人参皂苷水溶液热稳定性研究

目的 研究加热时间对人参皂苷水溶液的影响及受热后人参皂苷含量的变化情况。方法 红参须浓缩液加热不同时间,采用高效液相色谱法测定其人参皂苷Rg1、Re、Rb1、Rc和Rd的含量。结果 5种人参皂苷在加热6 h内,发生不同程度的降解反应,二醇类人参皂苷Rb1、Rc和Rd在加热2-3 h时,含量呈明显下降趋势,人参皂苷Rd降解速率最慢。三醇类人参皂苷Rg1和Re在加热3 h内含量快速下降,3 h后趋于平缓。结论 在常压受热条件下,人参皂苷水溶液主要成分人参皂苷Rg1、Re、Rb1、Rc和Rd的含量,随加热时间延长而不断下降,3 h后下降速率减缓。三醇类人参皂苷较二醇类对热更为敏感。     

三七总皂苷主要成分对人肝微粒体CYP3A4酶的抑制作用研究

目的：研究三七总皂苷主要成分人参皂苷Rg1、人参皂苷Rb1和三七皂苷R1对人肝微粒体CYP3A4酶的体外抑制作用.方法：采用人肝微粒体体外孵育法,在孵育体系中分别加入不同浓度的人参皂苷Rg1、人参皂苷Rb1和三七皂苷R1与探针底物睾酮共孵育,用LC-MS /MS 法测定代谢产物6β-羟基睾酮的生成量反映CYP3A4酶活性的影响.结果：人参皂苷Rg1在2～1 000 μmol·L-1的测试浓度范围内对CYP3A4未见剂量相关的抑制作用;人参皂苷Rb1在2～200 μmol·L-1的测试浓度范围内对CYP3A4未见剂量相关的抑制作用,在1 000 μmol·L-1的测试浓度下对CYP3A4具有轻微抑制作用;三七皂苷R1在2～1 000 μmol·L-1的测试浓度范围内对CYP3A4的IC50为126 μmol·L-1（＞100 μmol·L-1）.结论：三七总皂苷3个主要有效成分单体对CYP3A4酶的体外抑制作用不同,人参皂苷Rg1无抑制作用,人参皂苷Rb1高浓度下有轻微抑制作用,三七皂苷R1有弱抑制作用,三七总皂苷制剂与CYP3A4酶代谢相关的药物之间产生相互作用的可能性低.     

基于UPLC-Q-TOF/MS研究参附注射液的物质基础

目的对参附注射液中的人参皂苷和生物碱成分进行定性,并且对参附注射液中主要成分在大鼠体内的药物动力学过程进行研究。方法运用LC-MS对参附注射液中的成分进行定性,并测定大鼠血清中多种人参皂苷的浓度,计算其药动学参数。结果参附注射液中定性出16种人参皂苷成分和10种生物碱成分。人参皂苷Rd、Rg1、Rb1、Ro、Rc、Rb2的T12α分别为0.32±0.25、0.11±0.04、0.30±0.24、0.11±0.02、0.24±0.19、0.23±0.12 h,人参皂苷Rd、Rb1、Rc、Rb2的T12β分别10.25±0.39、21.31±3.64、13.95±2.56、15.06±1.54 h。结论该方法简便、灵敏、特异,适用于血清中6种人参皂苷成分的药物动力学研究;人参皂苷Rg1、Ro在大鼠体内的分布消除速度较快,Rd、Rb1、Rc、Rb2分布消除较慢。参附注射液体外、体内成分的分析为组分配伍研究建立了可靠的方法。     

三七总皂甙提取工艺研究进展

三七总皂甙（PNS）（又称三七总皂苷）为五加科人参属植物三七根部提取的有效成分,它含有人参皂甙Rb1、Rb2、Rc、Rd、Re、Rf、Rg1、Rg2、Rh、三七皂甙R1、1／2、R3、R4、R5、R6等20多种皂甙成分,其中以人参皂甙Rb1、Rg1、三七皂甙R1含量最高Ⅲ。三七总皂甙对中枢神经系统有明显的抑制作用和镇痛作用。     

