Chromatographic resolution of glucosidic compounds, ginsenosides on polyethersulphone membrane, and its application to the quantitative immunoassay for ginseng saponins

A method has been devised for the chromatographic resolution of glucosidic compounds, ginseng saponins, on polyethersulphone (PES) membrane. The method results in good resolution and quantitative immunoassay for ginsenoside Rb1 (G-Rb1), G-Rc, and G-Rd in crude extracts of various ginsengs. The newly established method is simpler and applies for quantitative analysis. Ginsenosides developed by acetonitrile-water-acetic acid solvent system on a PES membrane were directly treated with a NaIO4 solution followed by bovine serum albumin (BSA), resulting in a ginsenoside-BSA conjugate on a PES membrane. Anti-G-Rb1 monoclonal antibody (MAb) was bound, and then a second antibody labeled with peroxidase directed against the first antibody. Finally a substrate reacted to the enzyme and gave staining. The stained membrane was scanned, and spots were analyzed quantitatively using NIH Image software. At least 62.5 ng of G-Rb1, G-Rc, and G-Rd were clearly detectable individually. Three ginsenosides can be analyzed quantitatively between 0.125 and 2.0 microg.     

Opposite effects of synthetic auxins, 2,4-dichlorophenoxyacetic acid and 1-naphthalene acetic acid on growth of true ginseng cell culture and synthesis of ginsenosides

Effects of synthetic auxins (2,4-D and NAA) on growth of true ginseng (Panax ginseng C.A. Mey) suspension culture and ginsenoside synthesis were investigated. Cell suspensions were grown for 6–8 subcultures on media supplemented with various phytohormones. In all media supplemented with 2,4-D and cytokinins (benzyladenine or kinetin), the cell culture showed sustained growth both in the presence and absence of casein hydrolysate. The average growth index, determined from fresh weight increment over one subculture, equaled to 5.16 ± 0.90, and the maximum mitotic index was 2%. These cell populations having cell volume of 10–17 × 10⁴ μm³ were composed mostly (up to 60–80%) of 5-to 10-cell aggregates with unimodal distribution of nuclear DNA. These cell suspensions were suitable for isolation of protoplasts. The total average content of ginsenosides in the cell culture grown in the presence of 2,4-D constituted 0.18% of dry matter. In media supplemented with NAA, the cell growth was retarded irrespective of the cytokinin species and presence or absence of casein hydrolysate. The growth index (the ratio of final to initial fresh weights) was on average 2.15 ± 0.37, and the mitotic index did not exceed 0.13%. These suspensions, characterized by cell volume of 22–50 × 10⁴ μm³, were composed of large aggregates (> 50 cells). The attempts to isolate protoplasts from these suspensions were unsuccessful. About 25% of cells cultured in the presence of NAA had doubled nuclear DNA content by the end of the subculture. The total content of ginsenosides in cell cultures grown with NAA was on average 4.46% of cell dry matter. The results indicate that ginsenoside synthesis depends on the extent of differentiation in the population of true ginseng cells grown in suspension culture. A certain extent of cytodifferentiation in the cell culture was observed in the presence of NAA, whereas 2,4-D supported only cell proliferation in vitro.     

Rg3 inhibits gemcitabine-induced lung cancer cell invasiveness through ROS-dependent,NF-κB- and HIF-1α-mediated downregulation of PTX3

PTX3, a member of the long pentraxin subfamily, associated with innate immunity is indispensable for resistance to some cancer. Gemcitabine, an analog of cytosine arabinoside, has shown restrained benefits because of profound chemoresistance. The PTX3 expression on GEM in human lung cancer cells have not yet been clarified; the present study aimed to show reactive oxygen species (ROS) mediatory PTX3 expression through distinct mechanisms. Whereas ginsenoside Rg3 is a herbal medicine with strong antitumor activity. Furthermore, we tested the hypothesis; Rg3 abrogates GEM-induced production of ROS-mediated activation of Akt and extracellular signal-regulated kinase (ERK) pathways and inhibits nuclear piling-up of nuclear factor kappa B (NF-κB) and HIF-1α. On the basis of time and dose-dependent manner, our data demonstrated that GEM-induced PTX3 expression was dependent on ROS generation as it was abrogated by pretreatment of lung cancer cells with the free radical scavenger N-acetyl-l -cysteine. Our data demonstrated that PTX3 upregulation by GEM correlated with the time-dependent escalation of NF-κB and HIF-1α in the nucleus resulted from phosphorylation-induced degradation of IκBα, whereas HIF-1α upregulation was NF-κB-dependent. Increase in ROS expression in lung cancer cells on GEM treatment preceded the nuclear accumulation of NF-κB and HIF-1α and suppression of ROS diminished these effects. ERK1/2 and Akt activation mediated the effect of ROS on NF-κB and HIF-1α and their pharmacological inhibition suppressed GEM-induced PTX3. Our study findings reinforced the role regarding PTX3 signaling in GEM-induced resistance and pointed toward an unintended and undesired effect of chemotherapy and to get an active regimen; the synergy was associated with NF-κB downregulation in lung cancer.     

西洋参皂苷的HPLC测定及不同提取方法比较

建立西洋参中7种人参皂苷含量测定的分析方法,并以7种人参皂苷的提取率为指标,对西洋参皂苷不同提取溶剂和不同提取方法进行比较。采用梯度洗脱,使用Alltima C18色谱柱,乙腈-0.05%磷酸水溶液为流动相,流速为1.2 mL/min,柱温为35℃,检测波长为203 nm;分别选用不同的提取溶剂和不同的辅助提取方法提取西洋参皂苷。在选定的色谱条件下每种成分在各自的浓度范围内均具有较好的线性相关性,7种人参皂苷的加标回收率为94.1~97.9%。本法操作简便,重现性好,结果准确;各人参皂苷不同提取溶剂和不同提取方法的提取率有一定的差异。     

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.     

