DPPH-HPLC-QTOF-MS/MS快速筛选和鉴定杜仲黑茶中抗氧化活性成分

建立DPPH-HPLC-QTOF-MS/MS方法快速筛选、鉴定和测定杜仲黑茶中抗氧化活性成分。杜仲黑茶提取物与DPPH自由基反应30 min后,9个抗氧化活性成分的HPLC峰消失或峰面积明显减少,结合QTOF-MS/MS谱图及与标准品对照,确定抗氧化活性成分为:没食子酸,新绿原酸、原儿茶酸、绿原酸、咖啡酸、表没食子儿茶素没食子酸酯、槲皮素-3-O-桑布双糖苷、芦丁和异槲皮苷。并建立HPLC法同时测定9个抗氧化活性成分的含量。所建立的方法无须繁琐的分离纯化过程,可快速、灵敏、准确、高通量筛选复杂体系中的抗氧化活性成分,并为杜仲黑茶的质量控制提供科学依据。     

钩藤叶5种活性成分的测定研究

为建立钩藤Uncaria rhynchophylla(Miq.)Miq.ex Havil叶中5种活性成分含量测定方法,弥补钩藤叶质量控制方法的不足。本文通过对钩藤叶进行了抗氧化活性部位追踪,发现钩藤叶抗氧化活性主要与黄酮和多酚等成分相关;采用HPLC-PDA方法,以乙腈-0.1%磷酸水进行梯度洗脱,测定了钩藤叶中绿原酸、表儿茶素、芦丁、金丝桃苷和喜果苷等5种活性成分的含量。在该方法的条件下,5种成分分离良好,精密度、稳定性、重复性试验的RSD均3.0%,平均加样回收率为96.73%～103.5%。不同采集地点的12批样品的聚类分析表明5种成分的含量差异与产地地理分布相关。建立的HPLC方法操作简单,准确度好,精密度高,为钩藤叶的综合开发利用提供了基础。     

HPLC法测定大果山楂果实中八种酚酸类成分的含量

建立HPLC法同时测定大果山楂果实中原儿茶酸、绿原酸、二氢咖啡酸、咖啡酸、根皮酸、p-香豆酸、阿魏酸和肉桂酸的方法,并通过HPLC法分析这八种酚酸在不同产地大果山楂果实中的含量。采用ZORBAX SB-C18色谱柱,以甲醇/1.5%甲酸水溶液(v/v)为流动相,梯度洗脱,柱温35℃,流速1.0 m L·min~(-1),检测波长280、320 nm。结果表明:(1)在线性范围内八种酚酸质量浓度与色谱峰峰面积的线性关系良好,相关系数均0.997,检出限0.08～0.20μg·m L~(-1),定量下限0.27～0.67μg·m L~(-1),变异系数均5.0%,加标回收率99.3%～103.3%;(2) 10个不同产地的大果山楂果实中酚酸含量丰富,均检出原儿茶酸、绿原酸、二氢咖啡酸、咖啡酸、根皮酸、阿魏酸和肉桂酸七种酚酸,其中,二氢咖啡酸、根皮酸、阿魏酸均为首次检出,以绿原酸为主(8 410.2～13 826.7μg·g~(-1)),占总酚酸的80%以上,总酚酸的质量分数在10 187.8～15 583.9μg·g~(-1)之间,其中广西百色靖西和桂林恭城产的果实总酚酸质量分数相对较高,均大于15 000μg·g~(-1)。综上结果表明,该研究的HPLC法适用于大果山楂果实中酚酸含量的测定,可为大果山楂优良品种的选育、产品质量控制及深度开发利用提供方法和科学参考。     

