Traits of Panax ginseng hairy roots after cold storage and cryopreservation

Summary Panax ginseng hairy root cultures were established by infecting petiole segments with Agrobacterium rhizogenes strain 15834. Hairy root segments including root tips placed onto phytohormone-free 1/2 Murashige and Skoog solid medium and stored at 4 °C in the dark for 4 months, resumed elongation when the temperature was raised to 25 °C in the dark. For cryopreservation, a vitrification method was applied. Root tips precultured with 0.1 mg/l 2,4-D for 3 days and dehydrated with PVS2 solution for 8 minutes prior to immersion into liquid nitrogen had a survival rate of 60 % and could regenerate. The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside productivity and patterns as those of the control hairy roots cultured continuously at 25 °C. The conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.Abbreviations 1/2 MS a half strength Murashige and Skoog (1962) - B5 Gamborg B5 (Gamborg et al. 1968) - WP woody plant (Lloyd and McCown 1980) - RC root culture (Thomas and Davey 1982) - RCI root culture medium containing 100 mg/l myoinositol - HF phytohormone-free - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - PCR polymerase chain reaction - PVS2 plant vitrification solution 2 (Sakai et al., 1990) - FDA fluorecein diacetate     

Research Progress on Anti-Inflammatory Mechanisms of Black Ginseng

In recent years, black ginseng, a new type of processed ginseng product, has attracted the attention of scholars globally. Ginsenoside and ginseng polysaccharide, the main active substances of black ginseng, have been shown to carry curative effects for many diseases. This article focuses on the mechanism of their action in anti-inflammatory response, which is mainly divided into three aspects: activation of immune cells to exert immune regulatory response; participation in inflammatory response-related pathways and regulation of the expression level of inflammatory factors; effect on the metabolic activity of intestinal flora. This study identifies active anti-inflammatory components and an action mechanism of black ginseng showing multi-component, multi-target, and multi-channel characteristics, providing ideas and a basis for a follow-up in-depth study of its specific mechanism.     

Ginsenoside Rg1 protects H9c2 cells against nutritional stress-induced injury via aldolase /AMPK/PINK1 signalling

Insufficient nutrients supply will greatly affect the function of cardiac myocytes. The adaptive responses of cardiac myocytes to nutritional stress are not fully known. Ginsenoside Rg1 is one of the most pharmacologically active components in Panax Ginseng and possesses protective effects on cardiomyocyte. Here, we investigate the effects of ginsenoside Rg1 on H9c2 cells which were subjected to nutritional stress. Nutritional stress-induced by glucose deprivation strongly induced cell death and this response was inhibited by ginsenoside Rg1. Importantly, glucose deprivation decreased intracellular ATP levels and mitochondrial membrane potential. Ginsenoside Rg1 rescued ATP levels and mitochondrial membrane potential in nutrient-starved cells. For molecular mechanisms, ginsenoside Rg1 increased the expressions of PTEN-induced kinase 1 (PINK1) and p-AMPK in glucose deprivation treated H9c2 cells. Reducing the expression of aldolase in H9c2 cells inhibited ginsenoside Rg1′s actions on PINK1 and p-AMPK. Further, the nutritional stress mice were used to verify the mechanisms obtained in vitro. Ginsenoside Rg1 increased the expressions of aldolase, p-AMPK, and PINK1 in starved mice heart. Taken together, our results reveal that ginsenoside Rg1 limits nutritional stress-induced H9c2 cells injury by regulating the aldolase /AMP-activated protein kinase/PINK1 pathway.     

Ginsenoside Rg1 ameliorates diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress‐induced apoptosis in a streptozotocin‐induced diabetes rat model

Ginsenoside Rg1 has been demonstrated to have cardiovascular protective effects. However, whether the cardioprotective effects of ginsenoside Rg1 are mediated by endoplasmic reticulum (ER) stress‐induced apoptosis remain unclear. In this study, among 80 male Wistar rats, 15 rats were randomly selected as controls; the remaining 65 rats received a diet rich in fat and sugar content for 4 weeks, followed by intraperitoneal injection of streptozotocin (STZ, 40 mg/kg) to establish a diabetes model. Seven days after STZ injection, 10 rats were randomly selected as diabetic model (DM) controls, 45 eligible diabetic rats were randomized to three treatment groups and administered ginsenoside Rg1 in a dosage of 10, 15 or 20 mg/kg/day, respectively. After 12 weeks of treatment, rats were killed and serum samples obtained to determine cardiac troponin (cTn)‐I. Myocardial tissues were harvested for morphological analysis to detect myocardial cell apoptosis, and to analyse protein expression of glucose‐regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and Caspase‐12. Treatment with ginsenoside Rg1 (10–20 mg/kg) significantly reduced serum cTnI levels compared with DM control group (all P < 0.01). Ginsenoside Rg1 (15 and 20 mg/kg) significantly reduced the percentage of apoptotic myocardial cells and improved the parameters of cardiac function. Haematoxylin and eosin and Masson staining indicated that ginsenoside Rg1 could attenuate myocardial lesions and myocardial collagen volume fraction. Additionally, ginsenoside Rg1 significantly reduced GRP78, CHOP, and cleaved Caspase‐12 protein expression in a dose‐dependent manner. These findings suggest that ginsenoside Rg1 appeared to ameliorate diabetic cardiomyopathy by inhibiting ER stress‐induced apoptosis in diabetic rats.     

