HPLC法同时测定不同产地五味子中8种木脂素类成分

目的 测定产自辽宁、河北和黑龙江省的五味子中五味子醇甲、戈米辛J、五味子醇乙、戈米辛G、五味子酯甲、五味子甲素、五味子乙素和五味子丙素8种木脂素成分,为其质量控制提供检测手段.方法 五味子用甲醇超声提取,色谱柱为依利特ODS-C18(250 mm ×4.6 mm,5μm);流动相为乙腈(A)-水(B),梯度洗脱(0～17 min,A为50％;17 ～ 25 min,,A为50％ ～55％;25～30 min,A为55％～75％;30 ～ 35 min,A为75％;35 ～ 40 min,A为75％ ～65％;40～45 min,A为65％ ～50％);体积流量1.0 mL/min;柱温30℃;检测波长217 nm.结果 8种被测木脂素成分分离度良好;各成分质量浓度与峰面积在测定范围内均呈良好的线性关系(r ＞0.999 5);重复性良好;加样回收率(RSD)(n=9)分别为99.75％(0.38％)、100.69％ (2.31％)、100.46％( 1.39％)、99.87％( 1.15％)、100.22％( 1.45％)、100.15％(0.99％)、100.61％(0.25％)和101.31％(1.03％).结论 本实验所建立的HPLC方法稳定可靠、简便可行,可用于五味子中木脂素类成分的同时定量分析.     

HPLC测定五味子不同炮制品中6种木脂素类成分的含量

 目的 测定五味子及其不同炮制品中五味子醇甲、五味子醇乙、五味子酯甲、五味子甲素、五味子乙素和五味子丙素的含量。方法 色谱柱为依利特 ODS-C₁₈(4.6 mm×250 mm,5 μm);流动相为乙腈(A)-水(B),梯度洗脱(0～17 min,A为50%;17～25 min,A为50%～55%;25～30 min,A为55%～75%;30～35 min,A为75%;35～40 min,A为75%～65%;40～45 min,A为65%～50%);体积流量：1.0 mL·min-1;柱温：30 ℃;检测波长：217 nm。结果 与生品相比,五味子经醋制和酒制6种木脂素的含量均有所增加;且酒制品中的含量高于或等于醋制品中的含量。结论 本实验所建立的HPLC方法稳定可靠、简便可行,可用于五味子中木脂素类成分的同时定量分析,为五味子饮片及其炮制品的质量评价和质量控制奠定了基础。     

Gender-dependent pharmacokinetics of lignans in rats after single and multiple oral administration of Schisandra chinensis extract

Ethnopharmacological relevance

Schisandra chinensis (S. chinensis), a traditional Chinese medicine, has been widely used as sedatives and tonics in clinic. Schisandra lignans are believed to be the major bioactive components in S. chinensis. However, there is a lack of information about the effects of gender and repeated-dose on the pharmacokinetic properties of the schisandra lignans.

Aim of the study

The study was performed to investigate the influence of gender on the pharmacokinetics of schisandra lignans after administration of S. chinensis extract and to compare their pharmacokinetic behaviors between single and multiple administration.

Materials and methods

Two groups of rats (half male and half female) were received a single dose or multiple doses of S. chinensis extract, respectively. A liquid chromatography–tandem mass spectrometry method was developed and validated to determine the plasma concentrations of schisandra lignans.

Results

The pharmacokinetic parameters of schisandrin, schisandrol B, deoxyschisandrin, γ-schisandrin and schisantherin A were significantly different by gender difference. The t_1/2 of all the tested schisandra lignans in female rats were 2–9 times longer than the corresponding values in male rats. The C_max and AUC_0−t of these schisandra lignans except schisantherin A in female rats were 5–50 times higher than those in male rats. The pharmacokinetic profiles of schisandrin, schisandrol B, deoxyschisandrin and schisantherin A in both gender rats after multiple doses were similar to the corresponding profile after single dose.

Conclusion

All the tested schisandra lignans showed slower elimination and higher bioavailability in female rats after single or multiple administration of S. chinensis extract compared with male rats. Their pharmacokinetic profiles were not affected by repeated-dose except γ-schisandrin, which was eliminated more slowly in female rats after multiple administration.     

