CYP1A1体外诱导活性评价模型的建立及其在人参皂苷筛选中的应用

目的 建立hAhR介导的CYP1A1体外诱导活性评价模型,应用于体外快速筛选通过hAhR途径介导的对CYP1A1具有激活能力的药物。方法 利用双荧光素酶报告基因系统,将CYP1A1启动子序列插入报告基因质粒上游,构建pGL4.17-CYP1A1质粒,并与含有hAhR编码区序列的pcDNA3.1-hAhR表达质粒瞬时共转染HepG2细胞, pGL4.17-control、pcDNA3.1（+）分别为pGL4.17-CYP1A1、pcDNA3.1-hAhR的空载体,建立hAhR-CYP1A1报告基因模型;应用AhR的完全激动剂TCDD（2 nmol/L）验证该报告基因模型的可靠性,应用该模型考察人参皂苷Rf、Rc、Re、Rg₁、Rb、Rd、Rh₂和Rg₃（20 μmol/L）通过hAhR途径对CYP1A1的诱导作用。结果 经验证,报告基因模型构建成功;人参皂苷Rf、Re、Rg₁和Rc对AhR具有激活作用,其中与对照组比较,人参皂苷Rg₁、Rc对AhR激活效应显著（P<0.05、0.01）,人参皂苷Rb、Rd、Rh₂和Rg₃对AhR无激活作用。结论 本研究建立基于hAhR的CYP1A1体外诱导活性评价模型,可为具有潜在CYP1A1诱导作用的目标化合物的筛选提供有效、快速的体外筛选手段,人参皂苷Rg₁、Rc对AhR-CYP1A1激活效应显著。     

应用人肝细胞研究人参皂苷Rb1和Rg1、丹参素钠、冰片对CYP450药物代谢酶1A2、2B6和3A4的体外诱导作用

目的 使用原代培养的人肝细胞研究人参皂苷Rb₁和Rg₁、丹参素钠、冰片对细胞色素P450酶（CYP450）的诱导作用。方法 分别将3批次的冷冻原代人肝贴壁细胞进行接种培养,使用人参皂苷Rb₁和Rg₁、丹参素钠、冰片（30 μmol/L）对CYP1A2、CYP2B6和CYP3A4进行诱导,实时荧光定量PCR（qRT-PCR）法测定CYP450酶mRNA表达水平,评价4种单体成分对P450酶的诱导作用。阳性对照为20 μmol/L利福平（CYP3A4诱导剂）、50 μmol/L奥美拉唑钠（CYP1A2诱导剂）和1 mmol/L苯巴比妥钠（CYP2B6诱导剂）;阴性对照组为10 μmol/L红霉素。冰片（30 μmol/L）与利福平联合给药,观察冰片对利福平的CYP1A2、CYP2B6和CYP3A4诱导作用的影响。结果 3批次人肝细胞的诱导结果显示,阳性诱导剂奥美拉唑、苯巴比妥、利福平分别显著诱导CYP1A2、CYP2B6、CYP3A4的表达,阴性对照红霉素对CYP1A2、CYP2B6及CYP3A4的mRNA表达水平未见明显影响,人参皂苷Rb₁和Rg₁、丹参素钠、冰片在30 μmol/L浓度给药时,对3批次人肝细胞的CYP1A2、CYP2B6和CYP3A4 mRNA表达水平未见明显影响;30 μmol/L冰片与利福平联合给药,与利福平单独给药比较,3批次肝细胞的CYP2B6、CYP3A4 mRNA表达水平均减少,对CYP3A4的mRNA表达水平影响尤为显著。结论 人参皂苷Rb₁和Rg₁、丹参素钠、冰片在30 μmol/L浓度给药时,对3批次人肝细胞的CYP1A2、CYP2B6和CYP3A4均没有诱导作用。冰片与利福平联合用药时,可以阻断利福平对CYP2B6和CYP3A4的诱导作用,对CYP3A4的影响尤为显著。     

