北五味子及其不同炮制品中五味子甲素 和乙素的含量测定

目的:测定北五味子及其不同炮制品中五味子甲素、五味子乙素的含量。方法:采用高效液相色谱法,Spherisorb 10ODS1(4.6mm×250mm,10μm),以甲醇-水(70:30)为流动相,检测波长为254nm。线性范围0.1~0.5μg,相关系数为0.995.平均回收率为100.3%和100.4%,RSD=0.87%和2.85%(n=5)。结果:五味子甲素和乙素在生品、醋炙、炒制、酒炙、蒸炙、蜜炙品中的平均含量分别为0.13%,0.064%,0.052%,0.064%,0.11%,0.13%和0.21%,0.298%,0.238%,0.302%,0.216%,0.214%。结论:方法准确,快速,重现性好,可用于五味子各种炮制品的质量评价。     

不同贮藏条件对五味子质量的影响

基于多元活性成分同时测定结合多元统计分析探讨不同贮藏条件对五味子药材质量的影响。采用超快速液相色谱-三重四极杆/线性离子阱质谱(UFLC-QTRAP-MS/MS)同时测定不同贮藏条件(包装材料,贮藏温度)五味子中木脂素(五味子酯乙、五味子醇乙、五味子丙素、五味子乙素、五味子甲素、五味子酯甲、五味子醇甲、五味子酚、戈米辛D、戈米辛J、当归酰基戈米辛H)及有机酸(L-苹果酸、酒石酸、原儿茶酸、奎宁酸)共15种指标成分的含量;根据15种目标成分的含量,用灰色关联度分析和TOPSIS法对不同贮藏五味子进行综合评价。结果表明,15种化合物在一定浓度范围内均呈现良好的线性关系,相关系数均大于0.999 1;精密度、重复性和稳定性良好;平均加样回收率在96.64%～99.96%之间,RSD均小于5%。灰色关联度分析中r_i的最大差异较小为57.5%,TOPSIS法中C_i值的最大差异较大为81.3%,两种结果均显示S4、S3、S1的综合质量较好,五味子的适宜贮藏条件为以聚乙烯密封袋为外包装存放于阴凉库。所建立的方法准确、可靠,可用于五味子药材内在质量的综合评价,本研究可为五味子适宜储藏条件的优选提供基础资料。     

南五味子药材UPLC指纹图谱及一测多评法研究

建立南五味子药材超高效液相色谱(UPLC)指纹图谱和6种木脂素类成分一测多评法(QAMS),为南五味子药材质量标准提高提供参考。采用UPLC法建立南五味子药材指纹图谱,运用聚类分析(HCA)和正交偏最小二乘法-判别分析(OPLS-DA)对指纹图谱进行分析;以五味子酯甲为内参物,建立五味子酯丙、五味子酯乙、安五脂素、五味子甲素和五味子酯丁的一测多评法,并与外标法(ESM)测定结果对比,判断方法的准确性。14批南五味子药材指纹图谱确定了15个共有峰,通过对照品比对指认了其中7个成分,分别为原儿茶酸、五味子酯丙、五味子酯甲、五味子酯乙、安五脂素、五味子甲素、五味子酯丁,其指纹图谱相似度均在0.95以上;通过聚类分析(HCA)和正交偏最小二乘法-判别式分析(OPLS-DA)可以将南五味子药材分为4类,峰1(原儿茶酸)、峰2、峰4、峰6、峰7(五味子酯乙)、峰8、峰9、峰12、峰15共9个成分是导致产地差异性的主要标志物。一测多评法与外标法测定的五味子酯丙、五味子酯乙、安五脂素、五味子甲素和五味子酯丁含量结果无显著差异。所建立的方法简便、可靠,为南五味子药材质量评价提供参考。     

五味子乙素对大鼠成骨细胞增殖分化的影响

目的:探讨五味子乙素体外对大鼠成骨细胞增殖与分化的影响。方法:用改良的组织块法分离培养新生大鼠颅骨成骨细胞,五味子乙素以不同浓度加入细胞培养体系,作用不同时间后,用MTT法检测成骨细胞的增殖情况;用对硝基苯二钠基质动力学法(PNPP)测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量。结果:五味子乙素在0.75×10-4 mol/L 24 h,48 h及72 h,及0.75×10-5mol/L 24 h促进成骨细胞的增殖,在0.75×10-6mol/L24 h提高成骨细胞内碱性磷酸酶的活性。结论:五味子乙素体外能促进成骨细胞的增殖与分化。     