HPLC-MSn分析扶正化瘀方中五味子的入血成分

目的:分析扶正化瘀方(Fuzheng Huayu decoction,FZHY)灌胃大鼠后进入血液的五味子中木脂素类成分,以探讨部分体内效应成分.方法:应用液相色谱-电喷雾质谱联用(HPLC-ESI-MSn)技术,通过空白血清,口服FZHY方后含药血清,FZHY方和对照品之间的色谱图和质谱碎片信息的对比分析,结合文献报道,确定口服FZHY方后大鼠的入血成分.结果:FZHY方和口服FZHY方后含药血清中均含有五味子醇甲、五味子醇乙、五味子酯甲、五味子乙素,并在FZHY方中还发现含有五味子甲素和丙素.结论:五味子醇甲、五味子醇乙、五味子酯甲、五味子乙素是直接吸收入血的成分,可能是发挥抗肝纤维化的体内效应成分.     

基于多元统计分析和网络药理学的五味子醋制前后质量标志物预测分析

目的基于多元统计分析和网络药理学分析预测五味子醋制前后潜在的质量标志物。方法采用UPLC-Q/TOF-MS解析生、醋五味子饮片中主要的木脂素类成分,并运用多元统计方法筛选出炮制前后潜在的差异化学成分,即化学标记物。进一步通过网络药理学以及生物信息学分析显著差异成分相关的主要作用靶点和通路,构建"成分-靶点-通路"网络关系,预测生、醋五味子潜在的质量标志物。结果筛选了五味子醋制前后差异性成分40个,其中8个为生、醋五味子之间显著性差异成分(即化学标记物)。鉴定并确认了其中的5个化学成分,分别是5-羟甲基糠醛、五味子甲素及其同分异构体、五味子乙素和五味子酯丁。而其余3个化学标记物通过解析一级、二级质谱信息,推测它们很可能也属于木脂素类成分。网络药理学分析结果表明,鉴定并确认的5个潜在质量标志物与五味子的主要药理作用密切相关。最终五味子乙素和5-羟甲基糠醛被确定为最具代表性的潜在质量标志物。结论五味子醋制前后其化学成分发生了一系列复杂的变化,经研究分析确定五味子乙素和5-羟甲基糠醛可作为五味子醋制前后代表性的潜在质量标志物,并推测木脂素类成分可能为五味子醋制保肝的重要效应物质基础。     

Decursin attenuates the amyloid‐β‐induced inflammatory response in PC12 cells via MAPK and nuclear factor‐κB pathway

Decursin, the major bioactive component of Angelica gigas Nakai, exhibited neuroprotective properties. Our previous studies showed that decursin conferred neuroprotective effects in PC12 cells induced by Amyloid‐β (Aβ)_25–35 via antiapoptosis and antioxidant. In this study, the antiinflammatory effects of decursin against PC12 cells injury stimulated by Aβ_25–35 were assessed. Our results demonstrated that decursin suppressed the expression of cyclooxygenase‐2 protein and prostaglandin E2 content which was stimulated by Aβ_25–35 in PC12 cells. Meanwhile, the nuclear translocation of nuclear factor‐κB in Aβ_25–35‐treated PC12 cells was also inhibited by decursin. In addition, decursin suppressed phosphorylation of the two upstream pathway kinases, p38 and c‐Jun N‐terminal kinase. Overall, our findings indicate that decursin exerts protective effects against neuroinflammation stimulated by Aβ_25–35 in PC12 cells by abolishing cyclooxygenase‐2 protein expression through inactivation of nuclear factor‐κB via the upstream kinases including p38 and c‐Jun N‐terminal kinase. This work provides a new insight into the pharmacological mode of decursin and should facilitate its therapeutic application in treatment of inflammatory disorders.     