一测多评法测定复方人参片中的8种苷类成分

目的 建立一测多评法（quantitative analysis single-marker,QAMS）测定复方人参片中8种苷类成分的含量。方法 采用Agilent XDB-C₁₈色谱柱（4.6 mm×250 mm,5 μm）,流动相水（A）-乙腈（B）,梯度洗脱;体积流量1.0 mL·min^-1;检测波长203 nm;柱温：25℃。以人参皂苷Rb₁为内标,计算人参皂苷Rg₁、人参皂苷Re、淫羊藿苷、人参皂苷Rc、人参皂苷Rb₂、人参皂苷Rb₃、人参皂苷Rd的相对校正因子,测定其含量。结果 8种成分在各自范围内线性关系良好（r ≥ 0.999 0）,加样回收率为95.6%~104.4%,RSD为1.22%~2.73%,QAMS测定结果与外标法测定结果无显著性差异。结论 该法准确度、灵敏度高、专属性好、操作简单、重复性好。     

基于网络药理学预测三七治疗幽门螺杆菌相关疾病机制及实验验证

目的 基于网络药理学预测三七治疗幽门螺杆菌（Hp）相关疾病机制,研究三七中有效活性成分人参皂苷Rb₃对Hp造成的胃上皮细胞损伤的保护作用及机制。方法 使用Herb数据库收集“三七”的相关预测靶点,使用Gene Cards数据库收集Hp相关疾病的靶点;使用Draw Venn Diagram网站绘制Venn图,得到靶点交集;进行蛋白互作（PPI）网络分析、基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析。将GES-1细胞分为对照组、模型组及人参皂苷Rb₃低、中和高浓度（1、5、10 μ mol· L^-1）组,人参皂苷Rb₃组使用相应浓度的人参皂苷Rb₃预处理,培养过夜12 h至融合度为70%～80%。Hp悉尼株1 （SS1）按感染复数（MOI） 100加入细胞中制备损伤模型,人参皂苷Rb₃继续给药,共培养48 h。对照组不加SS1,对照组和模型组不加药。改良吉姆萨染色后通过光学显微镜观察细胞形态;结合Hoechst 33342荧光染色和Annexin V/PI双染流式细胞术检测细胞凋亡;试剂盒法检测活性氧（ROS）水平;采用实时荧光定量PCR（qRT-PCR）检测凋亡相关基因TP53、Bax、Bcl-2表达量;Western blotting法检测P53、p-Akt、cleaved/pro-Caspase 9、Bcl-2、Bax、cleaved/pro-Caspase 3的蛋白表达情况。结果 网络药理学结果表明三七治疗Hp相关疾病的靶点共16个,其PPI网络分析得到按度值大小排名前6位靶点为TP53、CASP3、PTGS2、IL6、TNF、IL1β。GO富集分析与KEGG富集分析结果均显示与凋亡相关。与模型组比较,经人参皂苷Rb₃处理后,GES-1细胞的细胞核染色质致密深染,破裂的细胞逐渐减少;Hoechst 33342荧光染色细胞核强荧光数目明显减少;细胞凋亡率显著降低（P＜0.05）;ROS水平显著降低（P＜0.05）;TP53与Bax的mRNA水平显著降低,Bcl-2 mRNA水平显著升高（P＜0.05）; p-Akt、Bcl-2蛋白表达显著升高（P＜0.05）,P53、Bax、cleaved/pro-Caspase 9与cleaved/pro-Caspase 3蛋白表达显著降低（P＜0.05）。结论 三七可能通过包括炎症及凋亡在内的多种途径治疗Hp相关疾病,人参皂苷Rb₃对Hp诱导的胃上皮细胞凋亡发挥显著改善作用,其可能机制是降低氧化应激水平,并调节Akt的磷酸化和P53的表达。     

