Protopanaxatriol-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.

Production of a Novel Protopanaxatriol-Type Ginsenoside by Yeast Cell Factories.

Ginsenosides, the main active compounds in Panax species, are glycosides of protopanaxadiol (PPD) or protopanaxatriol (PPT). PPT-type ginsenosides have unique pharmacological activities on the central nervous system and cardiovascular system. As an unnatural ginsenoside, 3,12-Di-O-β-D-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (3β,12β-Di-O-Glc-PPT) can be synthesized through enzymatic reactions but is limited by the expensive substrates and low catalytic efficiency. In the present study, we successfully produced 3β,12β-Di-O-Glc-PPT in Saccharomyces cerevisiae with a titer of 7.0 mg/L by expressing protopanaxatriol synthase (PPTS) from Panax ginseng and UGT109A1 from Bacillus subtilis in PPD-producing yeast. Then, we modified this engineered strain by replacing UGT109A1 with its mutant UGT109A1-K73A, overexpressing the cytochrome P450 reductase ATR2 from Arabidopsis thaliana and the key enzymes of UDP-glucose biosynthesis to increase the production of 3β,12β-Di-O-Glc-PPT, although these strategies did not show any positive effect on the yield of 3β,12β-Di-O-Glc-PPT. However, the unnatural ginsenoside 3β,12β-Di-O-Glc-PPT was produced in this study by constructing its biosynthetic pathway in yeast. To the best of our knowledge, this is the first report of producing 3β,12β-Di-O-Glc-PPT through yeast cell factories. Our work provides a viable route for the production of 3β,12β-Di-O-Glc-PPT, which lays a foundation for drug research and development.

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.

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.

Inhibitory Activities of Rare Ginsenoside Rg4 on Cecal Ligation and Puncture-Induced Sepsis.

Sepsis is an uncontrolled response to inflammatory infection and is associated with high levels of mortality and morbidity. Rg4 is a rare ginsenoside mainly found in the leaves of Panax ginseng C. A. Meyer and the major protopanaxatriol-type ginsenoside of black ginseng. In this study, we determined whether Rg4 affects cecal ligation and puncture (CLP)-induced sepsis. Animals were separated into the following six groups: control group, CLP-operated group, CLP plus maslinic acid (MA), and CLP plus Rg4 (5, 10, or 15 mg/kg). Survival rate, body weight changes, inflammatory cytokines, and histological analyses were assessed. Human endothelial cells were activated with the high-mobility group box 1 (HMGB1) protein and Rg4. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were used to assess inflammation and gene expression, respectively. After CLP surgery, the Rg4-administered group exhibited a higher survival rate and body weight compared with the untreated control group. Rg4 treatment reduced cytokine levels, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β, as well as nitric oxide (NO) levels and renal inflammation. After Rg4 treatment of HMGB1-activated cells, the expressions of toll-like receptor (TLR) 4 and TNF-α were decreased, and the activation of phosphoinositide 3-kinase (PI3K)/AKT signaling increased cell viability. In summary, Rg4 inhibited inflammation and exhibited a protective effect against CLP-induced sepsis, thereby reinforcing cell survival against septic responses.

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.

Highly Regioselective Biotransformation of Protopanaxadiol-type and Protopanaxatriol-type Ginsenosides in the Underground Parts of Panax notoginseng to 18 Minor Ginsenosides by Talaromyces flavus.

The transformation of major ginsenosides to minor ginsenosides by microorganisms was considered to be an environmentally friendly method. Compared with GRAS (generally recognized as safe) strains, non-food-grade microorganisms could transform polar ginsenosides to various minor ginsenosides. In this study, Talaromyces flavus screened from the P. notoginseng rhizosphere was capable of transforming PPD-type and PPT-type ginsenosides in the underground parts of P. notoginseng to 18 minor ginsenosides. The transformation reactions invovled deglycosylation, epimerization, and dehydration. To the best of our knowledge, this transformation characteristic of T. flavus was first reported in fungi. Its crude enzyme can efficiently hydrolyze the outer glucose linked to C-20 and C-3 in major ginsenosides Rb1, Rb2, Rb3, Rc, Rd, and 20(S)-Rg3 within 48 h. The transformation of major ginsenosides to minor ginsenosides by T. flavus will help raise the functional and economic value of P. notoginseng.

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.

