Properties of a novel β-glucosidase from Fusarium proliferatum ECU2042 that converts ginsenoside Rg3 into Rh2

A novel β-glucosidase from Fusarium proliferatum ECU2042 (FPG) was successfully purified to homogeneity with a 506-fold increase in specific activity. The molecular mass of the native purified enzyme (FPG) was estimated to be approximately 78.7 kDa, with two homogeneous subunits of 39.1 kDa, and the pI of this enzyme was 4.4, as measured by two-dimensional electrophoresis. The optimal activities of FPG occurred at pH 5.0 and 50 °C, respectively. The enzyme was stable at pH 4.0–6.5 and temperatures below 60 °C, and the deactivation energy (E_d) for FPG was 88.6 kJ mo1⁻¹. Moreover, it was interesting to find that although the purified enzyme exhibited a very low activity towards p-nitrophenyl β-d-glucoside (pNPG), and almost no activity towards cellobiose, a relatively high activity was observed on ginsenoside Rg₃. The enzyme hydrolyzed the 3-C, β-(1 → 2)-glucoside of ginsenoside Rg₃ to produce ginsenoside Rh₂, but did not sequentially hydrolyze the β-d-glucosidic bond of Rh₂. The K_m and V_max values of FPG for ginsenoside Rg₃ were 2.37 mM and 0.568 μmol (h mg protein)⁻¹, respectively. In addition, this enzyme also exhibited significant activities towards various alkyl glucosides, aryl glucosides and several natural glycosides.     

Determination of ginsenoside Rg3 in human plasma and urine by high performance liquid chromatography–tandem mass spectrometry

Here we report a method capable of quantifying ginsenoside Rg3 in human plasma and urine. The method was validated over linear range of 2.5–1000.0 ng mL⁻¹ for plasma and 2.0–20.0 ng mL⁻¹ for urine using ginsenoside Rg1 as I.S. Compounds were extracted with ethyl acetate and analyzed by HPLC/MS/MS (API-4000 system equipped with ESI⁻ interface and a C₁₈ column). The inter- and intra-day precision and accuracy of QC samples were ≤8.5% relative error and were ≤14.4% relative standard deviation for plasma; were ≤5.6% and ≤13.3% for urine. The Rg3 was stable after 24 h at room temperature, 3 freeze/thaw cycles and 131 days at −30 °C. This method has been applied to pharmacokinetic study of ginsenoside Rg3 in human.     

小剂量环磷酰胺联合参一胶囊治疗进展期非小细胞肺癌

目的：应用小剂量环磷酰胺（CTX）联合参一胶囊治疗Ⅲ、Ⅳ期非小细胞肺癌,观察其临床疗效及毒副作用。方法：Ⅲ、Ⅳ期非小细胞肺癌患者经标准化、放疗后随机分为两组,治疗组（38例）：CFX 50mg每日一次、参一胶囊20mg日两次口服,三至六个月;对照组（34例）：化疗后不用任何药物治疗。结果：联合治疗组患者外周血血管内皮生长因子（VEGF）表达下降。T淋巴细胞亚群CD4＋及CD4＋／CD8＋升高,病人生存质量及生存期提高。结论：Ⅲ、Ⅳ期NSCLC患者化疗后联合应用小剂量CTX联合参一胶囊治疗是一种安全有效的治疗方法,可以提高患者的生存质量及生存期。     

Ginsenoside Rg3 antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons

We previously reported that ginseng, a well-known herbal medicine, inhibited NMDA receptors in cultured hippocampal neurons. Here, we further examined the detailed mechanism of ginseng-mediated inhibition using its main active ingredient, ginsenoside Rg3. Co-application of ginsenoside Rg3 with increasing concentrations of NMDA did not change the EC50 of NMDA to the receptor, suggesting that ginsenoside Rg3 inhibits NMDA receptors without competing with the NMDA-binding site. Ginsenoside Rg3-mediated inhibition also occurred in a distinctive manner from the well-characterized NMDA receptor open channel blocker, MK-801. However, ginsenoside Rg3 produced its effect in a glycine concentration-dependent manner and shifted the glycine concentration-response curve to the right without changing the maximal response, suggesting the role of ginsenoside Rg3 as a competitive NMDA receptor antagonist. We also demonstrated that ginsenoside Rg3 significantly protected neurons against NMDA insults. Therefore, these results suggest that ginsenoside Rg3 protects NMDA-induced neuronal death via a competitive interaction with the glycine-binding site of NMDA receptors in cultured hippocampal neurons.     

用HPLC法研究不同提取方法对人参单体皂甙的提取效果

本文采用MPG-ODS色谱柱,以醋酸铵作HPLC流动相的改性荆在15 min以内较好地分离出单体皂甙Rg_2、Rb_1、Rc、Rd、Rg_1、Re等,首次比较了化学上醇提取方法和食用时水提取方法对单体皂甙及总皂甙提取效果的不同,结果表明,食用水对Rg组类皂甙的提取量高于Rb组,这将为人参的药理学研究及临床食用提供科学依据。     

In vitro characterization of the Ac locus in white clover (Trifolium repens L.)