UPLC-QE-MS结合UPLC表征绿萼梅不同发育时期的成分差异及抗氧化活性研究

梅花为蔷薇科植物梅Prunus mume(Sieb.) Sieb.et Zucc.的干燥花蕾,绿萼梅为临床入药的主要品种。该研究为明确不同发育时期绿萼梅的化学组成及差异,采用超高效液相色谱-四级杆静电场轨道阱质谱(UPLC-QE-MS)检测其化学组成,共检测得到328个化合物,包含67个黄酮、50个苯丙素、104个萜类、26个酚类、24个生物碱、11个有机酸及其衍生物、8个氨基酸衍生物及其他38个化合物。半定量结果显示绿萼梅中主要化学成分的含量随发育时期呈逐渐下降趋势。采用紫外-可见分光光度法对不同发育时期绿萼梅样品中总黄酮和总苯丙素含量进行测定,采用UPLC法测定不同发育时期8个指标性成分(新绿原酸、绿原酸、咖啡酸、芦丁、金丝桃苷、异槲皮苷、异绿原酸B、槲皮苷)含量,分析结果表明花蕾期与全开期之间成分含量差异显著,绿原酸可作为不同发育时期之间的差异质量标志物。DPPH自由基清除法检测不同发育时期绿萼梅的抗氧化活性,结果表明抗氧化能力与其成分含量密切相关,且随着花的发育逐渐减弱。该文结果可为梅花的化学成分动态变化规律和最佳采收期的确定提供分析方法和数据支撑。     

木芙蓉叶黄酮成分抗氧化及月周期规律研究

为探讨木芙蓉叶中总黄酮含量及其月周期变化规律。本文以Vc为对照,采用总抗氧化实验、DPPH法、ABTS法和PTIO法等4种方法测定木芙蓉叶总黄酮体外抗氧化活性,并采用UFLC法测定木芙蓉叶中4种黄酮的含量变化规律,同时分析与其抗氧化活性的相关性。结果表明木芙蓉叶黄酮抗氧化活性的月周期变化趋势在4种检测方法中有一定差异,但其抗氧化活性均与槲皮苷含量呈正相关性。综合分析木芙蓉叶中芦丁月周期变化规律及其抗氧化活性强弱,得出叶中芦丁成分可作为药效评价木芙蓉叶药材质量主要指标的结论。     

尾叶香茶菜水溶性部位化学成分研究

采用XAD16N大孔树脂、HW-40C凝胶柱和硅胶柱等色谱手段,从长白山产尾叶香茶菜甲醇提取物的水溶性部位中纯化得到山柰酚-3-O-芸香糖苷(1)、芦丁(2)、异槲皮苷(3)、槲皮素-7-O-鼠李糖苷(4)、金樱子皂苷A(5)、β-胡萝卜苷(6)、咖啡酸(7)、槲皮素(8)和熊果酸(9),其中化合物1,4,5,7,8为首次从尾叶香茶菜中分离得到,化合物1和5为首次从该属中分离得到。     

当归不同提取液中阿魏酸、咖啡酸含量及抗氧化作用的比较研究北大核心CSCD

为考察不同提取条件对当归提取液中阿魏酸和咖啡酸的含量及抗氧化作用的影响,本研究通过HPLC外标一点法检测当归提取液中阿魏酸和咖啡酸的含量,通过DPPH法和ABTS法检测当归提取液的抗氧化作用。研究结果表明,不同提取条件下当归提取液中阿魏酸的含量为0. 574～0. 707 mg/g,咖啡酸为0. 012～0. 082 mg/g;碱水溶液提取时阿魏酸与咖啡酸的含量最高,其抗氧化活性也最强,而95%乙醇提取时的含量最低,抗氧化活性最弱;提取溶剂随醇浓度的增加,咖啡酸的含量逐渐减少,阿魏酸的含量呈现先增加后减少的趋势,30%～50%乙醇提取时各组分有良好的协同抗氧化作用。     

