Protopanaxadiol-type Ginsenosides Research Articles

Effects of schisandra lignans on the absorption of protopanaxadiol-type ginsenosides mediated by P-glycoprotein and protopanaxatriol-type ginsenosides mediated by CYP3A4

Ethnopharmacological relevanceGinseng Radix et Rhizoma (GRR) and Schisandrae Chinensis Fructus (SCF) are frequently used as herb pairs in traditional herbal formulas especially for the synergetic beneficial effects on lung and heart. Shengmai-yin (SMY), a noted formula, was first published in the traditional Chinese medicine classic named Yixue Qiyuan written by Zhang Yuansu in the Jin Dynasty, and has been used for deficiency of both qi and yin, palpitation, shortness of breath and spontaneous sweating. In SMY, GRR, a sovereign herb, plays an essential role in tonifying lung and supplementing qi, and SCF as an adjuvant herb contributes to the effects of nourishing yin and promoting fluid production, both of which are traditionally used as invigorants in China, Korea, Japan, and Russia. However, the underlying compatibility mechanism of GRR-SCF has remained unknown. Aim of the studyIn order to explore the impact and underlying mechanism of schisandra chinensis extract (SCE) on the absorption of ginsenosides Rb1, Rc, Rb2 and Rd belonging to protopanaxdiol (PPD)-type and ginsenosides Rg1 and Re belonging to protopanaxtriol (PPT)-type, pharmacokinetic studies, molecular docking technique and single-pass intestinal perfusion (SPIP) experiment were conducted. Material and methodsPreliminarily, pharmacokinetic characteristics of ginseng extract (GE) in the presence and absence of SCE were studied. Thereafter, molecular docking was used to predict whether ginsenosides were P-glycoprotein (P-gp) or cytochrome P450 isoenzyme 3A4 (CYP3A4) substrates. Finally, the effects and underlying mechanism of SCE on the absorption of GE were further investigated by in situ SPIP experiment. ResultsOur findings indicated that SCE could increase exposure in vivo and the intestinal absorption of distinct ginsenosides. Additionally, we found that the PPD-type ginsenosides Rb1, Rc, Rb2, and Rd were substrates for P-gp, and the PPT-type ginsenosides Rg1 and Re were substrates for CYP3A4 rather than P-gp. SCE, which has been found with extensive inhibitory effects on P-gp and CYP3A4, could remarkably promote the intestinal absorption of ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd, obtaining similar effects comparable with ketoconazole known as a classic dual inhibitor of P-gp and CYP3A4. ConclusionsThe study demonstrated that SCE could improve the absorption of GE, and revealed the underlying compatibility mechanism of GRR and SCF from the perspective of P-gp and CYP3A4-mediated interactions to some extent, which provided a certain scientific reference for the compatibility and clinical practice of GRR-SCF as common herb pairs in traditional prescriptions such as SMY. Moreover, this study also furnished a strategy for improving the oral bioavailability of different types of ginsenosides by drug combinations.

Regulation of Nur77-TLR4/MyD88 signaling pathway is required for Ginsenoside Rc ameliorates hepatic fibrosis regression by deactivating hepatic stellate cells

HSCs (hepatic stellate cells) contribute to the excessive extracellular matrix (ECM) deposition plays a key role in the progression of hepatic fibrosis. The present study focused on the hepatoprotective effect of Ginsenoside Rc (Rc), one of the protopanaxadiol type ginsenoside, which has contributed to reverse activated HSCs to improve hepatic fibrosis via regulating Nur77-TLR4/MyD88 signaling pathway. We established the hepatic fibrosis model by intraperitoneal injection of carbon tetrachloride (CCl4). And HSCs were stimulated with TGF-β, followed by silencing of Nur77, and then incubated in Rc. Rc significantly alleviated histopathological changes, reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Rc could upregulate the Nur77 and downregulate fibrosis markers in the liver of mice, including decreasing the expressions of α-SMA, Collagen-I, the ratio of TIMP-1/MMP-13. Rc significantly increased the expression of Nur77 and suppressed the production of ECM in HSCs. Rc inhibited TLR4 signaling pathway, consequently reversing the inflammatory response, including the production of MyD88, IRAK1, IRAK4 and IL-23. When Nur77 was knocked in TGF-β-stimulated HSCs, TLR4 and α-SMA production were increased. Rc suppressed these activatory effects in Nur77 knockdown HSCs. Rc reduced inflammatory reaction by regulating the Nur77-TLR4 signaling pathway while suppressing the fibrogenesis suggesting, underscoring a promising approach of Rc for the treatment in hepatic fibrosis. Targeting Nur77-TLR4 signaling in HSCs would be the potential strategy for Rc against hepatic fibrosis.