Rubrisandrins A and B, lignans and related anti-HIV compounds from Schisandra rubriflora

Bioactivity-directed fractionation of an ethanolic extract of the fruits of Schisandra rubriflora led to the isolation and identification of dibenzocyclooctadiene lignans including the new lignans rubrisandrins A (1a + 1b) and B (2) and the known lignans gomisin J (3), (+/-)-gomisin M1 (4), (+)-gomisin M2 (5), schisanhenol (6), deoxyschisandrin, schisantherin B, schisandrin, tigloylgomisin P, gomisin O, angeloylgomisin P, and epigomisin O. Their structure and stereochemistry were determined by spectroscopic methods, including 2D-NMR techniques. Compounds 1 and 3-6 were active as anti-HIV agents. (+/-)-Gomisin M1 (4) exhibited the most potent anti-HIV activity, with EC50 and therapeutic index (TI) values of <0.65 microM and >68, respectively.     

高效液相色谱法测定五味子属药用植物木脂素的含量

目的 :对五味子属植物的木脂素成分进行含量测定 ,探讨五味子药物资源问题。方法 :用HPLC对五味子属 14个种 (变种 ) 18个样品 (果实、茎藤和根 )中木脂素成分五味子醇甲 (schisandrin) ,五味子醇乙 (gomisinA) ,五味子酯甲 (schisantherinA) ,五味子甲素 (deoxyschizandrin) ,表 加巴辛 (d-epigalbacine) ,安五脂素 [(+) anwulig nan],五味子乙素 (wuweizisuB) ,6O 苯甲酰戈米辛O(6-O-benzoylgomisinO)和五味子丙素 (wuweizisuC)进行含量测定。结果 :5种不同种的五味子果实中 ,9种木脂素总量为 0.52%～1.96% ,其中以五味子和华中五味子含量最高 ,11种 (包括变种 )茎的 9种木脂素总量为 0.02%～1.51% ;小花五味子果实含有与华中五味子相似的木脂素成分 ,其中甲素的含量较高 (0.59%) ;绿叶五味子与五味子成分相似 ,其醇甲、醇乙和乙素的含量均较高 ;翼梗五味子果实、华中五味子果实、小花五味子茎、毛脉五味子茎和二色五味子根的安五脂素含量较高 ,分别 0.77% ,0.42% ,0.50% ,0.26% ,0.38% ;毛脉五味子茎、毛叶五味子茎和金山五味子茎的表 加巴辛含量较高 (分别为0.89% ,1.51% ,0.17% )。结论 :五味子属植物果实的木脂素含量高于根和茎 ;翼梗五味子果实、小花五味子茎、华中五味子果实、二色五味子根、毛脉五味子和毛脉五味子茎、金山五味子茎可分别作为安五脂素和表-加巴辛的植物资源。     

HPLC法测定不同产地五味子中三种木脂素类成分的含量

目的：采用HPLC法同时测定五味子中3种木脂素类成分（五味子醇甲、五味子甲素、五味子乙素）含量。方法：KromasilTMC18柱（250mm×4.6mm,5μm）;流动相：甲醇0.05%磷酸水溶液（80∶20）,流速1.0mL/min;柱温25℃,检测波长250nm。结果：五味子醇甲、五味子甲素、五味子乙素3种成分的线性范围分别是191.8-2685.2ng、664-4648ng、356-4984ng;平均加样回收率分别为99.98%、100.37%、100.03%;三者RSD分别为0.68%、0.98%、1.06%。结论：该方法精密度、重复性和分离效果好,分析快速准确,可用于五味子药材的质量控制,并且可以作为目前五味子国家标准的有益补充。     

五味子乙素提纯工艺及药理作用研究进展

五味子乙素是中药五味子中的重要活性成分,属联苯环辛烯类木脂素。现代药理学研究表明,五味子乙素具有抗炎、抗氧化、抗肿瘤等多种药理活性,但其在提纯和检测方面受到多重因素的限制。对近年来五味子乙素的提取工艺、检测方法和药理作用的研究进展进行概述,为其进一步开发和利用提供参考。     

Apoptotic Effect of Galbanic Acid via Activation of Caspases and Inhibition of Mcl‐1 in H460 Non‐Small Lung Carcinoma Cells