当归不同提取液中阿魏酸、咖啡酸含量及抗氧化作用的比较研究

为考察不同提取条件对当归提取液中阿魏酸和咖啡酸的含量及抗氧化作用的影响,本研究通过HPLC外标一点法检测当归提取液中阿魏酸和咖啡酸的含量,通过DPPH法和ABTS法检测当归提取液的抗氧化作用。研究结果表明,不同提取条件下当归提取液中阿魏酸的含量为0. 574～0. 707 mg/g,咖啡酸为0. 012～0. 082 mg/g;碱水溶液提取时阿魏酸与咖啡酸的含量最高,其抗氧化活性也最强,而95%乙醇提取时的含量最低,抗氧化活性最弱;提取溶剂随醇浓度的增加,咖啡酸的含量逐渐减少,阿魏酸的含量呈现先增加后减少的趋势,30%～50%乙醇提取时各组分有良好的协同抗氧化作用。     

杜仲叶片6种主要活性成分的遗传变异研究

该研究采用方差分析、相关性分析和聚类分析等方法,对105份杜仲种质资源叶片6种主要活性成分含量进行比较分析,探讨其遗传变异特征,为叶用杜仲的良种选育及开发利用提供理论依据。结果显示:(1)不同种质间杜仲叶片6种活性成分含量的变异程度不同,异槲皮苷含量变异系数(34.42%)最大,变异幅度为1.16~6.92mg·g~(-1),总黄酮变异系数(19.35%)最低,变异幅度为6.70~22.53mg·g~(-1);6种活性成分多样性指数较高,均在2.0以上;不同种质间杜仲叶6种活性成分含量差异性均达到极显著水平(P0.01)。(2)不同地理来源间杜仲叶片的绿原酸、京尼平苷酸、车叶草苷、异槲皮苷和总黄酮含量存在显著差异。(3)相关性分析表明,3种环烯醚萜类化合物桃叶珊瑚苷、京尼平苷酸及车叶草苷的含量间呈极显著正相关关系;绿原酸与桃叶珊瑚苷、车叶草苷、异槲皮苷、总黄酮间呈极显著正相关关系;总黄酮与异槲皮苷呈极显著正相关关系。(4)根据杜仲叶片6种活性成分的含量聚类,将杜仲种质资源分为4大类群,其中类群Ⅳ为高含量类群,可为优良叶用杜仲资源筛选提供参考。研究表明,杜仲种质资源叶片主要活性成分存在较大变异,表现出丰富的多样性。     

几种天然有机多酚酸体外抗脂质过氧化研究

通过天然有机多酚酸对H2O2诱导小鼠肝匀浆脂质过氧化和自发脂质过氧化的保护作用的体外实验,比较研究绿原酸、咖啡酸、迷迭香酸、阿魏酸、丹参素、原儿茶醛和3-羟基肉桂酸体外抗氧化能力的强弱,探讨其抗氧化能力产生机理及构效关系。结果表明,7种多酚酸对H2O2诱导小鼠肝匀浆脂质过氧化的保护作用强弱依次为迷迭香酸咖啡酸原儿茶醛绿原酸阿魏酸丹参素3-羟基肉桂酸,其最大和最小抑制率相差2.4倍;对小鼠肝脏组织自发脂质过氧化的抑制率大小顺序为迷迭香酸、原儿茶醛、绿原酸、咖啡酸、阿魏酸、丹参素、3-羟基肉桂酸,最高和最低抑制率相差近90%。7种天然有机酸抗脂质过氧化作用因化合物结构不同存在显著差异。     