Protopanaxadiol-type ginsenoside hydrolases and their application in the preparation of ginsenoside Compound K: a review

Ginsenoside Compound K (CK) has anti-cancer and anti-inflammatory pharmacological activities. It has not been isolated from natural ginseng and is mainly prepared by deglycosylation of protopanaxadiol. Compared with the traditional physicochemical preparation methods, the preparation of CK by hydrolysis with protopanaxadiol-type (PPD-type) ginsenoside hydrolases has the advantages of high specificity, environmental-friendliness, high efficiency and high stability. In this review, the PPD-type ginsenoside hydrolases were classified into three categories based on the differences in the glycosyl-linked carbon atoms of the hydrolase action. It was found that most of the hydrolases that could prepare CK were PPD-type ginsenoside hydrolase type Ⅲ. In addition, the applications of hydrolases in the preparation of CK were summarized and evaluated to facilitate large-scale preparation of CK and its development in the food and pharmaceutical industries.

Effect of far-red light on the production and diversity of ginsenosides in leaves of Panax ginseng Meyer

Ginsenosides are the most valuable and pharmacologically active triterpenoid saponins found in Panax ginseng. Although light quality affects ginsenoside content, little is known about the underlying genetic and regulatory mechanisms. Additionally, the correlation between the adaptability of ginseng to shade and ginsenoside biosynthesis remains poorly understood. In the present study, transcriptome analysis of ginseng seedlings using RNA sequencing revealed that the expression of ginsenoside biosynthesis genes, including PgHMGR, PgFPS, PgSS, and PgUGT, was enhanced in shade conditions but downregulated by red light, indicating that far-red light might play an essential role in ginsenoside production. Further, gene expression analysis in adventitious roots and 2-year-old plants using qRT-PCR showed that the light quality-mediated expression patterns of ginsenoside genes varied with tissue and age. However, unlike the transcriptome, there was no difference in the total ginsenoside content in seedlings among various light conditions. Nevertheless, the amount of major protopanaxadiol-type ginsenosides increased under shade and red light conditions. Unlike seedlings and adventitious roots, there was a decrease in the expression of PgHMGR, PgFPS, PgSS, and PgDDS in 2-year-old plants, along with an increase in the ginsenoside content, under far-red light. Taken together, our findings suggest that far-red light is an important environmental factor for ginsenoside biosynthesis and diversification and provide information that can improve the quality of ginseng produced for medicinal purposes.

Comprehensive Identification of Ginsenosides in the Roots and Rhizomes of Panax ginseng Based on Their Molecular Features-Oriented Precursor Ions Selection and Targeted MS/MS Analysis.

Panax ginseng is widely used in Asian countries and its active constituents-ginsenosides-need to be systematically studied. However, only a small part of ginsenosides have been characterized in the roots and rhizomes of panax ginseng (RRPG) up to date, mainly because of a lack of the fragmentation ions of many more ginsenosides. In order to comprehensively identify ginsenosides in RRPG, molecular features of ginsenosides orienting precursor ions selection and targeted tandem mass spectrometry (MS/MS) analysis strategy were proposed in our study, in which the precursor ions were selected according to the molecular features of ginsenosides irrespective of their peak abundances, and targeted MS/MS analysis was then performed to obtain their fragmentation ions for substance characterization. Using this strategy, a total of 620 ginsenosides were successfully characterized in RRPG, including 309 protopanaxadiol-type ginsenosides, 258 protopanaxatriol-type ginsenosides and 53 oleanane-type ginsenosides. It is worth noting that, except for the known aglycones mass-to-charge ratio (m/z) 459, 475 and 455, twelve other aglycones, including m/z 509, 507, 493, 491, 489, 487, 477, 473, 461, 457, 443 and 441, were first reported in our experiment and they were probably the derivatizations of the protopanaxatriol and protopanaxadiol. Our study will not only help people to improve the cognition of ginsenosides in RRPG, but will also play a guiding and reference role for the isolation and characterization of potentially new ginsenosides from RRPG.