Single site mutations of glycosyltransferase with improved activity and regioselectivity for directed biosynthesis of unnatural protopanaxatriol-type ginsenoside product

Protopanaxatriol (PPT)-type ginsenosides are functional components in Panax species and exhibit diverse significant bioactivities. Glycosyltransferase Bs-YjiC from Bacillus subtilis 168 has been demonstrated to generate ginsenoside Rh1 and several unnatural PPT-type ginsenosides. However, the glycodiversification of ginsenosides and poor regioselectivity of glycosyltransferase might hamper the low-cost synthesis process of the desired product. The semi-rational design of Bs-YjiC is newly performed by structure-based guidance, and the small change of Bs-YjiC could target both activity improvement and regioselectivity enhancement. The created mutant F168M has 5.24 times of specific activity compared with WT and the increased conversion of 95.56% for PPT, and shows significantly improved regioselectivity of 87.28% for unnatural ginsenoside of 3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol, which is 8.83 times of the wild type (9.88%). The coupled catalytic strategy of mutant F168M and sucrose synthase AtSuSy (from Arabidopsis thaliana) is used for efficient regeneration of UDP-glucose, and unnatural PPT-type ginsenoside could reach a high titer of 8.66 g/L (regioselectivity 90.42%) in a fed-batch mode, which is 99 times of that by wild type (0.088 g/L). It represents the highest level of production reported to date, and achieves a maximum number of UDP-glucose regeneration cycles (RcMax = 39.92). This study exploits an effective biocatalyst for unnatural ginsenoside, thus facilitating study and medical application.

Inhibitory activities of 20(R, S)-protopanaxatriol against epidermal growth factor receptor tyrosine kinase

As major metabolites of protopanaxatriol-type ginsenosides, 20(R, S)-protopanaxatriol [20(R, S)-PPT] display multiple bioactivities. This work aimed to investigate the inhibitory activities of 20(R, S)-PPT against epidermal growth factor receptor tyrosine kinase and the potential mechanism. 20(R, S)-PPT inhibited the proliferation of HepG2 cells in a dose-dependent manner and blocked cell cycle progression at G1/G0 phase. Then 20(R, S)-PPT were found to influence the protein expressions involved in epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) signaling pathway. Molecular docking suggested that 20(R, S)-PPT could bind to the active sites of all target proteins in EGFR-MAPK pathway. It is worth noting that 20(R, S)-PPT showed stronger binding capacities with EGFR, compared with other proteins. Hence, this work further investigated the binding interactions and binding stabilities between 20(R, S)-PPT and EGFR. Both hydrophobic interactions and hydrogen bonds contributed to the 20(R, S)-PPT-EGFR binding. In addition, the in vitro inhibitory activities of 20(R, S)-PPT against EGFR tyrosine kinase were observed in a homogeneous time-resolved fluorescence assay, with the IC50 values of 24.10 ± 0.17 and 33.19 ± 0.19 μM respectively. Taken together with the above results, both of 20(R)-PPT and 20(S)-PPT might serve as potential EGFR tyrosine kinase inhibitors.

Ginsenoside-Rg2 affects cell growth via regulating ROS-mediated AMPK activation and cell cycle in MCF-7 cells

BackgroundGinsenoside-Rg2 (G-Rg2) is a protopanaxatriol-type ginsenoside isolated from ginseng. It has been found to exhibit various pharmacological effects, including antioxidant, anti-inflammatory, and anticancer effects. PurposeThis study aimed to investigate the anticancer effects of G-Rg2 on estrogen receptor-positive MCF-7 breast cancer (BC) cells, and the underlying mechanisms involving in reactive oxygen species (ROS) production. Study design/MethodsCell viability, cell cycle distribution, apoptosis, and ROS production were measured following exposure to G-Rg2. The protein expression levels of p-ERK1/2, p-Akt, PARP, p-Rb, cyclin D1, CDK6, and p-AMPK were quantified using western blot analysis. The in vivo activity of G-Rg2 was assessed in a xenograft model. Immunohistochemistry staining for p-Rb and p-AMPK was performed in tumor tissues. ResultsG-Rg2 significantly decreased cell viability but increased cell apoptosis. In MCF-7 cells, G-Rg2 increased ROS production by inhibiting ERK1/2 and Akt activation. G-Rg2-induced ROS induced G0/G1 cell cycle arrest and AMPK phosphorylation. In the xenograft model, the 5 mg/kg G-Rg2-treated group showed decreased tumor volume and weight, similar to the 5 mg/kg 4-OHT-treated group, compared to the control group. Immunohistochemistry staining showed that G-Rg2 treatment decreased Rb phosphorylation, while increasing AMPK phosphorylation in tumor tissues. ConclusionG-Rg2 has potential anticancer effects by increasing the ROS-AMPK signaling pathway and inhibiting ERK1/2 and Akt activation-mediated cell proliferation and cell cycle progression in MCF-7 BC cells.

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.

Characterization of a Novel Ginsenoside MT1 Produced by an Enzymatic Transrhamnosylation of Protopanaxatriol-Type Ginsenosides Re.