不同甜叶菊品种叶中绿原酸类成分的比较研究

该文以14个扦插培育的甜叶菊品种叶为材料,从8种不同型号的树脂中筛选出一种合适的大孔吸附树脂对甜叶菊叶中绿原酸类成分进行纯化前处理,采用HPLC法对不同甜叶菊品种叶中所含绿原酸类成分进行比较分析。结果表明:(1)在8种不同型号的树脂中,XAD~(-1)6对甜叶菊叶中绿原酸类成分吸附-解析性能最佳。(2)经优化,上样液浓度1.20 mg·mL~(-1)、样品溶液pH 3、解析液乙醇体积分数70%时XAD~(-1)6树脂对甜叶菊叶中绿原酸类成分具有较好的纯化效果。(3) HPLC检测分析表明,在14个品种中共检测出新绿原酸、绿原酸、隐绿原酸、异绿原酸B、异绿原酸A、异绿原酸C六种绿原酸类成分,其中主要成分均为异绿原酸A、绿原酸、异绿原酸C,而在品种3、5、13、14中没有检测出异绿原酸B。(4) 14个品种中6个绿原酸类成分的含量分别为异绿原酸A 20.55~54.3 mg·g~(-1)、绿原酸17.96~32.93 mg·g~(-1)、异绿原酸C 4.15~19.49 mg·g~(-1)、新原酸0.61~4.61 mg·g~(-1)、隐绿原酸0.52~3.11 mg·g~(-1)、异绿原酸B 0.0~3.17 mg·g~(-1),6种绿原酸类成分总量为43.9~97.8 mg·g~(-1)。可见,不同品种甜叶菊叶中绿原酸类成分含量有明显差异,富含绿原酸类成分的甜叶菊品种可用于开发获取绿原酸类物质。     

The Activity-Integrated Method for Quality Assessment of Reduning Injection by On-Line DPPH-CE-DAD

A sensitive on-line DPPH-CE-DAD method was developed and validated for both screening and determining the concentration of seven antioxidants of Reduning injection. The pH and concentrations of buffer solution, SDS, β-CD and organic modifier were studied for the detection of DPPH and seven antioxidants. By on-line mixing DPPH and sample solution, a DPPH-CE method for testing the antioxidant activity of the complex matrix was successfully established and used to screen the antioxidant components of Reduning injection. Then, antioxidant components including caffeic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid were quantified by the newly established CE–DAD method. Finally, the total antioxidant activity and the multiple active components were selected as markers to evaluate the quality of Reduning injection. The results demonstrated that the on-line DPPH-CE-DAD method was reagent-saving, rapid and feasible for on-line simultaneous determination of total pharmacological activity and contents of multi-components samples. It was also a powerful method for evaluating the quality control and mechanism of action of TCM injection.     

Altitudinal changes in secondary metabolite contents of Hypericum androsaemum and Hypericum polyphyllum

The genus Hypericum (Hypericaceae) has attracted scientific interest as its members have yielded many bioactive compounds. In the present study we investigated the content of hypericin, pseudohypericin, hyperforin, adhyperforin, chlorogenic acid, neochlorogenic acid, caffeic acid, 2,4-dihydroxybenzoic acid, 13,II8-biapigenin, hyperoside, isoquercitrin, quercitrin, quercetin, avicularin, rutin, (+)-catechin and (−)-epicatechin in aerial parts of plants from populations of H. androsaemum L. and H. polyphyllum Boiss. & Bal. from Turkey growing at different altitudes. The plant materials were dried and subsequently assayed for chemical content by HPLC. All the tested compounds were detected in both species at varying levels depending upon the altitude the plants were growing, except for hypercins and rutin which did not accumulate in H. androsaemum. It was observed that overall the compounds were more abundant in plants from higher altitudes. The differences in the levels of the compounds could contribute to the ability of the plants to deal with the abiotic stress of lower temperature and higher ultraviolet (UV)-B radiation which would be greater at higher altitudes compared to lower altitudes.     

苦丁茶中绿原酸及其异构体的提取变化分析

以富含绿原酸类成分的苦丁茶(Ilex kaushue)为材料,使用溶剂甲醇、乙醇、丙酮和水,结合超声波提取、水浴提取、回流提取等方法对绿原酸及其异构体的提取效率及提取后各异构体的变化进行分析。应用超高效液相色谱法可使苦丁茶的6种绿原酸类成分及咖啡酸在6 min内实现分离。提取结果表明,丙酮作为提取溶剂,在采用超声波和水浴提取时能获得较高的提取效率,易获得较多总量的绿原酸类成分和高含量异绿原酸A。而最常用的溶剂乙醇并未达到理想的提取效果。在不同溶剂的回流提取中,虽然提取的绿原酸类成分总量接近,但异绿原酸A和异绿原酸C含量有较大差异。醇溶液特别是乙醇溶液的回流提取使异绿原酸C的量大幅增加,而相应的异绿原酸A的量大幅减少,表明在醇加热条件下,异绿原酸A转化为异绿原酸C,这为获得抗氧化性更强的异绿原酸C提供了新思路。     