人参皂苷对电压门控离子通道的影响

离子通道是细胞膜和细胞器膜等生物膜上一类允许离子通透的蛋白质。许多离子通道,如大多数钠通道、钾通道、钙通道和部分氯通道受电压门控调节,这些电压门控离子通道具有广泛的生理功能。人参皂苷是中药人参、西洋参和三七等五加科人参属植物的主要活性成分,包括Ra1,Ra2,Rb1,Rb2,Rb3,Rc,Rd,Rg3和Rh2等原人参二醇,以及Re,Rf,Rg1,Rg2和Rh1等原人参三醇等。本文综述了人参皂苷多种成分对各种电压门控钠通道、钾通道、钙通道和氯通道的不同作用特点及可能的机制,提示人参皂苷多种成分不仅能直接作用于电压门控离子通道,还可以通过G蛋白和一氧化氮等信号通路途径,间接影响电压门控离子通道功能。     

HPLC法测定人参毛状根及不同人参样品中9种人参皂苷的含量

目的采用HPLC法测定不同人参样品中9种人参皂苷(Rc、Rb1、Rb2、Re、Rd、Rg1、Rg2、Rg3和Rh2)的含量。方法色谱条件:Zorbax SB C18柱(4.6mm×250mm,5μm),保护柱Extend-C18柱(4.6mm×12.5mm,5μm);以乙腈-水为流动相,梯度洗脱;流速:1.0ml/min;检测波长:203nm;柱温:35℃。结果 9种人参皂苷Rg1、Rb1、Re、Rc、Rg2、Rh2、Rg3、Rb2和Rd在120min内基线分离。方法学表明其线性关系良好,精密度、稳定性和重复性RSD均小于2.0%,加样回收率在98.3%~102%之间。测得人参叶和人参须根中的总皂苷含量最高,分别为48.9、23.6mg/g;人参毛状根中的总皂苷与人参主根和人参果的总皂苷含量差别不大,为7.47mg/g。结论该法准确性高,操作简便、快速,重复性好,精密度高,可用于不同人参样品中9种人参皂苷的含量测定。     

人参皂苷Rb_1、Rg_1对许旺细胞NGF表达的影响

目的:研究人参皂苷Rb1和Rg1影响许旺细胞神经生长因子(NGF)的表达程度。方法:分离纯化成人新鲜离体神经许旺细胞,置于人参皂苷条件培养基孵育48h;采用间接免疫荧光法标记细胞,流式细胞仪检测许旺细胞NGF表达率。结果:人参皂苷Rb1和Rg1各实验组许旺细胞表达NGF的百分率较对照组显著升高(P<0.05);人参皂苷Rb1与Rg1组之间许旺细胞表达NGF的百分率无显著差异。结论:人参皂苷Rb1和Rg1可以通过促进许旺细胞合成分泌NGF而具有潜在加速周围神经损伤修复的作用。     

组分配伍对人参总皂苷中主要成分体外吸收的影响

目的建立检测M199培养液中人参皂苷Rg1、Re、Rb1和Rd的LC—MS／MS分析方法,并探讨复方给药和单一组分给药对人参总皂苷四种成分Rg1、Re、Rb1和Rd在体外吸收的影响差异。方法采用大鼠肠囊外翻法探讨不同配伍对参皂苷四种成分吸收量的影响规律,采用LC／MS／MS方法测定药液浓度,并对实验结果进行统计分析。结果色谱采用SB-C18反相柱,流动相为甲醇和0．1％甲酸梯度洗脱,运行16rain。质谱采用MRM方式进行检测,人参皂苷Rg1、Re和Rb1的线性范围均为10-5000ng／ml,人参皂苷Rd的线性范围为4～2000ng／ml,提取回收率为78％到106％,方法的精密度、准确度和稳定性均符合要求。结论本方法操作简单、专属性强、灵敏度高、准确性好,可用于人参皂苷类成分的肠吸收的测定。肠吸收实验结果显示低剂量组复方配伍对皂苷类成分的吸收略有促进,中、高剂量影响没有显著性差异。     