Background: Ginsenosides, triterpene saponins of Panax species, are considered the main active ingredients responsible for various pharmacological activities. Herein, a new protopanaxatriol-type ginsenoside called “ginsenoside MT1” is described; it was accidentally found among the enzymatic conversion products of ginsenoside Re. Method: We analyzed the conversion mechanism and found that recombinant β-glucosidase (MT619) transglycosylated the outer rhamnopyranoside of Re at the C-6 position to glucopyranoside at C-20. The production of MT1 by trans-rhamnosylation was optimized and pure MT1 was obtained through various chromatographic processes. Results: The structure of MT1 was elucidated based on spectral data: (20S)-3β,6α,12β,20-tetrahydroxydammarene-20-O-[α-L-rhamnopyranosyl(1→2)-β-D-glucopyranoside]. This dammarane-type triterpene saponin was confirmed as a novel compound. Conclusion: Based on the functions of ginsenosides with similar structures, we believe that this ginsenoside MT1 may have great potential in the development of nutraceutical, pharmaceutical or cosmeceutical products.

Comparison of intramuscular and intravenous pharmacokinetics of ginsenosides in humans after dosing XueShuanTong, a lyophilized extract of Panax notoginseng roots

Ethnopharmacological relevanceMany bioactive constituents of Chinese herbal medicines have poor oral bioavailability. Besides oral administration, herbal medicines in China are also prepared for parenteral administration. Unlike for intravenous route, little is known about the intramuscular pharmacokinetics of herbal compounds. To facilitate rational use of herbal medicine, it is important to better understand such intramuscular pharmacokinetics. Aim of the studyBioactive constituents of XueShuanTong (a lyophilized extract of Panax notoginseng roots, extensively used in treatment of ischemic heart and cerebrovascular diseases) predominantly comprise ginsenosides Rb1 and Rd of 20(S)-protopanaxadiol-type and ginsenosides Rg1, and Re, and notoginsenoside R1 of 20(S)-protopanaxatriol-type; these saponins are poorly absorbed from the gastrointestinal tract. This study aimed to compare intramuscular and intravenous pharmacokinetics of these ginsenosides after dosing XueShuanTong. MethodsPharmacokinetics of ginsenosides was assessed in human volunteers receiving an intramuscular injection or 1.5-h intravenous infusion of XueShuanTong, both at 150 mg/person, and the plasma and urine samples were analyzed by liquid chromatography/mass spectrometry. ResultsLike after intravenous administration, the unchanged saponins were the major circulating forms after intramuscular administration, while their metabolites were poorly detected. These ginsenosides exhibited intramuscular bioavailability of 100%–112%, relative to the respective intravenous data. Similar to that after intravenous infusion, the 20(S)-protopanaxadiol-type ginsenosides after the intramuscular injection exhibited notably longer terminal half-lives (46–106 h) than the 20(S)-protopanaxatriol-type ginsenosides (1.1–1.4 h). ConclusionsIntramuscular route might be an effective alternative to intravenous route for XueShuanTong, from the pharmacokinetic perspective.

Formula: see text], a Rare Protopanaxatriol-Type Ginsenoside Fraction from Black Ginseng, Suppresses Inflammatory Gene iNOS via the Iinhibition of p-STAT-1 and NF-[Formula: see text]B.

Black ginseng (BG), which is ginseng that has been steamed and dried nine times, and its main protopanaxatriol-type ginsenosides Rg4, Rg6, Rh4, and Rg2 have been reported to exhibit various forms of biological activity, including antiseptic, antidiabetic, wound-healing, immune-stimulatory, and anti-oxidant activity. The aim of the this study was to examine the effects of [Formula: see text] (a rare protopanaxatriol-type ginsenoside fraction; Rg2, Rg4, Rg6, Rh1, and Rh4) on heme oxygenase-1 (HO-1) induction and on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-)2 in lipopolysaccharide (LPS)-activated human pulmonary artery endothelial cells (HPAECs). [Formula: see text] was tested to determine its effect on iNOS protein expression and inflammatory markers (interleukin [IL]-1[Formula: see text] and tumor necrosis factor [TNF]-[Formula: see text] in the lung tissue of LPS-treated mice. The results showed that [Formula: see text] induced the expression of HO-1, reduced LPS-activated NF-[Formula: see text]B-luciferase activity, and inhibited iNOS/NO and COX-2/PGE2, which contributed to the inhibition of STAT-1 phosphorylation. In particular, [Formula: see text] induced the translocation of Nrf2 from the cytosol to the nucleus by increasing Nrf2-ARE activity and decreased IL-1[Formula: see text] production in LPS-activated HPAECs. This reduction in iNOS/NO expression due to [Formula: see text] was reversed by siHO-1 RNA transfection. In LPS-treated mice, [Formula: see text] significantly reduced lung tissue iNOS protein levels and TNF-[Formula: see text] levels in the bronchoalveolar lavage fluid. In conclusion, these findings indicate that [Formula: see text] has a critical anti-inflammatory effect due to its ability to regulate iNOS via the inhibition of p-STAT-1 and NF-[Formula: see text]B, and thus it may be suitable for the treatment of inflammatory disease.