HPLC-ELSD法测定胃康颗粒中人参皂苷Rb1和黄芪甲苷的含量

目的 优化胃康颗粒中人参皂苷Rb₁和黄芪甲苷的提取方法,并建立其含量测定方法。方法 以人参皂苷Rb₁和黄芪甲苷的含量作为指标,采用单因素考察法对提取工艺进行优化;采用高效液相色谱-蒸发光散射法（HPLC-ELSD）,XBridge^®Shield RP₁₈（4.6 mm×250 mm,5 μm）为色谱柱;乙腈-水（32：68）为流动相;柱温为30 ℃;漂移管温度为60 ℃,载气流量为1.7 SLM,建立测定胃康颗粒中人参皂苷Rb₁和黄芪甲苷含量的方法。结果 当采用甲醇回流提取1.5 h,正丁醇提取5次,氨水洗涤2次时,人参皂苷Rb₁和黄芪甲苷的提取含量较高;建立的HPLC-ELSD法测定人参皂苷Rb₁和黄芪甲苷含量,线性关系良好（r > 0.9997）,日内日间精密度均小于1%,加样回收率分别为95.65%和100.57%,稳定性和重复性的RSD均小于3%,含量分别为2.8630 mg/g和0.2576 mg/g,RSD分别为0.62%和1.51%。结论 优化了胃康颗粒中人参皂苷Rb₁和黄芪甲苷的提取方法,建立了可靠、准确、重现性好的测定胃康颗粒中人参皂苷Rb₁和黄芪甲苷含量的HPLC-ELSD方法。     

基于一测多评定量控制联合灰色关联度分析的颈痛颗粒综合质量评价

目的 建立高效液相色谱一测多评（QAMS）法同时测定颈痛颗粒中三七皂苷R₁、人参皂苷Rg₁、人参皂苷Rb₁、3''-羟基葛根素、葛根素、3''-甲氧基葛根素、洋川芎内酯A、藁本内酯、延胡索乙素、去氢紫堇碱、紫堇碱、羌活醇和异欧前胡素含量,并通过灰色关联度分析法对13种成分含量检测结果进行分析评价。方法 采用Shimadzu C₁₈色谱柱;以乙腈-0.1%磷酸为流动相梯度洗脱;检测波长为203 nm（三七皂苷R₁、人参皂苷Rg₁和人参皂苷Rb₁）、280 nm（3''-羟基葛根素、葛根素、3''-甲氧基葛根素、洋川芎内酯A、藁本内酯、延胡索乙素、去氢紫堇碱和紫堇碱）和315 nm（羌活醇和异欧前胡素）;采用外标法（ESM）测定13种成分的含量。以葛根素为内参物质,分别计算其与三七皂苷R₁、人参皂苷Rg₁、人参皂苷Rb₁、3''-羟基葛根素、3''-甲氧基葛根素、洋川芎内酯A、藁本内酯、延胡索乙素、去氢紫堇碱、紫堇碱、羌活醇和异欧前胡素的相对校正因子（f）,并计算上述12种成分的含量,比较ESM和QAMS法检测结果的差异,验证一测多评法的准确性和可行性。基于灰色关联度分析不同批次颈痛颗粒中13种成分QAMS法含量检测数据,对15批次颈痛颗粒质量进行综合评价。结果 三七皂苷R₁、人参皂苷Rg₁、人参皂苷Rb₁、3''-羟基葛根素、葛根素、3''-甲氧基葛根素、洋川芎内酯A、藁本内酯、延胡索乙素、去氢紫堇碱、紫堇碱、羌活醇和异欧前胡素分别在各自范围内线性关系良好（r≥0.999 2）,平均加样回收率（n＝9）在96.86%～100.37%（RSD≤1.65%）。所建立的f耐用性良好,HPLC-QAMS法计算值和ESM实测值之间无明显差异。灰色关联度分析结果显示15批次颈痛颗粒相对关联度在0.332 4～0.600 4,表明颈痛颗粒批次间呈现一定质量差异。结论 QAMS多指标成分定量控制联合灰色关联度分析法操作便捷、结果准确,可用于颈痛颗粒的综合质量评价。     