Galbanic acid (GBA), a major compound of Ferula assafoetida, was known to have cytotoxic, anti‐angiogenic and apoptotic effects in prostate cancer and murine Lewis lung cancer cells; the underling apoptotic mechanism of GBA still remains unclear so far. Thus, in the present study, the apoptotic mechanism of GBA was investigated mainly in H460 non‐small cell lung carcinoma (NSCLC) cells because H460 cells were most susceptible to GBA than A549, PC‐9 and HCC827 NSCLC cells. Galbanic acid showed cytotoxicity in wild EGFR type H460 and A549 cells better than other mutant type PC‐9 and HCC827 NSCLC cells. Also, GBA significantly increased the number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells and sub G1 population in H460 cells. Western blotting revealed that GBA cleaved poly (ADP‐ribose) polymerase (PARP), activated Bax and caspase 9, attenuated the expression of Bcl‐2, Bcl‐x_L, and Myeloid cell leukemia 1 (Mcl‐1) in H460 cells. However, interestingly, overexpression of Mcl‐1 blocked the ability of GBA to exert cytotoxicity, activate caspase9 and Bax, cleave PARP, and increase sub G1 accumulation in H460 cells. Overall, these findings suggest that GBA induces apoptosis in H460 cells via caspase activation and Mcl‐1 inhibition in H460 cells as a potent anticancer agent for NSCLC treatment. Copyright © 2015 John Wiley & Sons, Ltd.     

生脉散复方化学动态变化与药效关系研究——生脉散中五味子化学动态变化(Ⅶ)

目的:探讨生脉散全方及其不同配伍组中五味子主要化合物———五味子醇甲的含量动态变化及原因。方法:采用HPLC-UV测定生脉散中全方及各配伍组五味子醇甲的含量。结果:人参、麦冬、人参总皂苷、人参皂苷Rg₁与五味子合煎均能提高五味子醇甲的含量。结论:皂苷类化合物的存在对五味子醇甲的煎出有促进作用。     

Genistein inhibits Aβ25–35‐induced SH‐SY5Y cell damage by modulating the expression of apoptosis‐related proteins and Ca2+ influx through ionotropic glutamate receptors

In this study, we investigated the protective effects of genistein against SH‐SY5Y cell damage induced by β‐amyloid 25–35 peptide (Aβ_25–35) and the underlying mechanisms. Aβ‐induced neuronal death, apoptosis, glutamate receptor subunit expression, Ca²⁺ ion concentration, amino acid transmitter concentration, and apoptosis‐related factor expression were evaluated to determine the effects of genistein on Aβ‐induced neuronal death and apoptosis. The results showed that genistein increased the survival of SH‐SY5Y cells and decreased the level of apoptosis induced by Aβ_25–35. In addition, genistein reversed the Aβ_25–35‐induced changes in amino acid transmitters, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptors, and N‐methyl‐d ‐aspartate (NMDA) receptor subunits in SH‐SY5Y cells. Aβ_25–35‐induced changes in Ca²⁺ and B‐cell lymphoma‐2 (Bcl‐2) and Bcl‐2‐associated X (Bax) protein and gene levels in cells were also reversed by genistein. Our data suggest that genistein protects against Aβ_25–35‐induced damage in SH‐SY5Y cells, possibly by regulating the expression of apoptosis‐related proteins and Ca²⁺ influx through ionotropic glutamate receptors.     

北五味子活性成分提取工艺的研究

目的探讨北五味子活性成分的最佳提取工艺。方法以北五味子提取物出率及其五味子甲素、乙素、总木脂素等指标对北五味子乙醇回流提取法、乙醇渗滤提取和超临界流体萃取进行了对比研究。结果3种提取方法对比显示，虽五味子浸膏率无显著变化，但超临界流体萃取方法总木脂素、乙素、甲素的含量均高于传统回流和渗滤提取方法。结论超临界流体萃取应用于北五味子活性成分的提取，具有省时、高效、节能等优点，可作为工业生产提取的理论依据。     