五味子属植物木脂素类资源的研究

本文调查了15个省区71个样品的15种五味子属植物。应用高效液相色谱法,对8种木脂素有效成分在五味子属植物中的含量进行了分析,这8种木脂素提五味子醇甲(schizandrol A)、醇乙(schizandrol B)、酯甲、酯乙(gomisinC,B)、五味子酚(schizanhenol)、甲素(deoxyschizandrin)、乙素(γ-schizandrin)及丙素(schizandrin C),同时对12种五味子的醇提物进行了主要药理作用的比较。根据研究结果,对各种五味子的质量作出评价以及可供开发利用的意见。     

不同产地五味子果实中甲素和乙素的差异性

研究五味子果实中的甲素和乙素含量是否存在产地闻差异,分析其影响因素,为中草药生产、加工企业选择五味子果实药效显著、品质优良的产地提供依据。收集了16个产地的五味子果实,采用高效液相色谱仪分别测定五味子甲素、乙素含量。结果表明,五味子甲素、乙素含量在产地间差异显著,二者含量最高的产地分别是最低产地的2．14倍和3．3倍。将各产地的五味子甲素、乙素含量与原产地的地理气候因子进行相关分析表明,甲素含量与主要气候因子中的温度、湿度呈显著正相关关系;乙素与温湿度相关不显著。根据主要药效成分含量,初步选择黑龙江省的苇河、吉林省的柳河为五味子品质优良产地。     

LC-MS/MS法分析北五味子中的木脂素成分

建立了同时测定北五味子果实中6种木脂素含量的高效液相色谱-离子阱质谱方法。样品经正己烷提取,提取液过滤后通过旋转蒸发仪浓缩近干,用甲醇溶解,然后采用LC-MS/MS进行测定。6种木脂素在0.005～10.0 mg/g范围内呈良好线性,相关系数为0.9907～0.9998。6种木脂素的定量限为4.0×10-4～1.5×10-3mg/g,平均添加回收率为81.01%～92.45%,相对标准偏差为3.05%～7.98%。采用本文所建立的方法对北五味子果实进行测定,获得了很好的分离效果,分析结果令人满意。     

五味子乙素对大鼠肝星状细胞增殖和胶原基因表达合成的影响

目的观察五味子乙素在大鼠肝星状细胞（HSCs）增殖,胶原表达合成方面的作用。方法经在体灌流消化大鼠肝脏分离HSCs,培养于DMEM培养基中,用^3H-TdR和^3H-pro掺入试验测定五味子乙素对HSCs的增殖和胶原合成影响,并用原位杂交方法探讨了其对HSCⅠ、Ⅲ型前胶原基因的表达作用。结果对于培养活化的HSCs,五味子乙素对HSCs摄取^3H-TdR没有明显影响,但在40μmol/L时,剂量依赖地抑制HSCs摄取^3H-proline,并在80μmol/L降低Ⅰ型前胶原基因表达（P〈0.05）。结论五味子乙素具有降低HSCs胶原基因表达合成作用。     

五味子乙素联合紫杉醇对非小细胞肺癌细胞上皮-间充质转化的作用

[目的]探讨五味子乙素联合紫杉醇是否通过调控PI3K/Akt/mTOR信号通路抑制非小细胞肺癌细胞上皮-间充质转化。[方法]选择人肺癌A549细胞,设PBS对照组、紫杉醇、五味子乙素、紫杉醇+五味子乙素组、紫杉醇+五味子乙素+PI3K抑制剂LY294002组。采用CCK-8检测细胞增殖;流式细胞术分析细胞凋亡及细胞周期;划痕实验检测细胞迁移能力;Transwell小室实验检测细胞侵袭能力;免疫荧光染色检测N-cadherin、Vimentin分布;qRT-PCR检测细胞E-Cadherin、N-Cadherin、Vimentin、Snail、Slug mRNA表达;Western Blotting检测E-Cadherin、N-Cadherin、Vimentin、Snail、Slug、p-PI3K、p-Akt、p-mTOR蛋白表达。[结果]与PBS对照组比较,紫杉醇+五味子乙素组细胞增殖率明显降低、细胞凋亡率明显增加(P<0.05),E-Cadherin mRNA和蛋白表达量明显增加,N-Cadherin、Vimentin、Snail、Slug mRNA和蛋白表达量明显降低(P&l...     

