固定化脂质体色谱研究中药复方的细胞膜通透性成分及其质量控制

提出了固定化脂质体色谱(immobilized liposome chromatography,ILC)研究中药复方的细胞膜通透性成分及其质量控制的方法。以当归补血汤及其组分为例,考察了它们在ILC色谱柱上的分离效果;当归补血汤水提取液和75％乙醇提取液在ILC色谱柱上各有8个保留峰,当归水提取液、甲醇提取液和75％乙醇提取液在ILC色谱柱上分别有3、6、9个保留峰,黄芪水提取液、甲醇提取液和75％乙醇提取液在ILC色谱柱上各有7个保留峰;建立了当归补血汤及其组分中阿魏酸和蒿本内酯的定量分析方法,当归补血汤水提取液和75％乙醇提取液中阿魏酸的含量分别为0．0743％和0．0688％,蒿本内酯的含量分别为0．0472％和0．457％,当归水提取液和75％乙醇提取液中阿魏酸的含量分别为0．0694％和0．0691％,蒿本内酯的含量分别为0．0781％和0．455％。     

联用色谱及多组分光谱相关色谱用于当归补血汤与单味药成分的比较研究

摘要利用HPLC-DAD提供的二维化学数据信息, 通过多组分光谱相关色谱方法及化学计量学分辨技术, 对相同试验条件下所得的当归补血汤及其单味药的色谱组分进行了比较和归属分析. 结果表明， 在当归补血汤色谱响应值较高的21个色谱峰中, 8个色谱峰的组分来自当归, 6个色谱峰的组分来自黄芪, 7个色谱峰的组分同时来源于当归和黄芪. 其中一些色谱峰簇中的其它组分发生了消长变化， 既有新物质的产生, 又有原有化学成分的消失， 说明中药复方配伍的药效物质基础不仅是各单味药化学成分的总和, 还有单味药在复方配伍过程中各种成分的相互作用. 本方法为中药复方与单味药化学组分的比较及归属分析, 尤其对重叠色谱峰簇的组分比较分析提供了方便、 快捷及可行的研究方法.     

褪色光度法测定Fenton反应产生的羟自由基及其应用

建立检测Fenton反应产生羟自由基的新方法。Fenton反应产生的羟自由基与苋菜红反应，颜色发生变化，用分光光度计测定其△A510值的变化，可间接测定羟自由基的生成量。通过对测定条件的研究，确定了体系最佳实验条件。抗氧化药物甘露醇、硫脲与羟自由基清除率具有明显的量效关系。测定了阿魏酸、芦丁等几种中药活性成分清除羟自由基的功能，此法可用于羟自由基清除剂的筛选及抗羟自由基机理研究。     

水杨基荧光酮荧光法测定钴（II）—过氧化氢系产生的羟自由基

本实验发现Co^2 与H2O2反应生羟自由基的产率比Fenton试剂的高100倍以上,采用水杨基荧光酮(salicyfluorone,SAF)-Co(II)-H2O2荧光法测定羟自由基的新体系,激发波长和发射波长为510nm和500nm,测定体系在反应前后的荧光变化,可间接测定羟自由基产生量,此法不需昂贵的仪器设备,方法灵敏,操作简单易行,清除率实难及ESR波谱法检测证明了该方法的准确可靠,对于医用筛选抗羟自由基药物及抗羟自由基机理研究等方面应用价值.     

药用百合提取液对羟自由基清除作用的研究

在Fenton反应产生羟自由基的稳定体系中,以溴邻苯三酚红为显色剂,用分光光度法测定吸光度的变化值,研究百合提取液在此体系中清除羟自由基的作用。通过常见抗氧化剂对羟自由基清除作用的对照试验表明,百合提取液对羟自由基有较好的清除效果,此法操作简便,稳定性好,测定快速,为开发百合复方保健品提供理论依据。     

罗丹明6G-锰(Ⅱ)-过氧化体系荧光法测定常见油性种子的抗氧化活性

碱性介质中罗丹明6G能产生特征荧光,其最大激发波长和发射波长分别为350 nm和550 nm;Mn2+-H2O2体系在碱性介质中产生的羟自由基可以迅速氧化罗丹明6G使其荧光猝灭,而油性种子的浸提物可以清除羟自由基,从而使溶液的荧光猝灭程度降低,据此建立了测定油性种子抗氧化性的新方法.实验观察到抗氧化剂维生素C和硫脲等在低浓度范围内对羟自由基的清除率与用量呈上升关系,而在大浓度下反而下降.讨论了样品的水、醋酸、乙醇溶剂浸提物对羟自由基的清除效率.用水、醋酸作提取剂,分别测试了7种常见油性种子的抗氧化活性.     