Production of ginsenoside compound K by microbial cell factory using synthetic biology-based strategy: a review

Ginsenoside compound K (CK) is a major intestinal bacterial metabolite of the protopanaxadiol-type ginsenoside family that can be absorbed in the systemic circulation. CK possesses diverse and important pharmacological properties. The low production and high cost of traditional manufacturing methods based on the extraction and biotransformation of total ginsenosides from ginseng have limited their medical application. However, considerable progress has been made in the area of de novo CK production via microbial cell factories using synthetic biology-based strategies. By introducing key enzymes responsible for CK biosynthesis into microbial cells, CK was produced via a series of in vivo enzymatic reactions that utilize the inherent precursors in microbial cells. After systematic optimization using various metabolic engineering strategies, the yield of CK increased significantly and exceeded the traditional plant extraction-biotransformation method, implying the commercial feasibility of this approach. This review summarizes recent novel advancements in the production of CK using microbial cell factories.

Anti-cancer effects of ginsenoside CK on acute myeloid leukemia in vitro and in vivo

ObjectivesAcute myeloid leukemia (AML) is a malignant disease characterized by clonal proliferation of myeloid cells, and its treatment continues to be a challenge due to high morbidity and mortality. Ginsenoside compound K, a major active metabolite of the protopanaxadiol-type ginsenosides, exhibits biological activities in various cancer cells and animal models. Here, we investigated the role of CK in anticancer potential in AML both in vitro and in vivo. Materials and methodsTo investigate the inhibitory effects of CK in AML cells, in vitro experiments, including cell viability assays, colony forming assays, and cell cycle and apoptosis assays were performed. AML animal experiment was established and quantitative analysis of lung tumor growth nodules and spleen weight and H&E staining were carried out to further determine the effects of CK on AML. In addition, the potential key genes induced and influenced by CK during treatment was identification by RNA-seq and qRT-PCR. ResultsCK suppressed AML cell activity and induced apoptosis and G1 cell cycle arrest based on the experiment results. Moreover, significantly down-regulated expression genes of BCL2, KIT, DNMT3A, MYC and CSF-1 and up-regulated expression gene of TET2 in CK treatment AML cells were discovered. ConclusionOur results demonstrated that CK could be used as an anti-AML drug with significant therapeutic efficacy and good biosafety.

Specific and efficient hydrolysis of all outer glucosyls in protopanaxadiol type and protopanaxatriol type ginsenosides by a β-glucosidase from Thermoclostridium stercorarium

To develop a new method for enzymatic preparation of minor ginsenosides, T. stercorarium β-glucosidase (Tsbgl) was characterized and its activities of deglycosylation towards natural ginsenosides were examined. The substrates of 1 mmol l−1 were incubated with the enzyme of 38.3 U ml−1 at 65 ℃ and pH 5.0. The Km values of Tsbgl for ginsenosides Rb1, Rg1 and pNPG were 0.37 ± 0.03, 3.26 ± 0.19, and 1.24 ± 0.03 mmol l−1, and the Vmax values were 183.63 ± 7.15, 85.03 ± 4.90, and 117.66 ± 1.96 μmol mg−1 min−1, respectively. The molar conversion of ginsenosides Rb1, Rb2, Rb3, Rc, Re, Rg1, and Rf by Tsbgl within 6 h was 100%, 50.1%, 42.7%, 92.0%, 57.3%, 67.9%, and 76.8%, respectively. The yield of aglycone protopanaxadiol was 35.5 μmol l−1 h−1 for Rb1, while the yields of aglycone protopanaxatriol were 64.2 and 70.4 μmol l−1 h−1 for Rg1 and Rf. Tsbgl with good organic solvent tolerance, mild reaction conditions and broad substrate specificity, could completely remove all outer glucosyls at the C-3 and C-20 hydroxyls of protopanaxadiol-type ginsenosides, and the C-6 and C-20 hydroxyls of protopanaxatriol-type ginsenosides through various pathways, providing a specific and efficient way to produce minor ginsenosides.