HPLC-MS/MS测定同济2号颗粒剂中黄芪甲苷、绿原酸、人参皂苷Rg1、人参皂苷Rb1和三七皂苷R1

目的 建立HPLC-MS/MS同时测定同济2号颗粒中5个主要活性成分黄芪甲苷、绿原酸、人参皂苷Rg₁、人参皂苷Rb₁及三七皂苷R₁的方法。方法 以水(0.1%甲酸)-乙腈(0.1%甲酸)为流动相,梯度洗脱,体积流量为0.4 mL/min。YMC-Pack Pro C₈色谱柱分离,采用电喷雾离子源(ESI源),负离子模式,以选择性离子监测模式(SIM)进行测定。监测离子分别是m/z 829(黄芪甲苷)、m/z 353(绿原酸)、m/z 845(人参皂苷Rg₁)、m/z 1 108(人参皂苷Rb₁)、m/z 932(三七皂苷R₁)。结果 黄芪甲苷、绿原酸、人参皂苷Rg₁、人参皂苷Rb₁和三七皂苷R₁分别在0.075～2.4、0.95～30.3、1.71～54.72、1.12～35.92、0.45～14.28 μg/mL与峰面积呈良好线性关系。结论 该方法专属性好,灵敏度高,准确快捷,适用于同济2号颗粒的快速检测,为该药的质量标准提供依据。     

HPLC同时测定藤珠胃康颗粒中4个皂苷类成分

目的 建立HPLC同时测定藤珠胃康颗粒中4个皂苷类成分（人参皂苷Re、人参皂苷Rb₁、人参皂苷Ro和竹节参皂苷Ⅳa）的方法。方法 采用Hypersil GOLD C₁₈色谱柱（250 mm×4.6 mm,5 μm）,流动相为乙腈-0.2 %磷酸水溶液,梯度洗脱（-5~0 min,19%乙腈;0~14 min,19%→26%乙腈;14~22 min,26%→29%乙腈;22~30 min,29%乙腈;30~40 min,29%→35%乙腈;40~55 min,35%乙腈）,体积流量1.0 mL·min^-1,检测波长203 nm,柱温30 ℃。结果 人参皂苷Re在0.080 4~1.608 μg（r=1）,人参皂苷Rb₁在0.108 8~2.176 μg（r=0.999 9）,人参皂苷Ro在0.288 8~5.776 μg（r=0.999 9）,竹节参皂苷Ⅳa在0.176 0~3.520 μg（r=0.999 9）内线性关系良好;平均回收率（n=6）分别为99.41%,101.92%,99.76%,100.31%,RSD值分别为2.22%,2.07%,0.33%,0.64%。结论 本实验所建立的方法简单,专属性强,重复性好,可用于藤珠胃康颗粒中人参皂苷Re、人参皂苷Rb₁、人参皂苷Ro和竹节参皂苷Ⅳa的测定。     

基于近红外光谱分析技术的注射用益气复脉(冻干)红参醇提过程在线监测

目的 利用近红外光谱分析技术建立注射用益气复脉（冻干）主要原料红参醇提过程中3种单体皂苷——人参皂苷Rg₁、Re和Rb₁的定量模型,实现提取过程中关键指标的快速检测。方法 在线采集红参醇提过程的近红外光谱,以超高效液相色谱（UPLC）法测定提取过程药液中人参皂苷Rg₁、Re和Rb₁的量为参考值,采用偏最小二乘法建立光谱与测定值之间的定量校正模型,进而对提取过程进行在线分析。结果 人参皂苷Rg₁和Re的建模波段均为9 403.7～7 498.3 cm^-1和6 102～5 446.3 cm^-1组合波段;人参皂苷Rb₁的建模波段为5 774.1～5 446.3 cm^-1。人参皂苷Rg₁、Re、Rb₁定量模型的交叉验证决定系数（R²）分别为99.40、99.44、99.41,交叉验证均方根误差分别为5.18、2.77、11.00。结论 所建立的3种单体皂苷定量模型预测性能良好,能够有效测定红参醇提过程中人参皂苷Rg₁、Re和Rb₁的量。     

