New staining methodology: Eastern blotting for glycosides in the field of Kampo medicines

Ginsenosides separated by silica gel TLC blotted to a polyvinylidene difluoride (PVDF) membrane treated with a NaIO₄ solution followed by bovine serum albumin (BSA) resulted in a ginsenoside–BSA conjugate on a PVDF membrane. The blotted spot were stained by antiginsenoside Rb1 (GRb1) and Rg1 (GRg1) monoclonal antibodies (MAbs). The newly established immunostaining method, Eastern blotting, was applied for the determination of ginsenosides possessing protopanaxadiol and/or protopanaxatriol in the Kampo medicines. In this method, we developed a new way to separate the ginsenoside molecule into two functional parts using a simple and well-known chemical reaction. The sugar parts were oxidized by sodium periodate to give dialdehydes, which reacted with amino groups of the protein and covalently bound to the adsorbent PVDF membrane. The MAb bound to the aglycone part of the ginsenoside molecule for immunostaining. Double staining of Eastern blotting for ginsenosides using anti-GRb1 and GRg1 MAbs promoted complete identification of ginsenosides in Panax species. This method was validated for immunocytolocalization of ginsenosides in fresh ginseng root. In addition, Eastern blotting for the detection of glycyrrhizin (GC) was also investigated. GC can be clearly determined by Eastern blotting in the Glycyrrhiza species. It is also possible for GC in rat serum to be surveyed by Eastern blotting by simple pretreatment as a further application.     

Phytochemical and analytical studies of Panax notoginseng (Burk.) F.H. Chen

Panax notoginseng (Burk.) F.H. Chen is distributed throughout the southwest of China, Burma and Nepal. The root of this plant, called notoginseng or sanchi, has a long history of use as a remedy in Oriental traditional medicine. Modern studies have found that extracts and compounds from notoginseng exert various physiological effects. The active constituents are mainly recognized as saponins. In this review, we summarized the discovery and analysis of chemical constituents in notoginseng. Fifty-six saponins from notoginseng were isolated and elucidated. All of them are dammarane saponins, 35 of which can be classified as belonging to the protopanaxadiols group, and 21 as belonging to the protopanaxatriols group. Evidence from phytochemical studies on notoginseng demonstrated that no oleanane-type saponin, which exists in Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), was found. Other types of compounds such as non-protein amino acids, polyacetylenes, phytosterols, flavonoids, and polysaccharides, many of which have pharmacological activities, were also isolated from notoginseng. Analytical studies on notoginseng were carried out based on botanical and phytochemical advances. In the qualitative studies, identification of the herbal materials and extracts was the main objective. The utilization of high-performance liquid chromatography (HPLC) fingerprint and molecular biological methods made the identification accurate and efficient. Spectral, chromatographic and immunoassay methods were used for the quantitative analysis. HPLC methods are the main authority regarding the determination of saponins and other types of constituents. The chromatographic conditions and detectors employed in the HPLC are discussed.     

西洋参药材高效液相色谱法指纹图谱的研究

目的建立西洋参药材的高效液相色谱法(HPLC)指纹图谱。方法采用中药色谱指纹图谱相似度评价系统分析不同产地西洋参药材的HPLC,建立西洋参指纹图谱分析指标。结果确立了国产和进口西洋参指纹图谱的技术参数,确定18个共有峰;不同产地西洋参药材指纹图谱与对照指纹图谱(R)的相似度均大于0.85。结论该方法建立的西洋参药材HPLC指纹图谱,为药材鉴别和品质评价、质量控制、制定质量标准提供依据。     

人参皂甙诱导HL-60细胞凋亡的研究

目的：观察人参皂甙（GS）是否具有诱导髓性白血病HL－60细胞株的凋亡作用。方法：取不同浓度的GS处理HL－60细胞，观察GS所致的细胞形态学的变化；流式细胞术分析细胞DNH含量的改变，并进行DNA片段分析（DNA Ladder);用Annexin V-FITC试验法分析细胞凋亡百分率。结果：人参皂甙能够抑制HL－60细胞生长，在一定剂量和时间范围内可引起细胞凋亡。结论：提示人参皂甙能特异性地诱导HL－60细胞凋亡，可为临床应用人参皂甙作为化疗药物的辅助剂治疗白血病提供实验依据。     