Identification and chemical profiling of anti-alcoholic liver disease biomarkers of ginseng Huang jiu using UPLC-Q-Orbitrap-HRMS and network pharmacology-based analyses

This study investigated the mechanism of characteristic non-volatile organic compounds (NVOCs) from ginseng Huang jiu (GH) in the treatment of alcoholic liver disease through UPLC-Q-Orbitrap-HRMS and network pharmacological analyses. Changes in NVOC contents in ginseng Huang jiu and ginseng-soaked wine fermented by different processing technologies were analyzed through liquid chromatography–mass spectrometry (LC-MS). A total of 96 ginsenosides were identified in ginseng Huang jiu throughout the fermentation process, which included 37 protopanaxadiol-type ginsenosides, 47 protopanaxatriol-type ginsenosides, and 4 oleanolic acid-type ginsenosides. Orthogonal partial least squares-discriminant analysis (OPLS-DA) revealed that 20(R)-Rg2, Gypenoside XVII, 20(S)-Rf3, CK, Rg5, Rh2, and other rare ginsenosides in ginseng Huang jiu could be the potential index for determining ginseng Huang jiu. In addition, ginseng Huang jiu could improve alcoholic liver disease by regulating the GSTP1, HRAS, AKR1B1, GSTA1, Androgen receptor (AR), GSR, and LDHB genes through bioinformatics analysis. This study provides new insights into improving the industrial production of ginseng Huang jiu and treating alcoholic liver disease with medicinal and food products.

Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene PAL promotes ginsenoside Rg3 accumulation in ginseng plant chassis.

The ginsenoside Rg3 found in Panax species has extensive pharmacological properties, in particular anti-cancer effects. However, its natural yield in Panax plants is limited. Here, we report a multi-modular strategy to improve yields of Rg3 in a Panax ginseng chassis, combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene, phenylalanine ammonia lyase (PAL). We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2, a glycosyltransferase that directly catalyzes the biosynthesis of Rg3 from Rh2 . Next, we used clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg3 . Overexpression of PAL accelerated the formation of the xylem structure, significantly improving ginsenoside Rg3 accumulation (to 6.19-fold higher than in the control). We combined overexpression of the ginsenoside aglycon synthetic genes squalene epoxidase, Pq3-O-UGT2, and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rg3 accumulation. Finally, we produced ginsenoside Rg3 at a yield of 83.6 mg/L in a shake flask (7.0 mg/g dry weight, 21.12-fold higher than with wild-type cultures). The high-production system established in this study could be a potential platform to produce the ginsenoside Rg3 commercially for pharmaceutical use.

Prevention Effect of Protopanaxadiol-Type Saponins Saponins and Protopanaxatriol-Type Saponins on Myelosuppression Mice Induced by Cyclophosphamide.

Ginsenosides from ginseng are used as a therapeutic agent for various diseases. They enhance the immunomodulatory effect in cyclophosphamide (CP)-treated tumor disease. The structural characteristics of steroidal saponins are mainly divided into protopanaxadiol-type saponin (PDS) and protopanaxatriol-type saponin (PTS). At present, few researchers have studied which kind of saponin plays a more important role, thus, we compared the prevention effect of PDS and PTS on myelosuppression mice induced by CP. The components and contents of saponin and monosaccharide were analyzed by using ultra high performance liquid chromatography-charged aerosol detector (UPLC-CAD) and reversed phase-high performance liquid chromatography (RP-HPLC), respectively. Thirty-two mice were randomly divided into four groups, including control, model (CP), CP+PDS, and CP+PTS. The mice were orally administered with PDS or PTS for 28 days and then injected with CP saline solution on 25, 26, 27, and 28 days at a dose of 50 mg × kg−1. After the end of modeling, the whole blood of mice from the ophthalmic venous plexus was collected to detect routine blood tests, inflammatory cytokines, and hematopoiesis-related cytokines. Cell cycle and the apoptosis of bone marrow in the right femur were detected. The spleen and thymus were used to calculate the organ index and histological examination, and splenocytes were used to detect the percentage of CD4+ and CD25+ T cells. In the saponins analysis, PDS mainly included the Rb1, Rc, Rb2, and Rd of protopanaxadiol-type ginsenosides (accounted for 91.64%), and PTS mainly included the Re, Rg1, and Rf of protopanaxatriol-type ginsenosides (accounted for 75.46%). The animal results showed that both PDS and PTS improved the most indicators of myelosuppression mice induced by CP, including increased weight, blood cell numbers, hematopoiesis-related cytokines, and inflammatory cytokines; promoted the cell cycle of bone marrow and inhibited the apoptosis of bone marrow; elevated the spleen and thymus indexes and CD4+ count of splenocytes. The prevention effect of PDS was better than PTS in some indicators, such as red blood cells, hemoglobin, interleukin (IL)-1β, IL-4, IL-10, tumor necrosis factor-α, CD4+, and thymus index. These results suggest both PDS and PTS can prevent myelosuppression of mice induced by CP. Meanwhile, PDS and its metabolite showed higher bioavailability and bioactivity compared with PTS.