人参皂苷Rb1调控MAPK通路改善小鼠脑缺血再灌注诱导血脑屏障损伤的作用研究

目的 探讨人参皂苷Rb₁对小鼠脑缺血再灌注诱导的血脑屏障（BBB）损伤的作用及机制。方法 将C57BL/6小鼠随机分为假手术组、模型组和人参皂苷Rb₁低、中、高剂量（5、10、20 mg·kg^-1）组,采用线栓法栓塞颈内动脉1 h后复灌建立脑缺血再灌注损伤模型,假手术组不栓塞,其余操作同模型小鼠。缺血1 h后ip相应药物,于再灌注24 h后处死取材。采用伊文思蓝染色法检测各组小鼠BBB损伤程度;采用实时荧光定量PCR （qRT-PCR）法检测各组小鼠脑组织中炎症因子白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）以及紧密连接蛋白1（ZO-1）、闭合蛋白（Occludin）的mRNA表达水平;同时采用Western blotting检测各组小鼠脑组织中ZO-1、Occludin蛋白,金属基质蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）以及MAPK通路相关蛋白磷酸化的表达水平。结果 与模型组相比,人参皂苷Rb₁可显著减少脑缺血再灌注小鼠脑组织中伊文思蓝的渗漏量（P<0.05）,显著降低脑组织中IL-1β、IL-6和TNF-α的mRNA转录水平（P<0.05、0.01）;显著上调ZO-1和Occludin的mRNA转录和蛋白表达水平（P<0.05、0.01）;显著降低MMP-2、MMP-9的蛋白表达水平（P<0.05、0.01）;显著抑制MAPK通路p38、JNK及ERK磷酸化蛋白的表达（P<0.05、0.01）。结论 人参皂苷Rb₁对小鼠脑缺血再灌注诱导的BBB损伤具有一定的改善作用,其作用机制可能与抑制MAPK信号通路激活,减少MMP-2、MMP-9蛋白的表达,进而减轻对ZO-1、Occludin等紧密连接蛋白的降解有关。     

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。     

Drug Metabolism: Intestinal Bacterial Hydrolysis is Required for the Appearance of Compound K in Rat Plasma after Oral Administration of Ginsenoside Rb1 from Panax ginseng

Ginsenoside Rb₁ from Panax ginseng root is transformed into compound K via ginsenosides Rd and F₂ by intestinal bacterial flora. Among 31 defined intestinal strains from man, only Eubacterium sp. A-44 transformed ginsenoside Rb₁ into compound K via ginsenoside Rd. The ginsenoside Rb₁-hydrolysing enzyme isolated from Eubacterium sp. A-44 was identical to a previously purified geniposide-hydrolysing β-D-glucosidase. When ginsenoside Rb₁ (200 mg kg^?1) was administered orally to germ-free rats, neither compound K nor any other metabolite was detected in the plasma, intestinal tract or cumulative faeces 7 or 15 h after administration. Most of the ginsenoside Rb₁ administered was recovered from the intestinal tract, especially the caeca, and cumulative faeces indicating poor absorption of ginsenoside Rb₁. When ginsenoside Rb₁ was administered orally to gnotobiote rats mono-associated with Eubacterium sp. A-44, a significant amount of compound K was detected in the plasma and considerable amounts were found in the caecal contents and cumulative faeces 7 and 15 h after administration. A small amount of ginsenoside Rb₁ was detected in the caecal contents only 7 h after administration. These results indicate that orally administered ginsenoside Rb₁ is poorly absorbed from the gut but that its metabolite compound K, produced by ginsenoside Rb₁-hydrolysing bacteria such as Eubacterium sp. A-44 in the lower part of intestine, is absorbed.     