外源人参皂苷对人参种子萌发和幼根抗氧化酶活性的影响

研究不同浓度外源人参皂苷(人参总皂苷,人参二醇组皂苷,人参三醇组皂苷, Rb族,Rb3,Re共4种皂苷混合物和两种单体皂苷)对人参种子萌发,幼苗根长、鲜重,幼根中抗氧化酶活性和MDA含量的影响.结果表明:所测试人参皂苷对人参种子萌发、人参幼苗根长生长和幼根鲜重增加均具有抑制化感效应,且抑制程度均随处理浓度的升高而增强;对人参幼根中抗氧化酶活性方面,不同浓度人参总皂苷,人参二醇组皂苷,人参三醇组皂苷处理后,人参根系中SOD,POD和CAT活性均有明显提高,呈现出各酶活性随浓度升高而逐渐增强的效应;人参皂苷Rb族处理后,SOD活性在低中浓度处理时,与对照差别不大,中高浓度处理后低于对照,POD活性在中高浓度处理后显著提高,高浓度处理后活性降幅较大难以恢复到对照水平,CAT活性均低于对照;人参皂苷Rb3处理后,SOD活性均低于对照水平,POD活性在低浓度处理时与对照相当,中高浓度处理后显著低于对照水平,CAT活性逐渐降低,在低中浓度处理时略高于对照,高浓度处理后低于对照水平;人参皂苷Re处理后,SOD和POD活性均显著低于对照.人参幼根中MDA含量均随着处理浓度的增加而升高.     

大孔吸附树脂分离纯化人参二醇类和三醇类皂甙

本实验研究大孔吸附树脂从人参根提取物中富集、分离人参二醇类和人参三醇类皂甙的工艺条件及参数。用不同浓度的乙醇洗脱,使人参二醇类和三醇类皂甙实现富集分离,人参二醇类皂甙富集在80％乙醇洗脱液部分,人参三醇类皂甙富集在40％洗脱液部分。得到含量大于25％的人参三醇类皂甙,含量大于50％的人参二醇类皂甙,总皂甙洗脱率在91％以上。此法能够较好地分离、纯化人参二醇类和三醇类皂甙。     

Optimization of dynamic-microwave assisted enzymatic hydrolysis extraction of total ginsenosides from stems and leaves of panax ginseng by response surface methodology

Abstract

Ginseng stems and leaves (GSAL) are abundant in ginsenosides compounds. For efficient utilization of GSAL and the enhancement of total ginsenosides (TG) compound yields in GSAL, TG from GSAL were extracted, using dynamic-microwave assisted extraction coupled with enzymatic hydrolysis (DMAE-EH) method. The extraction process has been simulated and its main influencing factors such as ethanol concentration, microwave temperature, microwave time and pump flow rate have been optimized by response surface methodology coupled with a Box-Behnken design(BBD). The experimental results indicated that optimal extraction conditions of TG from GSAL were as follows: ethanol concentration of 75%, microwave temperature of 60°C, microwave time of 20?min and pump flow rate of 38 r/min. After experimental verification, the experimental yields of TG was 60.62?±?0.85?mg?g^?1, which were well agreement with the predicted by the model. In general, the present results demonstrated that DMAE-EH method was successfully used to extract total ginsenosides in GSAL.     

Synthesis and Antitumor Activity of Panaxadiol Pyrazole and Isooxazole Derivatives

In this study, we designed and synthesized 19 nitrogen-containing heterocyclic derivatives of panaxadiol (PD). We first reported the antiproliferative activity of these compounds against four different tumor cells. The results of the MTT assay showed that the PD pyrazole derivative (compound 12b ) had the best antitumor activity and could significantly inhibit the proliferation of four tested tumor cells. For A549 cells, the IC₅₀ value was as low as 13.44±1.23 μM. Western blot analysis showed that the PD pyrazole derivative was a bifunctional regulator. On the one hand, it can down-regulate the expression of HIF-1α by acting on PI3 K/AKT signaling pathway in A549 cells. On the other hand, it can induce the decrease of CDKs protein family and E2F1 protein expression levels, thus playing a crucial role in cell cycle arrest. According to the results of molecular docking, we found that multiple hydrogen bonds were formed between the PD pyrazole derivative and two related proteins, and the docking score of the derivative was also significantly higher than that of the crude drug. In summary, the study of the PD pyrazole derivative laid a foundation for the development of ginsenoside as an antitumor agent.     

Identification of ginsenosides in roots of Panax ginseng by HPLC-APCI/MS

A new HPLC-APCI/MS method for the identification of ginsenosides has been developed. The analyses were performed on a reversed-phase C18 column using a binary eluent (acetonitrile and water) under gradient conditions. Although APCI is a high-temperature evaporative process, HPLC-APCI/MS could effectively identify thermo-labile ginsenosides. The [M-H]- ions and the thermal degradation ions of ginsenosides could be clearly observed under negative and positive ion conditions, respectively, and these were used to identify the molecular masses, the aglycone structures and the sugar groups of ginsenosides. APCI/MS can provide more explicit information than ESI/MS for identifying and distinguishing ginsenosides. Using the HPLC-APCI/MS method, 35 ginsenosides were identified in Panax ginseng.