Microsomal fractions from developing shoots of adult white clover plants (of genotype AcAc) and cotyledons of dark germinated clover seedlings can synthesize 2-hydroxy-2-methylpropanenitrile and 2-hydroxy-2-methylbutanenitrile, the aglycone precursors of the cyanogenic glucosides, linamarin and lotaustralin, from various precursors in the presence of NADPH. l-Valine, 2-methylpropanal oxime, and 2-methylpropanenitrile are converted to 2-hydroxy-2-methylpropanenitrile and are detected as cyanide and acetone. l-Isoleucine and 2-methylbutanal oxime are converted to 2-hydroxy-2-methylbutanenitrile and are detected as cyanide and 2-butanone. At least two steps in these conversions are missing in microsomes from plants of genotype acac.     

Production of a thermostable archaeal superoxide reductase in plant cells

Pyrococcus furiosus superoxide reductase (SOR) is a thermostable archaeal enzyme that reduces superoxide without producing oxygen. When produced as a fusion protein with the green fluorescent protein in plant cells, P. furiosus SOR is located in the cytosol and nucleus. The recombinant SOR enzyme retains its function and heat stability when assayed in vitro. Importantly, expressing SOR in plant cells enhances their survival at high temperature indicating that it functions in vivo. The archaeal SOR provides a novel mechanism to reduce superoxide and demonstrates the potential for using archaeal genes to alter eukaryotic metabolism.     

人参皂甙Rb1对大鼠脑缺血再灌注神经损伤后的修复作用

通过阻塞大鼠大脑动脉,制备短暂性脑缺血大鼠模型,将出现神经功能缺失症状的大鼠随机分组,实施再灌注后立即按(40mg/kg)进行腹腔注射人参皂甙Rb1。结果发现大鼠脑缺血再灌注后,人参皂甙Rb1通过促进NAIP、Bcl-2表达和抑制Bax表达发挥神经损伤后的修复作用。人参皂甙Rb1给药组在各时间点的胶质细胞源性神经营养因子(GDNF)阳性细胞数远远高于单纯脑缺血再灌注组(P<0.01)。GDNF的表达与缺血性损伤有一定的联系,可认为人参皂甙Rbl对神经系统有一定保护作用。     

人参皂苷Rb1通过上调PGC-1α缓解糖尿病心肌病

目的:研究人参皂苷Rb1对糖尿病心肌病的治疗作用并阐明其分子机制。方法:采用腹腔注射链脲佐菌素的方法,建立糖尿病心肌病动物模型。将小鼠分为3组:WT组,DM组,DM+Rb1组。超声心动图分析小鼠心功能;Western blot分析PGC-1α、cleaved caspase-3、bcl-2等蛋白表达;MitoSOX染色分析线粒体ROS含量;透射电镜分析线粒体数目。结果:与WT组相比,DM组小鼠心功能显著下降(LVEF,P<0.01),PGC-1α表达下调(P<0.01),线粒体数目减少(P<0.01);而Rb1处理后,显著改善了DM小鼠心功能(LVEF,P<0.01),恢复了PGC-1α表达(P<0.05),增加了线粒体数目(P<0.05)。同时,Rb1处理后,减少了糖尿病小鼠心肌线粒体ROS产生(P<0.01),恢复了bcl-2蛋白表达(P<0.01),降低了cleaved caspase-3蛋白表达(P<0.01),从而减少了高糖引起的细胞凋亡(P<0.05)。而siPGC-1α处理后,阻断了Rb1的上述作用。结论:人参皂苷Rb1通过上调PGC-1α改善糖尿病小鼠心功能,缓解糖尿病心肌病。其机制可能与人参皂苷Rb1降低心肌线粒体ROS产生并减少心肌细胞凋亡有关。     

Bioconversion of ginsenosides Rb(1), Rb(2), Rc and Rd by novel β-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: cloning, expression, and enzyme characterization

A new β-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344 bp (447 amino acid residues) with a predicted molecular mass of 49,399 Da. A BLAST search using the bglSp sequence revealed significant homology to that of glycoside hydrolase superfamily 1. This enzyme was overexpressed in Escherichia coli BL21 (DE3) using a pET21-MBP (TEV) vector system. Overexpressed recombinant enzymes which could convert the ginsenosides Rb₁, Rb₂, Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc₁ and ginsenoside F₂, respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for β-glucosidase showed the apparent K_m and V_max values of 2.9 ± 0.3 mM and 515.4 ± 38.3 μmol min⁻¹ mg of protein⁻¹ against p-nitrophenyl-β-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb₁, Rb₂, Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc₁ and F₂ quickly at optimal conditions of pH 5.0 and 37 °C. A little ginsenoside F₂ production from ginsenosides Gyp XVII, C-O, and C-Mc₁ was observed for the lengthy enzyme reaction caused by the side ability of the enzyme.