UPLC法评价硫磺熏蒸对西洋参皂苷类成分的影响

目的：评价硫磺熏蒸对西洋参皂苷类成分的影响。方法：建立超高效液相色谱法,测定并比较分析硫磺熏蒸前后西洋参中人参皂苷Rg₁、Re和Rb₁的含量。结果：硫磺熏蒸后西洋参中人参皂苷Rg₁、Re和Rb₁总量仍符合《中国药典》规定;但当硫磺熏蒸致二氧化硫残留量大于400 mg·kg^-1时,人参皂苷Re、Rb₁的含量及人参皂苷Rg₁、Re和Rb₁三者的总量显著降低。当二氧化硫残留量不大于150 mg·kg^-1时,人参皂苷Rg₁、Re和Rb₁的含量及其总量基本不受影响。结论：《中国药典》规定的西洋参二氧化硫残留量不得大于150 mg·kg^-1有其科学合理性,硫磺过度熏蒸西洋参（二氧化硫残留量大于400mg·kg^-1）对西洋参中皂苷类成分的含量有显著影响。     

Ginsenoside Rb3 Inhibits Angiotensin II‐Induced Vascular Smooth Muscle Cells Proliferation

Abstract: This study was designed to examine the effect of ginsenoside Rb₃ on angiotensin (Ang) II‐induced proliferation of cultured rat vascular smooth muscle cells (VSMCs). VSMCs proliferation was evaluated by [³H]Thymidine incorporation. The cell cycle was examined by flow cytometry. The expression of mRNA of proto‐oncogene c‐myc, c‐fos and c‐jun was observed by RT‐PCR. Ginsenoside Rb₃ had no effects on VSMCs proliferation in physiological condition. Ang II significantly increased the proliferation of VSMCs and the expression of mRNA of proto‐oncogene c‐myc, c‐fos and c‐jun. Ginsenoside Rb₃ markedly inhibited Ang II‐induced VSMCs proliferation. Concomitantly, ginsenoside Rb₃ decreased cell cycle progression from G₀/G₁ to S phase. Furthermore, ginsenoside Rb₃ significantly attenuated the expression of mRNA of proto‐oncogene c‐myc, c‐fos and c‐jun. This study showed that ginsenoside Rb₃ inhibited Ang II‐induced VSMCs proliferation, at least in part by inhibiting Ang II‐induced G₀/G₁ to S phase transition and attenuating the expression of mRNA of c‐fos, c‐jun and c‐myc. The findings may explain the beneficial effects of ginsenoside Rb₃ in cardiovascular diseases, and it will be useful to develop prevention and therapeutics of cardiovascular diseases.     

Pharmacokinetic interaction of aconitine,liquiritin and 6-gingerol in a traditional Chinese herbal formula,Sini Decoction

1.?This study aimed to investigate the pharmacokinetic interaction of the three ingredients in a traditional Chinese herbal formulation, Sini Decoction, and provide evidence for its compatibility mechanism.

2.?First, the effect of liquiritin and 6-gingerol on the pharmacokinetic parameters of aconitine was investigated in rats by using a sensitive and reliable LC–MS/MS method. Then the Caco-2 cell monolayer model and Rhodamine-123 uptake assay were used to investigate the effect of liquiritin and 6-gingerol on the absorption of aconitine and the activity of P-gp.

3.?The C_max of aconitine increased significantly (p?C_max and AUC_(0–t) of aconitine increased approximately twofold, and while t_1/2 only increased 1.2-fold. The Caco-2 cell monolayer model and Rhodamine-123 uptake assay indicated that both liquiritin and 6-gingerol could increase the absorption of aconitine by inhibiting the activity of P-gp.

4.?These results indicated that both liquiritin and 6-gingerol could promote the absorption of aconitine and increase its drug concentration in blood by inhibiting the activity of P-gp, and it could also provide evidence for compatibility mechanism of the traditional Chinese herbal formula, Sini Decoction.     

Effects of ginseng saponins isolated from red ginseng on ultraviolet B-induced skin aging in hairless mice