人参皂苷Rg1对家兔离体小肠平滑肌自发收缩活动的影响

目的观察人参皂苷Rg1对家兔离体小肠平滑肌收缩活动的影响,并探讨其作用机制。方法试验采用经典的离体小肠灌流技术,观察人参皂苷Rg1对家兔小肠平滑肌自发收缩活动的影响,并探讨其作用机制。结果人参皂苷Rg1剂量依赖性的抑制家兔离体小肠平滑肌收缩的幅度;L型钙通道开放剂BayK8644和左旋硝基精氨酸甲酯(L-NAME)均可完全阻断人参皂苷Rg1对家兔小肠平滑肌收缩活动的抑制作用;在无钙台式液中,人参皂苷Rg1抑制rynodine引起的细胞内钙释放所导致的收缩活动。结论人参皂苷Rg1抑制家兔小肠平滑肌的收缩活动,其抑制收缩活动机制可能是:增加小肠平滑肌NO浓度,从而抑制细胞外钙内流和内钙释放。     

三七总皂苷和灯盏花素单用与联用时主要成分的药动学比较

目的考察三七总皂苷(主要成分为三七皂苷R1、人参皂苷Rg1、人参皂苷Re、人参皂苷Rb1、人参皂苷Rd)和灯盏花素(主要成分为野黄芩苷)复方粉针制剂在健康比格犬体内的药动学,并与单独使用三七总皂苷粉针或灯盏花素粉针时的药动学参数进行比较。方法建立检测给药后不同时间点的比格犬血样中三七总皂苷各组分及野黄芩苷浓度的液相色谱-串联质谱联用(LC-MS/MS)方法,计算各成分的药动学参数。结果单次给予复方粉针后,三七皂苷R1、人参皂苷Rg1、人参皂苷Re、人参皂苷Rb1、人参皂苷Rd及野黄芩苷的血浆消除半衰期(t1/2)分别为(1.08±0.30),(0.95±0.16),(1.40±0.39),(51.08±10.42),(64.84±17.70)及(2.00±0.88)h;峰浓度(Cmax)分别为(4641.00±758.84),(11325.00±1418.62),(1822.00±253.37),(39380.00±5644.03),(12964.00±2738.41)及(2669.00±841.79)ng·mL-1;血药浓度-时间曲线下面积(AUC0-∞)分别为(2832.31±308.38),(3454.00±473.08),(1210.80±161.06),(1360410.90±277244.88),(320529.65±101345.47),(450.68±90.50)ng·mL-1·h。与三七总皂苷粉针或灯盏花素粉针单次给药相比,复方粉针单次给药后,血药浓度-时间曲线相似;三七皂苷R1、人参皂苷Rg1、人参皂苷Re、人参皂苷Rd的Cmax显著升高(P<0.05);人参皂苷Rb1和野黄芩苷Cmax差异无统计学意义(P>0.05);t1/2、AUC0-∞差异无统计学意义(P>0.05)。结论三七总皂苷粉针和灯盏花素粉针联合用药可提高三七总皂苷部分组分的Cmax,对野黄芩苷的药动学没有明显影响。     

Ginsenosides Rg3 and Rh2 inhibit the activation of AP-1 and protein kinase A pathway in lipopolysaccharide/interferon-gamma-stimulated BV-2 microglial cells