北五味子活性成分提取工艺的研究

目的探讨北五味子活性成分的最佳提取工艺。方法以北五味子提取物出率及其五味子甲素、乙素、总木脂素等指标对北五味子乙醇回流提取法、乙醇渗滤提取和超临界流体萃取进行了对比研究。结果3种提取方法对比显示,虽五味子浸膏率无显著变化,但超临界流体萃取方法总木脂素、乙素、甲素的含量均高于传统回流和渗滤提取方法。结论超临界流体萃取应用于北五味子活性成分的提取,具有省时、高效、节能等优点,可作为工业生产提取的理论依据。     

Inhibition of UDP‐Glucuronosyltransferases (UGTs) Activity by constituents of Schisandra chinensis

Structure–activity relationship for the inhibition of Schisandra chinensis's ingredients toward (Uridine‐Diphosphate) UDP‐glucuronosyltransferases (UGTs) activity was performed in the present study. In vitro incubation system was employed to screen the inhibition capability of S. chinensis's ingredients, and in silico molecular docking method was carried out to explain possible mechanisms. At 100 μM of compounds, the activity of UGTs was inhibited by less than 90% by schisandrol A, schisandrol B, schisandrin, schisandrin C, schisantherin A, gomisin D, and gomisin G. Schisandrin A exerted strong inhibition toward UGT1A1 and UGT1A3, with the residual activity to be 7.9% and 0% of control activity. Schisanhenol exhibited strong inhibition toward UGT2B7, with the residual activity to be 7.9% of control activity. Gomisin J of 100 μM inhibited 91.8% and 93.1% of activity of UGT1A1 and UGT1A9, respectively. Molecular docking prediction indicated different hydrogen bonds interaction resulted in the different inhibition potential induced by subtle structure alteration among schisandrin A, schisandrin, and schisandrin C toward UGT1A1 and UGT1A3: schisandrin A > schisandrin > schisandrin C. The detailed inhibition kinetic evaluation showed the strong inhibition of gomisin J toward UGT1A9 with the inhibition kinetic parameter (K_i) to be 0.7 μM. Based on the concentrations of gomisin J in the plasma of the rats given with S. chinensis, high herb–drug interaction existed between S. chinensis and drugs mainly undergoing UGT1A9‐mediated metabolism. In conclusion, in silico‐in vitro method was used to give the inhibition information and possible inhibition mechanism for S. chinensis's components toward UGTs, which guide the clinical application of S. chinensis. Copyright © 2015 John Wiley & Sons, Ltd.     

Schisandrin B stereoisomers protect against hypoxia/reoxygenation‐induced apoptosis and associated changes in the Ca2+‐induced mitochondrial permeability transition and mitochondrial membrane potential in AML12 hepatocytes

The effects of the schisandrin B stereoisomers, (±)γ‐schisandrin [(±)γ‐Sch] and (?)schisandrin B [(?)Sch B], on hypoxia/reoxygenation‐induced apoptosis were investigated in AML12 hepatocytes. Changes in cellular reduced glutathione (GSH) levels, Ca²⁺‐induced mitochondrial permeability transitions (MPTs) and mitochondrial membrane potentials (Δψ_m values) were also examined in (±)γ‐Sch‐ and (?)Sch B‐treated cells, without or with hypoxia/reoxygenation challenge. The (±)γ‐Sch/(?)Sch B pretreatments (2.5–5.0 µm ) protected against hypoxia/reoxygenation‐induced apoptosis in AML12 cells in a concentration‐dependent manner, with the (?)Sch B effect being more potent. Drug antiapoptotic effects were further evidenced by suppression of hypoxia/reoxygenation‐induced mitochondrial cytochrome c release and subsequent cleavage of caspase 3 and poly‐ADP‐ribose polymerase by (?)Sch B pretreatment. Whereas hypoxia/reoxygenation challenge increased the extent of Ca²⁺‐induced MPT pore opening, and Δψ_m, in AML12 hepatocytes, cytoprotection afforded by (±)γ‐Sch/(?)Sch B pretreatment against hypoxia/reoxygenation‐induced apoptosis was associated with a decreased sensitivity to Ca²⁺‐induced MPT and an increased Δψ_m in both unchallenged and challenged cells, compared with the drug‐free control. The results indicate that (±)γ‐Sch/(?)Sch B pretreatment protected against hypoxia/reoxygenation‐induced apoptosis in AML12 hepatocytes and that the cytoprotection afforded by (±)γ‐Sch/(?)Sch B may at least in part be mediated by a decrease in sensitivity to Ca²⁺‐induced MPT, which may in turn result from enhancement of cellular GSH levels by drug pretreatments. Copyright © 2009 John Wiley & Sons, Ltd.     

