounds, are metabolized by gastric juices and intestinal microorganisms to produce ginsenoside metabolites. However, little is known about how ginsenoside metabolites regulate NMDA receptor channel activity. In the present study, we investigated the effects of ginsenoside metabolites, such as compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT), on oocytes that heterologously express the rat NMDA receptor. NMDA receptor-mediated ion current (INMDA) was measured using the 2-electrode voltage clamp technique. In oocytes injected with cRNAs encoding NMDA receptor subunits, PPT, but not CK or PPD, reversibly inhibited INMDA in a concentration-dependent manner. The IC50 for PPT on INMDA was 48.1±4.6 µM, was non-competitive with NMDA, and was independent of the membrane holding potential. These results demonstrate the possibility that PPT interacts with the NMDA receptor, although not at the NMDA binding site, and that the inhibitory effects of PPT on INMDA could be related to ginseng-mediated neuroprotection.