The anti-inflammatory effect of ginsenosides Rg3 and Rh2, which improves ischemic brain injury induced by middle cerebral artery occlusion, was investigated in lipopolysaccharide (LPS) and IFN-gamma-induced murine BV-2 microglial cells. Ginsenoside Rh2 inhibited the production of NO, with an IC50 value of 17 microM. The inhibitory effect of Rh2 on NO correlates with the decreased protein and mRNA expression of an inducible NO synthase (iNOS) gene. Additionally, ginsenoside Rh2 inhibited the expression of COX-2, pro-inflammatory TNF-alpha and IL-1beta in BV-2 cells induced by LPS/IFN-gamma, while it increased the expression of the anti-inflammatory cytokine IL-10. Electrophoretic mobility shift assays revealed that ginsenoside Rh2 significantly inhibited the LPS/IFN-gamma-induced AP-1 DNA binding activity, while it enhanced the protein binding to CRE sequences. However, it did not affect NF-kappaB binding activity. Thus, the anti-inflammatory effect of Rh2 appears to depend on the AP-1 and protein kinase A (PKA) pathway. The anti-inflammatory effect of ginsenoside Rg3 against LPS/IFN-gamma-activated BV-2 cells was less potent than that of ginsenoside Rh2. These findings suggest that the in vivo anti-ischemic effect of ginsenoside Rg3 may originate from ginsenoside Rh2, which is a main metabolite of ginsenoside Rg3 by intestinal microflora, and that of ginsenoside Rh2 may be due to its anti-inflammatory effect in brain microglia.     

Inhibitory effects of ginsenoside Rg1 and Rb1 on rat brain microsomal Na+,K(+)-ATPase activity

Rat brain microsomal Na+, K(+)-ATPase activity was inhibited significantly and rapidly by ginsenoside Rb1. The IC50 of Rb1 for Na+,K(+)-ATPase was 6.3 +/- 1.0 mumol/L. The inhibition was enhanced with increasing the concentration of Rb1 or decreasing that of Na+ and K+. Kinetic analysis revealed that ginsenoside was an uncompetitive inhibitor with respect to ATP. The inhibitory effect of Rg1 on rat brain microsomal Na+,K(+)-ATPase was much weaker than that of Rb1.     

人参皂苷Rg3对乳腺癌MCF-7细胞增殖和侵袭的影响

目的 观察人参皂苷Rg3对雌激素受体阳性的乳腺癌细胞MCF-7增殖和侵袭的影响,并探讨其可能的作用机制。方法 采用MTT法检测细胞的增殖能力,流式细胞仪分析细胞周期分布以及凋亡比率,通过Transwell小室观察细胞侵袭力,RT-PCR法检测细胞中的MMP-9 mRNA的表达。结果 与对照组相比,人参皂苷Rg3能显著抑制MCF-7细胞的增殖;G₀/G₁期及S期细胞比例减少,而G₂/M期细胞比例显著增加;同时细胞凋亡比率亦明显提升,而细胞侵袭指数降低,且呈现良好的剂量、时间依赖性。同时人参皂苷Rg3还能显著抑制细胞中MMP-9 mRNA的表达水平(P<0.05)。结论 人参皂苷Rg3能抑制MCF-7细胞的增殖和侵袭,其作用机制可能与其能降低MMP-9基因的表达有关。     

人参皂苷Rg3差异调节前列腺癌细胞PC3和DU145增殖

目的 探讨人参皂苷Rg3对前列腺癌细胞PC3和DU145细胞增殖的调节作用。方法 100 µmol/L人参皂苷 Rg3处理体外培养的前列腺癌细胞 PC3和 DU145,使用 CCK-8实验检测细胞的增殖能力;DCFH-DA活性氧荧光探针试剂盒检测人参皂苷 Rg3处理的 DU145细胞中活性氧（ROS）水平;JC-1法检测 PC3和 DU145细胞线粒体膜电位的去极化;RT-PCR和免疫印迹法检测PC3和DU145细胞中氧化还原相关蛋白的表达差异。结果 CCK-8实验结果表明,100 µmol/L人参皂苷 Rg3能够显著抑制 PC3细胞增殖,而对 DU145细胞的增殖没有显著调节作用;DCFHDA活性氧荧光探针试剂盒检测结果表明,人参皂苷 Rg3能够显著上调 DU145细胞和 PC3细胞中 ROS水平;JC-1检测实验表明,人参皂苷Rg3能够诱导PC3和DU145细胞中线粒体膜电位去极化;RT-PCR和免疫印迹实验表明,抗氧化蛋白谷胱甘肽过氧化物酶-1（GPX-1）和超氧化物歧化酶2（SOD2）在DU145细胞中的表达显著高于PC3细胞。结论 人参皂苷Rg3对PC3和DU145细胞的增殖表现出不同的调节作用,可能与细胞中抗氧化蛋白的表达差异有关。     