It is well-known that chronic ultraviolet B (UVB) exposure at low-dose causes skin photoaging including increases in skin thickness and wrinkle formation and reduction in skin elasticity. This study examined the effects of total saponins and ginsenoside Rb₁ isolated from Red Ginseng roots on skin thickness, elasticity, and wrinkle formation caused by long-term, low-dose UVB irradiation in hairless mice. The topical application of total ginseng saponins (10 pg or 100 ng/mouse) and ginsenoside Rb₁ (100 fg, 10 pg, or 1 ng/mouse) significantly inhibited increases in skin thickness and wrinkle formation and the reduction in skin elasticity induced by long-term UVB irradiation. Furthermore, we examined the histological effects of total saponins and ginsenoside Rb₁ in the skin of UVB-irradiated hairless mice. The increases in apoptotic, Ki-67-, and 8-hydroxy-2′-deoxyguanosine-positive cells induced by UVB exposure were prevented by the topical application of total saponins and ginsenoside Rb₁. Furthermore, total saponins and ginsenoside Rb₁ prevented the disruption of collagen fibers induced by the long-term UVB irradiation. Ginsenoside Rb₁ (100 fg, 10 pg, and 1 ng/ml) increased the Bcl-2 expression level in UVB-treated human keratinocytes. The protective effect of ginsenoside Rb₁ on UVB-mediated apoptosis may be due to the up-regulation of Bcl-2 expression. These results suggest that the protective effect of ginsenoside Rb₁ on skin photoaging induced by chronic UVB exposure may be due to the increase in collagen synthesis and/or the inhibition of matrix metalloproteinase expression in dermal fibroblasts.     

Three new dammarane-type triterpene saponins from the leaves of Panax ginseng C.A. Meyer

Three new dammarane-type triterpene ginsenosides, together with six known ginsenosides, were isolated from the leaves of Panax ginseng C.A. Meyer. The new saponins were named as ginsenoside Rh₁₁, ginsenoside Rh₁₂, and ginsenoside Rh₁₃. Their structures were elucidated as (20S)-3β,6α,12β,20-tetrahydroxydammara-25-ene-24-one 20-O-β-d-glucopyranoside (1), (20S)-3β,12β,20,24,25-pentahydroxydammarane 20-O-β-d-glucopyranoside (2), and (20S,23E)-3β,12β,20,25-tetrahydroxydammara-23-ene 20-O-β-d-glucopyranoside (3) on the basis of 1D and 2D NMR experiments and mass spectra. The known ginsenosides were identified as ginsenoside M_7cd, ginsenoside Rg₆, ginsenoside Rb₃, gypenoside XVII, gypenoside IX, and 20-(E)-ginsenoside F₄.     

Protective effects of ginsenoside Rb3 on oxygen and glucose deprivation-induced ischemic injury in PC12 cells

Aim:

To investigate the protective effects of ginsenoside Rb₃, a triterpenoid saponin isolated from the leaves of Panax notoginseng, on ischemic and reperfusion injury model of PC12 cells and elucidate the related mechanisms.

Methods:

PC12 cells exposed to oxygen and glucose deprivation (OGD) and restoration (OGD-Rep) were used as an in vitro model of ischemia and reperfusion. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) leakage were used to evaluate the protective effects of ginsenoside Rb₃. Cellular apoptosis and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. Intracellular calcium ion concentration ([Ca²⁺]_i) was detected using fluorophotometer system. Caspase-3, -8, and -9 activities were measured using assay kits with an ELISA reader. Western blotting assay was used to evaluate the release of cytochrome c and expression of caspase-3, Bcl-2 and Bax proteins.

Results:

It was shown that ginsenoside Rb₃ (0.1–10 μmol/L) significantly increased cell viability and inhibited LDH release in a dose-dependent manner on the ischemic model. In addition, ginsenoside Rb₃ also significantly inhibited ischemic injury-induced apoptosis, [Ca²⁺]_i elevation, and decrease of MMP. Meanwhile, pretreatment with ginsenoside Rb₃ significantly induced an increase of Bcl-2 protein expression and a decrease of cytosolic cytochrome c, cleaved-caspase 3 and Bax protein expression, the caspase-3, -8, and -9 activity were also inhibited.

Conclusion:

The results indicated that ginsenoside Rb₃ could markedly protected OGD-Rep induced ischemic injury and the mechanisms maybe related to its suppression of the intracellular Ca²⁺ elevation and inhibition of apoptosis and caspase activity. Ginsenoside Rb₃ could be a promising candidate in the development of a novel class of anti-ischemic agent.