大鼠在体肠灌注研究五味子有效成分肠吸收

目的:考察五味子醇提取物中五味子醇甲、五味子酯甲、五味子甲素和五味子乙素四种有效成分在大鼠体内肠吸收机制。方法:通过建立大鼠在体循环肠灌注模型并运用HPLC法检测四种有效成分在不同浓度下以及在P-gp抑制剂共同作用下的吸收差异。结果:五味子四种有效成分,在考察的浓度范围以内浓度增加一倍,吸收速率常数Ka和表观渗透系数却没有显著性差异(P>0.05)。加入P-gp抑制剂维拉帕米(VP)与正常组相比,五味子酯甲、甲素和乙素的吸收速率常数(Ka)和表观渗透系数(Papp)都显著增加(P<0.05)。结论:五味子四种有效成分的吸收并非简单的被动扩散,而是载体媒介转运,其中五味子酯甲、五味子甲素和五味子乙素为P-gp的底物,通过加入P-gp抑制剂可促进这三种成分的吸收。     

淫羊藿苷通过Wnt/β-catenin信号通路抑制淀粉样蛋白Aβ 25-35所诱导的神经毒性

 目的 研究淫羊藿苷（icariin）对β-淀粉样蛋白（Aβ_25-35）所致的大鼠肾上腺嗜铬细胞瘤细胞株（PC12细胞）神经毒性的抑制作用和潜在机制。 方法 将PC12细胞分为正常对照组、Aβ_25-35损伤组、淫羊藿苷与Aβ_25-35共同给药组、淫羊藿苷单独给药组,然后考察淫羊藿苷对PC12细胞损伤的保护作用,以及淫羊藿苷对Wnt/β-catenin信号通路的调节作用。结果 淫羊藿苷可以显著的抑制Aβ_25-35所诱导的PC12细胞神经毒性,提高细胞存活率,此外其还可以降低凋亡相关蛋白caspase-3和PARP的激活。机制研究发现,淫羊藿苷可以激活Wnt/β-catenin信号通路中的关键蛋白β-catenin的活性,抑制其磷酸化,并促进该蛋白向细胞核中的转运。此外,淫羊藿苷还可以通过调节不同的磷酸化位点来抑制GSK-3β信号蛋白的活化。结论 淫羊藿苷可显著抑制Aβ_25-35所致的PC12细胞的毒性作用,其潜在机制可能是通过激活Wnt/β-catenin信号通路来实现的。     

Difference in antioxidation for schisandrins and schisantherin between bio- and chemo- systems

The difference in antioxidation and the mechanism of action of schisandrins and schisantherin in biological and chemical systems was investigated. The antioxidative activities of schisandrin A (SA), schisandrin B (SB) and schisantherin D (SD) isolated from Schisandra chinensis Baill were studied on haemolysis of mouse erythrocytes and on autooxidation of linoleic acid induced by a water-soluble free radical initiator, 2,2′-azobis(2-amidino-propane)dihydrochloride. It was found that SA, SB and SD inhibited haemolysis of erythrocytes but did not inhibit peroxidation of linoleic acid in micelles. Chlorhexidine, an inhibitor of demethylase, significantly inhibited the antioxidative activities of SA, SB and SD on haemolysis. The results indicated that the methoxy groups in SA, SB and SD may be demethylated by demethylase contained in erythrocytes and metabolized to produce phenolic hydroxyl groups which inhibit the peroxidation of erythrocyte membranes. © 1997 John Wiley & Sons, Ltd.