Ginsenoside Rh2 reduces ischemic brain injury in rats

Ginseng was incubated under mildly acidic conditions and its inhibitory effect on a rat ischemia-reperfusion model was investigated. When ginseng was treated with 0.1% hydrochloric acid at 60 degrees C, its protopanaxadiol saponins were transformed to diasteromeric ginsenoside Rg3 and Delta20-ginsenoside Rg3. When the transformed ginseng extract, of which the main component was ginsenosides Rg3, was treated with human intestinal microflora, the main metabolite was ginsenoside Rh2. Orally administered acid-treated ginseng (AG) extract and ginsenoside Rh2 potently protect ischemia-reperfusion brain injury. The ginsenoside Rh2 also inhibited prostaglandin-E2 synthesis in lipopolysaccharide-stimulated RAW264.7 cells, but showed no in vitro antioxidant activity. These results suggest that AG and ginsenoside Rh2 can improve ischemic brain injury.     

Ginsenoside Rg1 inhibits proliferation of vascular smooth muscle cells stimulated by tumor necrosis factor-α

AIM: To investigate the proliferation of vascular smooth muscle cells (VSMC) affected by ginsenoside Rg1 and further explore the molecular mechanism of ginsenoside Rg1 using proteomics. METHODS: The proliferation of VSMC was measured by MTS assay kit and flow cytometry. Proteomic alterations were analyzed using two-dimensional electrophoresis and peptide mass fingerprinting. Differential proteins found in proteomics were confirmed by RT-PCR. RESULTS: The proliferation of VSMC was enhanced significantly after tumor necrosis factor-alpha (TNF-alpha) treatment, and ginsenoside Rg1 treatment inhibited proliferation in a dose-dependent manner. Proteomic analysis showed 24 protein spots were changed, including 17 spots that were increased and 7 spots that were decreased. Ginsenoside Rg1 could restore the expression levels of these proteins, at least partly, to basic levels of untreated cells. The expression of G-protein coupled receptor kinase, protein kinase C (PKC)-zeta, N-ras protein were decreased, while cycle related protein p21 was increased by ginsenoside Rg1 in TNF-alpha treated VSMC. CONCLUSION: PKC-zeta and p21 pathway might be the mechanism for inhibitory effects of ginsenoside Rg1 on proliferation of VSMC.     

人参皂苷 Rg3 对人乳腺癌 MCF-7 细胞血管生成拟态的影响

摘要： 目的 探讨人参皂苷 Rg3 对人乳腺癌 MCF-7 细胞体外血管生成拟态的抑制作用及其机制。方法 取 MCF-7 对数生长期细胞分为实验组和对照组, 实验组加入含有终浓度为 10、 20、 50、 100、 150 及 300 mg/L 的 Rg3 完 全培养基; 对照组加入含 DMSO 完全培养基。CCK-8 法分析 Rg3 对 MCF-7 细胞增殖的影响, 分析实验组各浓度下 的抑制率和 Rg3 半数抑制浓度 （IC50） 并确定实验浓度。体外管道形成抑制实验观察 Rg3 对 MCF-7 细胞血管生成拟 态的影响。实时荧光定量 PCR 法检测 Rg3 对细胞血管内皮生长因子-A（VEGF-A）和基质金属蛋白酶（MMP） 9 mRNA 的表达水平的影响。蛋白免疫印迹法检测 Rg3 对细胞低氧诱导因子 （HIF） -1α、 VEGF-A 和 MMP9 蛋白表达 的影响。双抗夹心酶联免疫法测定 Rg3 对细胞培养上清液中 VEGF-A 、 MMP9 蛋白表达的影响。结果 人参皂苷 Rg3 可以有效抑制 MCF-7 细胞增殖, 其平均 IC50为 （115.34±8.50） mg/L; 选取 50、 100 和 150 mg/L 的 Rg3 作为实验浓 度。50、 100 和 150 mg/L 组形成的管状结构数目分别为 （19.0±1.0）、（15.0±1.5）、（10.0±1.7） 个/视野, 呈逐渐降低趋势; 且均较对照组（22.0±1.8）减少（均 P < 0.05）。对照组、 50 mg/L 组、 100 mg/L 组及 150 mg/L 组细胞中的 VEGF-A 和 MMP9 的 mRNA 和蛋白表达水平、 HIF-1α 的蛋白表达水平均呈逐渐下降趋势 （P < 0.05）。对照组、 50 mg/L 组、 100 mg/ L 组及 150 mg/L 组细胞培养上清液中的 VEGF-A 蛋白呈逐渐下降趋势 （P < 0.05）; 后 3 组的 MMP9 蛋白表达水平均呈 逐渐下降趋势 （P < 0.05）, 但 50 mg/L 组与对照组的 MMP9 蛋白表达水平差异无统计学意义。结论 人参皂苷 Rg3 可以抑制 MCF-7细胞体外血管形成拟态的能力, 其分子机制可能与抑制 MCF-7细胞中 HIF-1α、 VEGF-A和 MMP9的蛋白表达有关     