罗丹明6G-锰（Ⅱ）-过氧化氢体系荧光法测定常见油性种子的抗氧化活性

碱性介质中罗丹明6G能产生特征荧光，其最大激发波长和发射波长分别为350nm和550nm；Mn^2 -H2O2体系在碱性介质中产生的羟自由基可以迅速氧化罗丹明6G使其荧光猝灭，而油性种子的浸提物可以清除羟自由基，从而使溶液的荧光猝灭程度降低，据此建立了测定油性种子抗氧化性的新方法。实验观察到：抗氧化剂维生素C和硫脲等在低浓度范围内对羟自由基的清除率与用量呈上升关系，而在大浓度下反而下降。讨论了样品的水、醋酸、乙醇溶剂浸提物对羟自由基的清除效率。用水、醋酸作提取剂，分别测试了7种常见油性种子的抗氧化活性。     

鸡足山耳蕨总黄酮的提取及抗氧化性研究

研究鸡足山耳蕨中总黄酮的提取及其抗氧化性.采用70％乙醇提取鸡足山耳蕨中总黄酮,用NaNO2Al(NO3)3-NaOH分光光度法测定黄酮含量,将提取液采用Fenton体系、普鲁士蓝法进行体外抗氧化活性研究,用硫代巴比妥酸(TBA)分光光度法研究其对羟自由基·OH引发DNA氧化损伤的抑制作用.结果表明样品中总黄酮含量为4.98％,回收率99.78％( RSD=1.06％,n=5).总黄酮浓度为90μg/mL时,对·OH的清除率可达36.2％;浓度为87.5μg/mL时,对羟自由基引发DNA损伤的抑制率可达93.0％.说明鸡足山蕨中总黄酮对羟自由基有较好的清除能力,对DNA氧化损伤有显著抑制作用.     

亮绿褪色光度法检测Fenton反应产生的羟自由基

建立了Fg^2＋-H2O2-亮绿的分析新体系,并用于羟自由基的检测。该法利用Fenton反应产生羟自由基,并加入亮绿显色剂,羟自由基使亮绿褪色。在632nm处测其△A值的变化,可间接测定羟自由基的产生量。探讨了最佳实验条件,并对苯甲酸与丹参等中草药的抗羟自由基氧化性能作了比较,以验证本法的有效性,得到较好的清除结果。该法稳定,可作为一种简便的筛选抗氧化剂的方法。     

中药配伍的统一分析方法研究 Ⅱ.拟合定量

 配伍过程中物质组分的浓度变化需要进行定量 ,但复方中大量的重叠色谱峰影响定量的准确性。而在色谱理论基础上发展起来的拟合定量技术 ,可以对重叠色谱峰进行拟合定量。在统一分离和复方中单味药的特征物质组分得到识别的基础上 ,以黄芪、当归用量变化时 ,复方“当归补血汤”中黄芪、当归的特征物质组分的拟合定量为实例 ,介绍了中药配伍过程中定量的手段和研究方法     

当归多糖ASP3的甲基化分析

通过部分酸水解和甲基化分析, 结合GC-MS测试手段, 对当归多糖ASP3的糖链结构进行了研究. 采用0.2 mol/L三氟乙酸(Trifluoroacetic acid, TFA)对ASP3进行了部分酸水解, 对水解前后的多糖组分进行甲基化分析. 结果显示: ASP3的糖残基主要由D-GalpA, D-Galp, L-Araf和L-Rhap组成, 主链由1,4-D-GalpA连接, 形成“光滑区”半乳糖醛酸聚糖, 由1,4-D-GalpA通过O-4位与1,2-; 1,2,4-L-Rhap的O-2位交替连接形成含有较多分支的“毛发区”鼠李半乳糖醛酸聚糖. 58.8%的Rhap残基发生O-4位取代(1,2,4-L-Rhap). 由T-, 1,5-, 1,3,5-Araf和T-, 1,3-, 1,3,6-, 1,4-, 1,4,6-D-Galp聚合形成的阿拉伯半乳聚糖、半乳聚糖以及阿拉伯聚糖是ASP3侧链的主要组成, 通过Rhap残基的O-4位与主链相连.     