Predominance of oleanane-type ginsenoside R0 and malonyl esters of protopanaxadiol-type ginsenosides in the 20-year-old suspension cell culture of Panax japonicus C.A. Meyer

Cell culture of Panax spp. is a potent sustainable source of biologically active triterpene glycosides (ginsenosides) specific for this genus. In this study, growth and biosynthetic profiles were investigated for P. japonicus suspension cell culture maintained for over 20 years by periodic subcultures in flasks. Cell culture demonstrated intensive growth evidenced by high accumulation of dry weight (DW, 9.8 gL−1), viability (80–90%) and specific growth rate (0.12 day−1) that were comparable to those recorded at the year of culture induction. Nineteen structurally different ginsenosides were identified in cell biomass using UPLC-Q-Exactive-Orbitrap HRMS and HPLC-ESI-Q-MS. Ginsenoside R0 and malonyl-ginsenosides (MalGs) accounted for over 80% of the total ginsenoside content in cells during the subculture cycle while the sum of neutral ginsenosides of protopanaxatriol (PPT: Re, Rg1, Rf) and protopanaxadiol (PPD: Rb1, Rb2, Rc, Rd) groups constituted less than 20%. Ginsenoside contents were maximized during growth decline phase as 35.3 mg/gDW for R0, 48.7 mg/gDW for MalGs, 10.4 mg/gDW for PPD and 5.2 mg/gDW for PPT. These data confirmed that 20-year-old cell culture of P. japonicus retained the ability for intensive growth and accumulation of wide spectrum of ginsenosides which opens the door for wide application of this culture.

Enhanced biotransformation of the minor ginsenosides in red ginseng extract by Penicillium decumbens β-glucosidase

Compound K (C-K) and Rh2, which are present at low levels in ginseng and ginseng extracts, have higher intestinal absorption rates than other ginsenosides. Here, we attempted to convert ginsenoside Rb1 to C-K using a β-glucosidase from Penicillium decumbens. Ten commercially available enzymes were screened to identify enzymes that can convert ginsenoside Rb1 to C-K, resulting in the selection of a P. decumbens-derived β-glucosidase. β-Glucosidase showed maximum activity at pH 4.0 and 60 °C; its substrate specificity for ginsenoside Rb1 was investigated. The main glucoside-hydrolyzing pathways were as follows: ginsenoside Rb1 or Rd → gypenoside XVII → F2 → C-K and ginsenoside Rg3 → Rh2. The P. decumbens-derived β-glucosidase was used to generate C-K and Rh2 using protopanaxadiol-type ginsenosides as substrates. Additionally, to apply this enzyme to the commercialized red ginseng extract products, the contents of C-K and Rh2 in the total ginsenosides significantly (p < 0.05) increased up to 36-fold and 8.9-fold, respectively, higher than prior to subjecting to biotransformation. To the best of our knowledge, this is the first report of the dual biotransformation of C-K and Rh2 by a food-grade commercial enzyme.This study demonstrates that the use of a specific β-glucosidase may increase C-K and Rh2 contents in the ginseng extract through a simple biotransformation process and, thus, enhance its health benefits.

Antioxidant, Anti-Inflammatory and Antithrombotic Effects of Ginsenoside Compound K Enriched Extract Derived from Ginseng Sprouts

Partially purified ginsenoside extract (PGE) and compound K enriched extract (CKE) were prepared from ginseng sprouts, and their antioxidant, anti-inflammatory and antithrombotic effects were investigated. Compared to the 6-year-old ginseng roots, ginseng sprouts were found to have a higher content of phenolic compounds, saponin and protopanaxadiol-type ginsenoside by about 56%, 36% and 43%, respectively. PGE was prepared using a macroporous adsorption resin, and compound K(CK) was converted and enriched from the PGE by enzymatic hydrolysis with a conversion rate of 75%. PGE showed higher effects than CKE on radical scavenging activity in antioxidant assays. On the other hand, CKE reduced nitric oxide levels more effectively than PGE in RAW 264.7 cells. CKE also reduced pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β and IL-6 than PGE. Tail bleeding time and volume were investigated after administration of CKE at 70–150 mg/kg/day to mice. CKE administered group showed a significant increase or increased tendency in bleeding time than the control group. Bleeding volume in the CKE group increased than the control group, but not as much as in the aspirin group. In conclusion, ginseng sprouts could be an efficient source of ginsenoside, and CKE converted from the ginsenosides showed antioxidant, anti-inflammatory and antithrombotic effects. However, it was estimated that the CKE might play an essential role in anti-inflammatory effects rather than antioxidant effects.