High-density immobilization of a ginsenoside-transforming \u03b2-glucosidase for enhanced food-grade production of minor ginsenosides

Use of recombinant glycosidases is a promising approach for the production of minor ginsenosides, e.g., Compound K (CK) and F1, which have potential applications in the food industry. However, application of these recombinant enzymes for food-grade preparation of minor ginsenosides are limited by the lack of suitable expression hosts and low productivity. In this study, Corynebacterium glutamicum ATCC13032, a GRAS strain that has been used extensively for the industrial-grade production of additives for foodstuffs, was employed to express a novel β-glucosidase (MT619) from Microbacterium testaceum ATCC 15829 with high ginsenoside-transforming activity. A cellulose-binding module was additionally fused to the N-terminus of MT619 for immobilization on cellulose, which is an abundant and safe material. Via one-step immobilization, the fusion protein in cell lysates was efficiently immobilized on regenerated amorphous cellulose at a high density (maximum 984 mg/g cellulose), increasing the enzyme concentration by 286-fold. The concentrated and immobilized enzyme showed strong conversion activities against protopanaxadiol- and protopanaxatriol-type ginsenosides for the production of CK and F1. Using gram-scale ginseng extracts as substrates, the immobilized enzyme produced 7.59 g/L CK and 9.42 g/L F1 in 24 h. To the best of our knowledge, these are the highest reported product concentrations of CK and F1, and this is the first time that a recombinant enzyme has been immobilized on cellulose for the preparation of minor ginsenosides. This safe, convenient, and efficient production method could also be effectively exploited in the preparation of food-processing recombinant enzymes in the pharmaceutical, functional food, and cosmetics industries.

Pulmonary Protective Functions of Rare Ginsenoside Rg4 on Particulate Matter-induced Inflammatory Responses

Inhalation of fine particulate matter (PM2.5) is associated with an increase in lung injury caused by the loss of integrity of the vascular barrier. The rare ginsenoside Rg4 is a main protopanaxatriol type ginsenoside of black ginseng (BG). The aim of this study was to investigate the beneficial effects of Rg4 on PM-induced lung endothelial cell (EC) barrier disruption and pulmonary inflammation. Permeability, leukocyte migration, activation of proinflammatory proteins, generation of reactive oxygen species (ROS), and histology were examined in PM2.5-treated EC and mice. Rg4 significantly scavenged PM2.5-induced ROS, inhibited ROS-induced activation of p38 mitogen-activated protein kinase (MAPK), activated Akt in purified pulmonary endothelial cells, which helped maintain endothelial integrity. Further, Rg4 reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in bronchoalveolar lavage fluids in PM-induced mouse lung tissues. Data suggested that Rg4 might exhibit protective effects in PM-induced inflammatory lung injury and vascular hyperpermeability.

Inhibitory effects of protopanaxatriol type ginsenoside fraction (Rgx365) on particulate matter-induced pulmonary injury

ABSTRACTInhalation of fine particulate matter (PM2.5) is associated with elevated pulmonary injury attributed to the loss of vascular barrier integrity. Black ginseng (BG), steamed 9 times and dried ginseng, and its major protopanaxatriol type ginsenosides (ginsenoside Rg4, Rg6, Rh4, Rh1, and Rg2) exhibited various biological activities including anti-septic, anti-diabetic, wound healing, immune-stimulatory, and anti-antioxidant activity. The aim of this study was to investigate the beneficial effects of Rgx365 (a protopanaxatriol type rare ginsenosides fraction) on PM-induced lung endothelial cell (EC) barrier disruption and pulmonary inflammation. Permeability, leukocyte migration, activation of proinflammatory proteins, generation of reactive oxygen species (ROS), and histology were examined in PM2.5-treated EC and mice. Rgx365 significantly scavenged PM2.5-induced ROS, inhibited ROS-induced activation of p38 mitogen-activated protein kinase (MAPK), activated Akt in purified pulmonary EC, which helped maintain endothelial integrity. Further, Rgx365 reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in bronchoalveolar lavage fluids in PM-induced mouse lung tissues. Data suggested that Rgx365 might exhibit protective effects in PM-induced inflammatory lung injury and vascular hyperpermeability.