Metabonomic analysis of the anti‐inflammatory effects of volatile oils of Angelica sinensis on rat model of acute inflammation

Metabonomics based on GC‐MS was used to study the possible anti‐inflammatory mechanisms of volatile oils of Angelica sinensis (VOAS) in rats with acute inflammation. Acute inflammation was induced by subcutaneous injection of carrageenan in rats. The levels of prostaglandin E₂ (PGE₂), histamine (HIS) and 5‐hydroxytryptamine (5‐HT) in the inflammatory fluid were detected. Principal component analysis and orthogonal partial least squares‐discriminant analysis models were performed for pattern recognition analysis. After the administration of VOAS, the levels of PGE₂, HIS, and 5‐HT returned to levels observed in normal group. According to GC‐MS analysis, the intervention of VOAS in rats with acute inflammation induced substantial and characteristic changes in their metabolic profiles. Fourteen metabolite biomarkers, namely, lactic acid, malic acid, citric acid, trans‐dehydroandrosterone, aldosterone, linoleic acid, hexadecanoic acid, pregnenolone, octadecenoic acid, myristic acid, l ‐histidine, octadecanoic acid, arachidonic acid (AA) and l ‐tryptophan, were detected in the inflammatory fluid. The levels of all biomarkers either increased or decreased significantly in model groups. VOAS possibly intervened in the metabolic process of inflammation by altering histidine metabolism, tryptophan metabolism, AA metabolism, steroid hormone biosynthesis, fatty acid metabolism and energy metabolism. Metabonomics was used to reflect an organism's physiological and metabolic state comprehensively, and it is a potentially powerful tool that reveals the anti‐acute‐inflammatory mechanism of VOAS. Copyright © 2014 John Wiley & Sons, Ltd.     

超高效液相色谱-串联质谱法测定白芷中51种禁用农药的残留量

将白芷药材粉碎至通过孔径为(250±9.9)μm的网筛,并称取此白芷粉末样品2.00g于聚丙烯管中与水10mL混匀,再加入含1%(体积分数)乙酸的乙腈10.0mL,涡旋1min后加入无水硫酸镁4g和乙酸钠1g,剧烈振荡3min,离心5min,取其上清液5.0mL置于已盛有无水硫酸镁900mg、N-丙基乙二胺(PSA)450mg和硅胶300mg的净化管中,剧烈振荡5min使净化完全,离心5min。移取上清液3.0mL于40℃水浴中减压蒸缩至近干。加入50μg·L^-1的磷酸三苯酯内标溶液300μL,加入乙腈定容至1.0mL,经0.20μm滤膜过滤,取其滤液,按仪器工作条件进行超高效液相色谱-串联质谱法分析。选用Eclipse Plus C18色谱柱和以不同比例的(A)含10mmol·L^-1甲酸铵的0.1%(体积分数)甲酸溶液和(B)乙腈的混合液为流动相,按梯度洗脱程序对白芷中可能残留的51种禁用农药进行色谱分离,并在电喷雾离子源正、负离子(ESI+和ESI-)电离方式和动态多反应监测(dMRM)模式条件下进行串联质谱法测定。采用基质匹配标准曲线法定量。51种农药的质量浓度在一定范围内与其对应的峰面积呈线性关系,并测得其检出限(3S/N)为0.3~5.0μg·kg^-1。标准加入法回收试验的结果显示,其中大部分农药的回收率在71%以上,测定值的相对标准偏差(n=6)均小于20%。按此方法分析了70批白芷样品,共检出16种农药,其中甲拌磷亚砜的检出率达95%,最高检出量为15.2μg·kg^-1;甲拌磷砜和毒死蜱的检出率均为10%,最高检出量依次为3.5,45.0μg·kg^-1,其余13种农药的检出率均低于5%,且检出量均较低。     