Ginsenosides Rb1 and Rg1 effects on survival and neurite growth of MPP<Superscript>+</Superscript>-affected mesencephalic dopaminergic cells

Summary. Ginsenosides Rb1 and Rg1 are the main active ingredients of Panax ginseng C.A. Meyer (Araliaceae). They appear to exert protection against ischaemia and anoxic damage in animal models, suggesting an antioxidative and cytoprotective role. In our study, primary cultures from embryonic mouse mesencephalon are applied to examine the effects of these two ginsenosides on neuritic growth of dopaminergic cells and their survival affected by 1-methyl-4-phenylpyridinium-iodide (MPP⁺). Ginsenoside Rb1 (at 10µM) enhanced the survival of dopaminergic neurons by 19% compared to untreated control. MPP⁺ (at 1µM) significantly reduced the number of dopaminergic neurons and severely affected neuronal processes. Both ginsenosides counteracted these degenerations and significantly protected lengths and numbers of neurites of TH⁺ cells. Both compounds however could not prevent the cell loss caused by MPP⁺. Our study thus indicates partial neurotrophic and neuroprotective actions of ginsenosides Rb1 and Rg1 in dopaminergic cell culture.     

复方双参口腔崩解片的制备工艺研究

目的：制备复方双参口腔崩解片,使用星点设计-效应面优化法对处方工艺进行优化筛选。方法：以丹参水提物和人参皂苷为主药,采用粉末直接压片法制备复方双参口腔崩解片,选择MCC／L—HPC（8：2）和PVPP作为联合崩解剂。以MCC／L—HPC的用量及PVPP的用量为考察因素,崩解时限作为评价指标,用线性方程和二次及三次多项式描述崩解时限和两个影响因素之间的数学关系,根据最佳数学模型描绘效应面及等高线图,选择最佳处方,并进行预测分析。结果：当MCC／L—HPC含量25％,PVPP含量8．2％时,片剂的崩解时间最短。各指标的三项式拟合方程均优于多元线形回归方程,建立的数学模型的预测值与实际值符合较好。结论：用星点设计-效应面法优化复方双参口腔崩解片处方工艺预测性良好。     

人参皂苷Rg3对小鼠B16黑素瘤细胞侵袭、转移及MMP-9表达的影响

目的:观察人参皂苷Rg3(简称Rg3)对小鼠黑素瘤细胞B16的肺转移、侵袭能力及基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)表达的影响,探讨其抗肿瘤转移的作用机制。方法:体内建立小鼠自发肺转移和实体瘤模型,观察腹腔注射不同剂量的Rg3(对照组给予0.9%氯化钠溶液)后,肺部肿瘤转移灶的数目,并检测实体瘤中MMP-9蛋白的表达情况。采用Boyden小室侵袭实验及免疫细胞化学染色法检测Rg3对肿瘤细胞体处侵袭能力及MMP-9表达的影响。结果:采用不同剂量的Rg3(0.3、1.0和3.0mg/kg)治疗后,小鼠肺部转移灶的数目均较少,肿瘤组织中MMP-9的表达水平降低,与对照组比较差异有统计学意义(P<0.05)。在体外,2.5和5.0μg/mLRg3治疗组中侵袭穿过人工基膜的B16细胞数目明显少于对照组(P<0.01),且5.0μg/mLRg3可抑制肿瘤细胞中MMP-9的表达。结论:Rg3能够抑制小鼠黑素瘤细胞的肺转移,其抗肿瘤转移作用可能是通过降低肿瘤细胞中MMP-9的表达水平及细胞侵袭能力来实现的。     

Identification of ginsenoside interaction sites in 5-HT3A receptors

We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.     

磷脂存在下人参皂甙的含量测定

探讨了磷脂对人参皂甙含量测定的影响。实验结果表明：当以氯仿脱脂时，由于磷脂的增溶作用，增加了人参皂甙在非极性有机溶剂中的溶解度，造成含量测定结果偏低；从而建立了以大孔吸附树脂分出磷脂后进行比色测定的方法。平均回收率为１０１．３８％，ＲＳＤ为１．２２％。