正交设计优选五味子果实中总木脂素提取工艺

目的:比较不同方法对北五味子总木脂素提取的影响,优选最佳提取工艺方法。研究五味子木脂素抗微波辐射损伤作用。方法:以干燥北五味子果实为原料,分别采用传统加热回流法、超声提取法、微波提取法、微波—超声提取法提取北五味子总木脂素,对这四种提取方法分别进行正交试验分析,以确定最佳提取工艺。结果:通过结果比较分析,以微波提取工艺结果最佳,在微波提取功率为200W,乙醇体积分数为80%,提取时间为40min,料液比为1:12的提取条件下,北五味子总木脂素的产量达到了16.44mg/g,使得木脂素的提取产量较传统加热回流法、超声提取法、微波—超声提取法得到了显著的提高。     

Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC₂₀ values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca²⁺-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl₂ by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca²⁺-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca²⁺ influx through L-type Ca²⁺ channels and intracellular Ca²⁺ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.     

Effect of Tacrolimus on the pharmacokinetics of bioactive lignans of Wuzhi tablet (Schisandra sphenanthera extract) and the potential roles of CYP3A and P-gp

We recently reported that Wuzhi tablet (WZ), a preparation of the ethanol extract of Wuweizi (Schisandra sphenanthera), had significant effects on blood concentrations of Tacrolimus (FK506) in renal transplant recipients and rats. The active lignans in WZ are schisandrin A, schisandrin B, schisandrin C, schisandrol A, schisandrol B, schisantherin A, and schisantherin B. Until now, whether the pharmacokinetics of these lignans in WZ would be affected by FK506 remained unknown. Therefore, this study aimed to investigate whether and how FK506 affected pharmacokinetics of lignans in WZ in rats and the potential roles of CYP3A and P-gp. After a single oral co-administration of FK506 and WZ, the blood concentration of lignans in WZ was decreased by FK506; furthermore, the AUC of schisantherin A, schisandrin A, schisandrol A and schisandrol B was only 64.5%, 47.2%, 55.1% and 57.4% of that of WZ alone group, respectively. Transport study in Caco-2 cells showed that these lignans were not substrates of P-gp, suggesting decreased blood concentration of lignans by FK506 was not via P-gp pathway. Metabolism study in the human recombinant CYP 3A showed that these lignans had higher affinity to CYP3A than that of FK506, and thus had a stronger CYP3A-mediated metabolism. It was concluded that the blood concentrations of these lignans were decreased and their CYP3A-mediated metabolisms were increased in the presence of FK506 since these lignans had higher affinity to CYP3A.     

Schisandrin B--a novel inhibitor of P-glycoprotein

P-glycoprotein-mediated drug efflux is one of the major causes of the cancer multidrug resistance (MDR). Inhibition of P-glycoprotein could reverse cancer MDR. Here, we show that schisandrin B, a naturally occurring compound from Schisandra chinensis (Turcz.) Baill, bears strong potency to inhibit P-glycoprotein. Schisandrin B reversed the drug resistance of four MDR cell lines characterized with overexpression of P-glycoprotein and fully restored the intracellular drug accumulation by interacting with P-glycoprotein. Schisandrin B has a core structure of dibenzocyclooctadiene, representing a novel P-glycoprotein inhibitor. To our best knowledge, the role of schisandrin B to inhibit P-glycoprotein has not been reported.     

Simultaneous quantification of four active schisandra lignans from a traditional Chinese medicine Schisandra chinensis(Wuweizi) in rat plasma using liquid chromatography/mass spectrometry

A simple, rapid and sensitive method was developed for the simultaneous quantification of four active schisandra lignans (schisandrin, schisantherin A, deoxyshisandrin and gamma-schisandrin) from a traditional Chinese medicine Schisandra chinensis(Wuweizi) in rat plasma using a high-performance liquid chromatography system coupled to a positive ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of three volumes of methanol followed by centrifugation. The analytes and internal standard (IS) bicyclol were separated on a Zorbax SB-C18 column (3.5 microm, 2.1 mm x 100 mm) with mobile phase of methanol/water (70:30, v/v) containing 0.1% formic acid at a flow rate of 0.2 mL/min with an operating temperature of 25 degrees C. Detection was performed on a Trap XCT mass spectrometer equipped with an electrospray ionization (ESI) source operated in selected ion monitoring (SIM) mode. Positive ion ESI was used to form sodium adduct molecular ions at m/z 455 for schisandrin, m/z 559 for schisantherin A, m/z 439 for deoxyshisandrin, m/z 423 for gamma-schisandrin, and m/z 413 for the internal standard bicyclol. Linear detection responses were obtained for the four test compounds ranging from 0.010 to 2.0 microg/mL and the lower limits of quantitation (LLOQs) for four lignans were 0.010 microg/mL. The intra- and inter-day precisions (R.S.D.%) were within 12.5% for all analytes, while the deviation of assay accuracies was within +/-13.0%. The average recoveries of analytes were greater than 80.0%. All analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic study of the four lignans after oral administration of Schisandra chinensis extraction to rats.     