Altitudinal changes in the content of bioactive substances in Hypericum orientale and Hypericum pallens

Altitudinal changes in the content of hypericin, pseudohypericin, hyperforin, adhyperforin, chlorogenic acid, neochlorogenic acid, caffeic acid, 2,4-Dihydroxybenzoic acid, amentoflavone, hyperoside, isoquercitrin, quercitrin, quercetin, avicularin, rutin, (+)-catechin and (?)-epicatechin among Hypericum orientale L. and Hypericum pallens Banks and Sol. populations from Northern Turkey were investigated for the first time. Thirty flowering individuals were collected from five different altitudes (400, 950, 1,150, 1,620 and 2,150 m) for H. pallens and six different altitudes (500, 1,150, 1,650, 2,100, 2,720 and 3,250 m) for H. orientale. The plant materials were dried at room temperature and subsequently assayed for chemical contents by HPLC. All chemicals were detected in both species at various levels depending on altitude of growing sites except for caffeic acid which was absent in H. pallens. It was found that plants from higher altitudes produced significantly higher amount of the bioactive compounds tested. The results were discussed as a possible protective response of plants to the different abiotic stress factors as high ultraviolet (UV)-B radiation and low temperature which were prevalent in higher altitudes.     

Phenolic composition and antioxidant activities of two Phlomis species: A correlation study

Two traditional Chinese medicines (Phlomis umbrosa Turcz. and Phlomis megalantha Diels), as well as five pure phenolic compounds (protocatechic, chlorogenic, benzoic, rosmarinic acid, and rutin) have been studied for antioxidant activities in acetone and methanol extracts from leaves. An HPLC method was developed to quantify the amounts of 14 phenolic compounds in the leaf extracts. The antioxidant capacities of the studied species are high. Almost all samples were capable of directly scavenging DPPH and superoxide free radicals, inhibiting linoleic acid oxidation, acting as reducing agents, and reducing plasmid DNA damage induced by hydroxyl radicals. Among different extracts, the acetone extract of P. megalantha exhibited the highest antioxidant activity. The major phenolic compounds identified were protocatechic, chlorogenic, caffeic, rosmarinic acid, and (−)-epicatechin. Antioxidant activities of pure compounds and correlation analysis indicated that protocatechic and rosmarinic acids were the major contributors to the observed antioxidant activities of the investigated Phlomis extracts. To cite this article: Y. Zhang, Z.-z. Wang, C. R. Biologies 332 (2009).     

Chemical composition of Hypericum species from the Taeniocarpium and Drosanthe sections

The presence of several phytochemicals, namely naphthodianthrones hypericin and pseudohypericin, phloroglucinol derivatives hyperforin and adhyperforin, the phenolic acids as chlorogenic acid, neochlorogenic acid, caffeic acid and 2,4-dihydroxybenzoic acid, the flavonols, hyperoside, isoquercitrin, quercitrin, quercetin, avicularin, rutin, and flavanols (+)-catechin and (?)-epicatechin, as well as biflavonoid amentoflavone was investigated in seven Turkish species of Hypericum from Taeniocarpium and Drosanthe sections. Plants were harvested at flowering, dried at room temperature, dissected into different tissues and assayed for chemical contents by HPLC. All chemicals were detected at various levels depending on species and plant parts. Despite the observed quantitative variation in the chemical content of plant material, it was found that phytochemical profiles of the species from the same section were very similar. The present data could be helpful in selecting the future targets for phytochemical and biological studies as well as enriching our current chemical knowledge about Hypericum species. Such kind of data could also be useful for elucidation of the chemotaxonomical relationships among the sections of Hypericum genus.     