20(S)-Protopanaxadiol Alleviates Myocardial Ischemia/Reperfusion Injury in Rats Through Suppression of Oxidative Stress and Apoptosis

20(S)-protopanaxadiol (PPD) is an active natural product which is transformed from protopanaxadiol-type ginsenosides. The present study was conducted to evaluate the effects of PPD on myocardial ischemia/reperfusion (I/R) injury in a rat model. PPD (20mg/kg) or positive-control drug Diltiazem (10mg/kg) was administered daily for 7 days before left anterior descending I/R operation. After 2-hour reperfusion, changes of cardiac morphology, structure, and function were evaluated by HE staining and echocardiography. Myocardial infarct size was assessed using nitroblue tetrazolium staining. The activities of cardiac enzymes in serum were also evaluated. Cardiomyocyte apoptosis was detected using the terminal dUTP nick end labelling (TUNEL) assay. The extent of oxidative stress was evaluated according to the activities of superoxide dismutase (SOD) and glutathione per oxidase (GPx) and the levels of malondialdehyde (MDA). Western blot and immunohistochemistry were used to determine the expression of apoptosis associated proteins, including Bcl-2, Bax, cleaved Caspase-3, cleaved Caspase-9, and cytochrome C. According to the results, PPD reduced I/R‑induced increases in myocardial infarct size and improved cardiac function. Furthermore, PPD decreased cardiomyocyte apoptosis on TUNEL staining, which was verified by increased Bcl-2, and decreased expression of Bax, cytochrome C, cleaved Caspase-9, and cleaved Caspase-3 in I/R rat myocardium. Additionally, PPD reduced MDA levels and increased the anti-oxidative capacity by upregulating the activities of SOD and GPx. Taken together, the results suggest that PPD serves a protective role against oxidative stress and cardiomyocyte apoptosis during myocardial ischemia/reperfusion injury.

Changes in the Leaf Physiological Characteristics and Tissue-Specific Distribution of Ginsenosides in Panax ginseng During Flowering Stage Under Cold Stress.

Panax ginseng is a valuable traditional herbal medicine material with numerous applications. Ginsenosides are the key bioactive compounds in ginseng. Cold stress can activate stress tolerance mechanisms that regulate biomass and biosynthesis in ginseng tissue. In this study, the effects of short- and long-term cold stress (5°C) on the physiological characteristics, tissue-specific ginsenoside distributions, and ginsenoside synthesis gene expressions of 3-year-old P. ginseng during the flowering period were investigated. Short-term cold stress significantly reduced ginseng biomass (root fresh weight and dry weight), and increased malondialdehyde, proline, soluble sugar, and soluble protein concentrations. Superoxide dismutase, peroxidase, and catalase activities also increased significantly under cold stress. With prolongation of the cold stress period, all antioxidant enzyme activity decreased. The protopanaxatriol-type ginsenoside concentrations in the taproots (phloem and xylem) and fibrous roots, as well as the protopanaxadiol-type ginsenoside concentrations in the leaves, increased significantly under short-term cold stress. The key genes (SE, DS-II, CYP716A52v2, and CYP716A53v2) involved in the ginsenoside biosynthesis pathway were significantly positively correlated with the ginsenoside accumulation trends. Thus, short-term cold stress can stimulate membrane lipid peroxidation, in turn stimulating the antioxidant enzyme system to alleviate oxidative damage and increasing the expression of key enzyme genes involved in ginsenoside biosynthesis. During agricultural production, protopanaxadiol/protopanaxatriol ratios could be manipulated by low-temperature storage or treatments.