Rapid discrimination of Schisandra sphenanthera and Schisandra chinensis using electronic tongue and ultra-performance liquid chromatography coupled with chemometrics

Both Schisandra sphenanthera (S. sphenanthera) and Schisandra chinensis (S. chinensis) are used as traditional Chinese medicines, but they have different medicinal properties. Because S. sphenanthera is cheaper, it is often used as a counterfeit product for S. chinensis. In the present study, an electronic tongue (e-tongue) was used for discrimination of the two Schisandraceae species. In addition, the contents of schisandrin, schizandrol B, schisantherin A, deoxyschizandrin, and schisandrin B were determined simultaneously by ultra-performance liquid chromatography. Principal component analysis (PCA) and discriminant factor analysis (DFA) were used to establish the mathematical models for species identification, and the classification rates for both methods reached 100%. The e-tongue coupled with multivariate analysis exhibited the excellent performance and classification accuracy, and this was validated by the ultra-performance liquid chromatography results. This simple e-tongue technique could be useful for rapid and accurate identification of S. sphenanthera and S. chinensis.     

Dibenzocyclooctadiene lignans: a class of novel inhibitors of multidrug resistance-associated protein 1

We recently reported that dibenzocyclooctadiene lignans were a novel class of P-glycoprotein (P-gp) inhibitors. In this study, we demonstrated that the lignans of this class were also effective inhibitors of multidrug resistance-associated protein 1 (MRP1). The activities of 5 dibenzocyclooctadiene lignans (schisandrin A, schisandrin B, schisantherin A, schisandrol A, and schisandrol B) to reverse MRP1-mediated drug resistance were tested using HL60/Adriamycin (ADR) and HL60/Multidrug resistance-associated protein (MRP), two human promyelocytic leukemia cell lines with overexpression of MRP1 but not P-gp. The five lignans could effectively reverse drug resistance of the two cell lines to vincristine, daunorubicin, and VP-16. This study, together with our previous reports, proves that dibenzocyclooctadiene lignans have multiple activities against cancer multidrug resistance, including inhibition of P-gp and MRP1, and enhancement of apoptosis. Considering that cancer multidrug resistance (MDR) is multifactorial, agents with broad activities are preferable to the use of combination of several specific modulators to prevent drug-drug interaction and cumulative toxicity.     

Splice isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4: Expression and hypoxic regulation

Using an ex vivo model of isolated–perfused rat hearts and cultured H9c2 cells, the structure–activity relationships of schisandrin B (Sch B), and analogs lacking either the methylendioxy group or cyclooctadiene ring, schisandrin A (Sch A) and dimethyl diphenyl bicarboxylate (DDB), respectively, were investigated. Pretreatment with Sch B, but not with Sch A or DDB, protected against myocardial ischemia–reperfusion (I-R) injury in rats. Although Sch B pretreatment largely prevented H9c2 cells from menadione-induced cytotoxicity, Sch A pretreatment produced only a marginal protection. However, DDB pretreatment did not cause any detectable effect. The myocardial and cellular protection afforded by Sch B pretreatment correlated with increases in mitochondrial ATP generation capacity and/or reduced glutathione level as well as heat shock protein (Hsp)25/70 expression, under both control and oxidative stress conditions. The results indicate that the methylenedioxy group and the cyclooctadiene ring are important structural determinants of Sch B in enhancing mitochondrial functional ability and glutathione status, as well as tissue Hsp25/70 expression, thereby protecting the myocardium against I-R injury.     

Schisandrin B protects against menadione-induced hepatotoxicity by enhancing DT-diaphorase activity

Pretreating mice with schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, at a daily dose of 1 mmol/kg for 3 days protected against menadione-induced hepatic oxidative damage in mice, as evidenced by decreases in plasma alanine aminotransferase activity (78%) and hepatic malondialdehyde level (70%), when compared with the menadione intoxicated control. In order to define the biochemical mechanism involved in the hepatoprotection afforded by Sch B pretreatment, we examined the activity of DT-diaphorase (DTD) in hepatocytes isolated from Sch B pretreated rats. Hepatocytes isolated from Sch B pretreated (a daily dose of 1 mmol/kg for 3 days) rats showed a significant increase (25%) in DTD activity. The increase in DTD activity was associated with the enhanced rate of menadione elimination in the hepatocyte culture. The ensemble of results suggests that the ability of Sch B pretreatment to enhance hepatocellular DTD activity may at least in part be attributed to the protection against menadione hepatotoxicity.