Analysis of marker compounds with anti‐platelet aggregation effects in mailuoning injection using platelet binding assay combined with HPLC‐DAD‐ESI‐MS and solid‐phase extraction technique

Introduction – Mailuoning is prepared from a traditional formula of Chinese medicines and widely used as an antithrombotic agent. In this study, the platelet binding assay was used as a novel biospecific separation and analysis method to explore its active constituents, which could be considered as marker compounds for quality control. Objective – To establish a rapid and simple method to predict marker compounds in herbal medicine injection and evaluate the effects of those compounds. Material and methods – Platelets were used to bind and separate constituents. Binding constituents were analysed and taken as potential active compounds for further evaluation. Solid‐phase‐extraction was adopted to improve sensitivity. HPLC‐DAD and ESI‐MS were used to determine the binding constituents. Results – Five compounds were extracted through the platelet binding process and identified as neochlorogenic acid, caffeic acid, isochlorogenic acid and their isomers. Caffeic acid was selected for the flow cytometric assay to test its effect on platelets activation, which was determined by CD62P (P‐selectin) expression. The results indicated that caffeic acid could significantly inhibit platelet activation while chlorogenic acid did not. Conclusion – Caffeic acid could be considered as a marker compound of Mailuoning injection due to its anti‐platelet effect. The study also suggested that platelet binding assay combined with some preconcentration technique could be efficiently used to predict anti‐platelet compounds in complicated herbal medicines. Copyright © 2010 John Wiley & Sons, Ltd.     

Urinary excretion of hydroxycinnamates and flavonoids after oral and intravenous administration.

The urinary recoveries of the hydroxycinnamates, ferulic acid (3-methoxy, 4-hydroxy cinnamic acid), and chlorogenic acid (the quinic acid ester of 3,4-dihydroxycinnamic acid), and three structurally related flavonoids were studied in the rat. For the latter, the aglycone quercetin was compared with its 3-glucoside (isoquercitrin) and 3-rhamnoglucoside (rutin). Doses of 50 mg/kg were administered via the oral and intravenous routes and urine collected over the subsequent 24-h period. Reverse phase HPLC with photo-diode array detection was used to analyze the unchanged compound and their metabolites excreted in the urine. Ferulic acid and isoquercitrin were orally absorbed (5.4 and 0.48% of administered dose, respectively) and are therefore bioavailable. In contrast, neither unchanged chlorogenic acid, rutin, quercetin, nor the conjugated metabolites in the form of glucuronide or sulphate were detected in the urine after oral dosing. All the flavonoids studied produced low total urinary recoveries after intravenous administration, 9.2% for quercetin-3-rhamnoglucoside, 6.7% for the 3-glucoside, and 2.4% for the aglycone, indicating that extensive metabolism to low molecular weight compounds or excretion via other routes may be occurring. Overall it can be stated that renal excretion is not a major pathway of elimination for intact flavonoids and hydroxycinnamates in the rat.     

Phenolic compounds of Sophora flavescens Soland. of Russian origin

A qualitative composition and a quantitative content of phenolic compounds of underground and above-ground parts of Sophora flavescens Soland. (the Fabaceae family) growing in Russia (Transbaikalia, Primorsky Krai, Aga Buryat Autonomous District) were studied. Eleven compounds were isolated from the roots and rhizomes: kushenol A, isokurarinone, kuraridine, sophoraflavanone G, kurarinone, isoxanthohumol, umbeliferon, and, for the first time, scopoletin, ferulic, caffeic, and chlorogenic acids. Ten phenolic compounds were identified in the herb of S. flavescens: cynaroside, cosmosiin, caffeic acid, and, for the first time, apigenin, luteolin, quercetin, umbelliferone, rutin, chlorogenic, and neochlorogenic acids. Dominant compounds in the underground part were kurarinone and sophoraflavanone G, and in the above-ground part, cynaroside and rutin. It was shown that the maximum content of flavonoids in the underground part of S. flavescens was accumulated in the epidermal layers of rhizomes. The dynamics of flavonoids accumulation in S. flavescens was studied.