Highly regioselective hydrolysis of the glycosidic bonds in ginsenosides catalyzed by snailase

Minor ginsenosides display higher physiological activities than those of major ginsenosides, but their low abundance limits their clinical application. In this study, snailase was employed to transform protopanaxadiol-type (PPD) ginsenosides, protopanaxatriol-type (PPT) ginsenosides, and Ro to minor ginsenosides. The results showed that snailase hydrolyzed the saccharide chains at the C-3 position of Rb1, Rc, Rb2 Rg3 and Ro and the saccharide chains at the C-28 position of Ro efficiently. The end glucosyl unit of the saccharide chain at the C-6 position of Rf was hydrolyzed entirely. However, the rhamnosyl of Re was intact. The end glucosyl unit of the ginsenoside Rb1 was hydrolyzed entirely, but both arabinopyranosyl of Rb2 and arabinofuranosyl of Rc were not hydrolyzed. So snailase is selective for glycoside bond locations and saccharide types of ginsenoside, and it can be used to produce Rg3, CK, Rh1 and so on.

Complete Bioconversion of Protopanaxadiol-Type Ginsenosides to Compound K by Extracellular Enzymes from the Isolated Strain Aspergillus tubingensis.

A compound K-producing fungus was isolated from meju (fermented soybean brick) and identified as the generally recognized as safe (GRAS) strain Aspergillus tubingensis. The extracellular enzymes obtained after the cultivation of 6 days in the medium with 20 g/L citrus pectin as an inducer showed the highest compound K-producing activity among the inducers tested. Under the optimized conditions of 0.05 mM MgSO4, 55 °C, pH 4.0, 13.4 mM protopanaxadiol (PPD)-type ginsenosides, and 11 mg/mL enzymes, the extracellular enzymes from A. tubingensis completely converted PPD-type ginsenosides in the ginseng extract to 13.4 mM (8.35 mg/mL) compound K after 20 h, with the highest concentration and productivity among the results reported so far. As far as we know, this is the first GRAS enzyme to completely convert all PPD-type ginsenosides to compound K.

Biotransformation of Protopanaxadiol-Type Ginsenosides in Korean Ginseng Extract into Food-Available Compound K by an Extracellular Enzyme from Aspergillus niger.

Compound K (C-K) is one of the most pharmaceutically effective ginsenosides, but it is absent in natural ginseng. However, C-K can be obtained through the hydrolysis of protopanaxadiol-type ginsenosides (PPDGs) in natural ginseng. The aim of this study was to obtain the high concentration of food-available C-K using PPDGs in Korean ginseng extract by an extracellular enzyme from Aspergillus niger KACC 46495. A. niger was cultivated in the culture medium containing the inducer carboxymethyl cellulose (CMC) for 6 days. The extracellular enzyme extracted from A. niger was prepared from the culture broth by filtration, ammonium sulfate, and dialysis. The extracellular enzyme was used for C-K production using PPDGs. The glycoside-hydrolyzing pathways for converting PPDGs into C-K by the extracellular enzyme were Rb1 → Rd → F2 → C-K, Rb2 → Rd or compound O → F2 or compound Y → C-K, and Rc → Rd or compound Mc1 → F2 or compound Mc → C-K. The extracellular enzyme from A. niger at 8.0 mg/ml, which was obtained by the induction of CMC during the cultivation, converted 6.0 mg/ml (5.6 mM) PPDGs in Korean ginseng extract into 2.8 mg/ml (4.5 mM) food-available C-K in 9 h, with a productivity of 313 mg/l/h and a molar conversion of 80%. To the best of our knowledge, the productivity and concentration of C-K of the extracellular enzyme are the highest among those by crude enzymes from wild-type microorganisms.

Advances in Saponin Diversity of Panax ginseng.

Ginsenosides are the major bioactive constituents of Panax ginseng, which have pharmacological effects. Although there are several reviews in regards to ginsenosides, new ginsenosides have been detected continually in recent years. This review updates the ginsenoside list from P. ginseng to 170 by the end of 2019, and aims to highlight the diversity of ginsenosides in multiple dimensions, including chemical structure, tissue spatial distribution, time, and isomeride. Protopanaxadiol, protopanaxatriol and C17 side-chain varied (C17SCV) manners are the major types of ginsenosides, and the constitute of ginsenosides varied significantly among different parts. Only 16 ginsenosides commonly exist in all parts of a ginseng plant. Protopanaxadiol-type ginsenoside is dominant in root, rhizome, leaf, stem, and fruit, whereas malonyl- and C17SCV-type ginsenosides occupy a greater proportion in the flower and flower bud compared with other parts. In respects of isomeride, there are 69 molecular formulas corresponding to 170 ginsenosides, and the median of isomers is 2. This is the first review on diversity of ginsenosides, providing information for reasonable utilization of whole ginseng plant, and the perspective on studying the physiological functions of ginsenoside for the ginseng plant itself is also proposed.