Urinary metabolomics analysis reveals the effect of volatile oil from Angelica sinensis on LPS‐induced inflammation rats

Lipopolysaccharide (LPS)‐induced inflammation occurs commonly and volatile oil from Angelica sinensis (VOAS) can be used as an anti‐inflammatory agent. The molecular mechanisms that allow the anti‐inflammatory factors to be expressed are still unknown. In this paper, we applied gas chromatography–mass spectrometry (GC–MS) and high‐performance liquid chromatography–time‐of‐flight mass spectrometry (LC‐Q/TOF–MS) based on a metabolomics platform coupled with a network approach to analyze urine samples in three groups of rats: one with LPS‐induced inflammation (MI); one with intervention with VOAS; and normal controls (NC). Our study found definite metabolic footprints of inflammation and showed that all three groups of rats, MI, intervention with VOAS and NC have distinct metabolic profiles in urine. The concentrations of 48 metabolites differed significantly among the three groups. The metabolites in urine were screened by the GC–MS and LC‐Q/TOF–MS methods. The significantly changed metabolites (p < 0.05, variable importance in projection > 1.5) between MI, NC and VOAS were included in the metabolic networks. Finally, hub metabolites were screened, including glycine, arachidonic acid, l ‐glutamate, pyruvate and succinate, which have high values of degree (k). the Results suggest that disorders of glycine, arachidonic acid, l ‐glutamate, pyruvate and succinate metabolism might play an important part in the predisposition and development of LPS‐induced inflammation. By applying metabolomics with network methods, the mechanisms of diseases are clearly elucidated.     

Preparation and Identification of Angelica sinensis Polysaccharide-iron Complex

Angelica sinensis polysaccharide(ASP) was extracted from Angelica sinensis by boiling water. An Angelica sinensis polysaccharide-iron complex(APC) was prepared under the alkaline condition by adding a ferric chloride solution to the ASP solution. Then some identifiable properties of the complex were studied. The content of iron (Ⅲ) in the complex was determined with iodometry. The thermal property, the microscopic structure, the spectral characteristics, and N, C, H contents of the complex were examined by a variety of techniques including DSC, TEM, IR,NMR, and elemental analysis. The content of iron (Ⅲ) in the complex ranges from 10% to 40%. The DSC result shows that the melting point of the complex is about 450 ℃. The TEM result shows that the complex has an iron(Ⅲ)core(β-FeOOH core) linked by hydroxy and oxy bridges, with the polysaccharide chains attached to the surface of the core. The IR and NMR results also show that there is aβ-FeOOH core in the complex. The elemental analysis shows that the contents of N, C , H in the complex are, respectively, lower than those of N, C, H in ASP. All our studies indicate that the APC consists of aβ-FeOOH core surrounded by ASP.     

Simultaneous determination of eight components in Angelica sinensis based on UHPLC‐ESI‐MS/MS method for quality evaluation

A method employing ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) for determination of eight components including ferulic acid, senkyunolide A, butylphthalide, ligustilide, butylidenephalide, senkyunolide I, senkyunolide H and levistolide A in Angelica sinensis was established. The separation was carried out using a Waters ACQUITY UHPLC BEH C₁₈ column with gradient elution with 0.1% formic acid aqueous and acetonitrile at a flow rate of 0.4 mL/min. Good linearity was attained with R² of 0.9983–0.9998 in wide concentration ranges. The method had limit of detection (LOD) and quantitation (LOQ) in the range of 0.42–6.98 ng/mL and 1.39–23.28 ng/mL, respectively. Intra‐ and inter‐day precisions varied with relative standard deviations (RSDs) from 0.33% to 0.88% and 0.37% to 1.04%, respectively. Moreover, the average recoveries were in a satisfactory range of 92.7%–102.1% with RSDs of less than 3.60%. Finally, the method was successfully applied to analyze 19 batches of A. sinensis samples grown in Min County, Gansu province, China, as well as that collected in other regions. The findings indicated that the established method is reliable and may thus be applied as a powerful tool for qualitative and quantitative analysis of components in A. sinensis, which has its implications in quality control of A. sinensis.     