Effects of various ginseng saponins on 5-hydroxytryptamine release and aggregation in human platelets

The effects of various ginseng saponins isolated from red ginseng roots, on aggregation and 5-hydroxytryptamine release (5-HT) human platelets have been investigated. Among the six saponins tested, only ginsenoside Rg1 inhibited adrenaline- and thrombin-induced platelet aggregation and 5-HT release dose-dependently, at concentrations of 5 to 500 micrograms ML-1. Ginsenoside Rg1 had no effect on adrenaline- and thrombin-induced arachidonic acid release and diacylglycerol production. But it did reduce the elevation of cytosolic free calcium concentration (Ca2+)i shown in the second phase induced by adrenaline and thrombin, at concentrations of 10 to 500 micrograms mL-1. Those data suggest that the inhibitory effects of ginsenoside Rg1 on 5-HT release from, and aggregation of, platelets might be due to the reduction of (Ca2+)i elevation at the second phase induced by adrenaline and thrombin. The results suggest that ginsenoside Rg1 in red ginseng roots may be active as a drug in the treatment of artheroscleorosis and thrombosis.     

Inhibitory mechanisms of dihydroginsenoside Rg3 in platelet aggregation: critical roles of ERK2 and cAMP

Ginsenoside Rg3, a single ginseng saponin, is known to be a major anti-platelet component of protopanaxadiol that is isolated from Korean red ginseng. In this study, we investigated whether dihydroginsenoside Rg3, a stable chemical derivative of ginsenoside Rg3, also demonstrated anti-platelet activity. Dihydroginsenoside Rg3 inhibited thrombin-induced platelet aggregation in a concentration-dependent manner with an IC50 (concentration producing 50% inhibition) of 18.8 +/- 0.4 microM. Ginsenoside Rg3 inhibited platelet aggregation which was induced by thrombin (0.1 U mL(-1)) with an IC50 of 40.2 +/- 0.9 microM. We next determined whether dihydroginsenoside Rg3 affected different types of ligand-induced platelet aggregation. We found that dihydroginsenoside Rg3 inhibited collagen-induced platelet aggregation with an IC50 of 20.0 +/- 0.9 microM. To elucidate the inhibitory mechanism of dihydroginsenoside Rg3 on aggregation, we analysed its downstream signalling pathway. It was interesting to note that dihydroginsenoside Rg3 elevated cyclic AMP production in resting platelets, but did not affect cyclic GMP production. In addition, we found that dihydroginsenoside Rg3 potently suppressed phosphorylation of extracellular signal-regulated kinase 2 (ERK2), which was stimulated by collagen (2.5 microg mL(-1)), but not of p38 mitogen-activated protein kinase. Taken together, our results indicate that dihydroginsenoside Rg3 potently inhibited platelet aggregation via the modulation of downstream signalling components such as cAMP and ERK2.