Enzymatic transglycosylation of ginsenoside Rg1 by rice seed α-glucosidase

Six α-monoglucosyl derivatives of ginsenoside Rg1 (G-Rg1) were synthesized by transglycosylation reaction of rice seed α-glucosidase in the reaction mixture containing maltose as a glucosyl donor and G-Rg1 as an acceptor. Their chemical structures were identified by spectroscopic analysis, and the effects of reaction time, pH, and glycosyl donors on transglycosylation reaction were investigated. The results showed that rice seed α-glucosidase transfers α-glucosyl group from maltose to G-Rg1 by forming either α-1,3 (α-nigerosyl)-, α-1,4 (α-maltosyl)-, or α-1,6 (α-isomaltosyl)-glucosidic linkages in β-glucose moieties linked at the C6- and C20-position of protopanaxatriol (PPT)-type aglycone. The optimum pH range for the transglycosylation reaction was between 5.0 and 6.0. Rice seed α-glucosidase acted on maltose, soluble starch, and PNP α-D-glucopyranoside as glycosyl donors, but not on glucose, sucrose, or trehalose. These α-monoglucosyl derivatives of G-Rg1 were easily hydrolyzed to G-Rg1 by rat small intestinal and liver α-glucosidase in vitro.     

Ginsenoside Rb1 ameliorates CKD‐associated vascular calcification by inhibiting the Wnt/β‐catenin pathway

Vascular calcification (VC) is a pathological process underpinning major cardiovascular conditions and has attracted public attention due to its high morbidity and mortality. Chronic kidney disease (CKD) is a common disease related to VC. Ginsenoside Rb1 (Rb1) has been reported to protect the cardiovascular system against vascular diseases, yet its role in VC and the underlying mechanisms remain unclear. In this study, we established a CKD‐associated VC rat model and a β‐glycerophosphate (β‐GP)‐induced vascular smooth muscle cell (VSMC) calcification model to investigate the effects of Rb1 on VC. Our results demonstrated that Rb1 ameliorated calcium deposition and VSMC osteogenic transdifferentiation both in vivo and in vitro. Rb1 treatment inhibited the Wnt/β‐catenin pathway by activating peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), and confocal microscopy was used to show that Rb1 inhibited β‐catenin nuclear translocation in VSMCs. Furthermore, SKL2001, an agonist of the Wnt/β‐catenin pathway, compromised the vascular protective effect of Rb1. GW9662, a PPAR‐γ antagonist, reversed Rb1's inhibitory effect on β‐catenin. These results indicate that Rb1 exerted anticalcific properties through PPAR‐γ/Wnt/β‐catenin axis, which provides new insights into the potential theraputics of VC.     

基于UPLC法测定离体大鼠及人源肠道菌对三七中人参皂苷代谢的差异

为探究人与大鼠肠道菌群对三七水煎液中三醇型人参皂苷Rg1、Re及二醇型人参皂苷Rb1、Rd体外代谢的差异性及发现其代谢产物原人参二醇PPD与原人参三醇PPT,实验利用UPLC方法测定三七水煎液分别与人、大鼠肠道菌群在厌氧条件下共培养24h后的孵育液中4种皂苷的含量及代谢产物PPD与PPT的含量。结果表明三七中含有三醇型人参皂苷Rg19.4500mg/g、Re1.8872mg/g,二醇型人参皂苷Rb18.5816mg/g、Rd1.9456mg/g。与人源肠道菌共培养后,三七中含有的二醇型、三醇型人参皂苷含量显著降低,重要的是,在培养液中检测到代谢产物PPD和PPT的存在,含量分别为0.2136mg/g及0.0344mg/g,与大鼠肠道菌共培养后,三七中含有的二醇型皂苷含量有轻微降低,而三醇型皂苷含量未见明显变化,但有少量PPT(0.0184mg/g)的生成。由此可见:在体外条件下,三七水煎液中人参皂苷会被人肠道菌群降解生成代谢产物PPD和PPT,而大鼠肠道菌群的降解产物却仅有PPT生成,二者存在种属差异。     

Cloning,overexpression and characterization of a thermostable β-xylosidase from Thermotoga petrophila and cooperated transformation of ginsenoside extract to ginsenoside 20(S)-Rg3 with a β-glucosidase

A thermostable β-xylosidase gene Tpexyl3 from Thermotoga petrophila DSM 13,995 was cloned and overexpressed by Escherichia coli. Recombinant Tpexyl3 was purified, and its molecular weight was approximately 86.7 kDa. Its optimal activity was exhibited at pH 6.0 and 90 °C. It had broad specificity to xylopyranosyl, arabinopyranosyl, arabinofuranosyl and glucopyranosyl moieties. The β-xylosidase activity of the recombinant Tpexyl3 was 6.81 U/mL in the LB medium and 151.4 U/mL in a 7.5 L bio-reactor. It was applied to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, which was combined with thermostable β-glucosidase Tpebgl3. After transforming under optimal condition, the 20 g/L of ginsenoside extract was transformed into 6.28 g/L of Rg3 within 90 min, with a corresponding molar conversion of 95.0% and Rg3 productivity of 1793.49 mg/L/h, respectively. This study is the highest report of a GH3 family glycosidase with arabinopyranosidase activity and also the first report on the high substrate concentration bioconversion of ginsenoside extract to ginsenoside 20(S)-Rg3 by using two thermostable glycosidases.