明日叶水溶性总糖含量的测定

采用苯酚-硫酸比色法测定明日叶水溶性总糖含量.以葡萄糖为标准品,6％苯酚溶液和浓硫酸作显色剂,应用分光光度法在490 mn波长处测定样品的吸光度,按标准曲线计算总糖含量.实验结果表明,明日叶水溶性总糖提取物中总糖含量（质量分数）为17.82％,实验精密度及重复性好.高、中、低三种不同浓度加样量加标回收率分别为101.55％、100.42％、101.26％,相对标准偏差（RSD）分别为：1.67％、0.76％、1.38％,回收率高.此方法操作简便,重复性、稳定性好,准确度高,可为明日叶水溶性总糖含量测定提供可靠的定量检测方法,为明日叶进一步研究开发提供科学依据.     

Identification and quantification of 13 components in Angelica sinensis (Danggui) by gas chromatography-mass spectrometry coupled with pressurized liquid extraction

Angelica sinensis (Danggui in Chinese), a well-known traditional Chinese medicine, is also used as a health food product for women's care in Europe and America. Therefore, the demand for Danggui is enormous throughout the world. Due to the shortage of Angelica sinensis, Angelica acutiloba and Angelica gigas are commonly used as the substitutes of Danggui in the market of southeast Asia. However, the three common Angelica roots showed variation in their genetic and chemical composition. Up to date, it is thought that ferulic acid, ligustilide and other phthalides such as butylidenephthalide are the biologically active components of Danggui. In this paper, the contents of 13 compounds including ferulic acid, Z-ligustilide, E-ligustilide, Z-butylidenephthalide, E-butylidenephthalide, 3-butylphthalide, 3-butylidene-4-hydroxyphthalide, senkyunolide A, 6,7-epoxyligustilide, senkyunolide F, senkyunolide H, senkyunolide I, and 6,7-dihydroxyligustilide were determined or estimated by using gas chromatography-mass spectrometry (GC-MS) coupled with pressurized liquid extraction (PLE). The results showed that GC-MS coupled with PLE offered a simple, rapid and high sensitive method to analysis of components in Angelica root. And the contents of investigated compounds in Angelica sinensis, Angelica acutiloba and Angelica gigas, which are used as Danggui in China, Japan and Korea, respectively, were highly variant. It is thought that interaction of multiple chemical compounds contributes to the therapeutic effects of Chinese medicines. However, the overall clinical efficacy of these different Danggui has not been determined. Therefore, comparison of chemical components and pharmacological activities of different Angelica root is helpful to elucidate the mechanism of therapeutic effects of Danggui.     

A new method providing complementary explanation of the blood-enriching function and mechanism of unprocessed Angelica sinensis and its four kinds of processed products based on tissue-integrated metabolomics and confirmatory analysis

Angelica sinensis (AS) is a common Traditional Chinese Medicine used for tonifying blood in China. Unprocessed AS and its four kinds of processed products (ASs) are used to treat blood deficiency syndrome in the country. The different blood-tonifying mechanisms of ASs remain unclear. In this work, a novel method integrating metabolomics and hematological and biochemical parameters was established to provide a complementary explanation of blood supplementation mechanism of ASs. Our results revealed that different ASs exhibited various blood supplementation effect, and that AS parched with alcohol demonstrated the best blood supplementation effect. Eight metabolites from liver tissue and 12 metabolites from spleen tissue were considered to be potential biomarkers. These biomarkers were involved in four metabolic pathways. Correlation analysis results showed that l -aspartic acid and l -alanine (spleen tissue), linoleic acid, and l -cystathionine (liver tissue) exhibited a high positive or negative correlation with the aforesaid biochemical indicators. The blood-supplementation effect mechanism of ASs were related to four metabolic pathways. l -Aspartic acid and l -alanine (spleen tissue), linoleic acid, and l -cystathionine (liver tissue) were the four key metabolites associated with the blood supplementation effect of ASs. This study gives a complementary explanation of the blood supplementation effect and mechanism of action of ASs.