液质联用技术研究人参与干姜或赤芍配伍前后人参皂苷及其抗氧化活性的变化

采用高效液相色谱与电喷雾质谱联用技术(HPLC-ESI-MS),对不同质量比的人参与干姜或赤芍配伍过程中人参皂苷的变化进行了研究,发现随加入的干姜量增加,共煎液中的人参皂苷含量依次降低;少量的赤芍可以使各皂苷的溶出量增加;同时测定了人参单煎液、人参与干姜、赤芍共煎液中正丁醇提取物和水提物的抗氧化活性。 以抗坏血酸(500 μmol/L)作对照,人参与干姜、赤芍配伍溶液的抗氧化活性比人参单煎液要好,同时人参与干姜、赤芍共煎液中正丁醇提取物的FRAP(铁离子还原/抗氧化能力测定)值分别为1562.29和2969.78 μmol/L,高于人参与2种药单煎液（1260.27和2502.07 μmol/L）之和。     

人参与附子、黄连配伍的HPLC-ESI-MS研究及抗氧化活性测定

采用高效液相色谱与电喷雾质谱联用技术(HPLC-ESI-MS)对不同比例的人参与附子或黄连的水煎液、药渣和沉淀进行研究, 共鉴定了8种人参皂苷, 发现加入附子或黄连后, 溶液中人参皂苷含量明显减少, 进一步研究证明附子或黄连中的某些成分阻止了皂苷的溶出; 同时以抗坏血酸作对照, 应用铁离子还原/抗氧化能力测定法(FRAP)测定了人参单煎液及人参与附子、黄连共煎液中正丁醇提取物和水提取物的抗氧化活性. 结果证明, 共煎液中正丁醇提取物的抗氧化活性高于人参单煎液, 同时也高于人参与附子或黄连单煎混合液的抗氧化活性, 但水提取物抗氧化活性的变化规律有所不同, 这可能与煎煮过程中物质之间的相互作用有关.     

液质联用测定人参与五灵脂、莱菔子配伍的人参皂苷

采用液质联用(HPLC-ESI-MSn)技术,对不同比例的人参、五灵脂和人参、莱菔子的水煎液、沉淀和药渣进行研究,共鉴定了10种人参皂苷,并对这10种人参皂苷配伍前后的变化进行了系统的研究。实验结果表明,在共煎液中,除Ro外,其它皂苷的含量都有所下降。五灵脂或莱菔子的加入对人参皂苷的影响不同,莱菔子影响人参皂苷的溶出,而与五灵脂配伍,部分人参皂苷生成沉淀。     

田七花提取物中22种皂苷类成分的液相色谱-质谱测定

在甲酸体系中以高效液相色谱负离子模式电喷雾电离质谱以及碰撞诱导裂解技术研究了12种人参皂苷(Re、Rg1、Rg2、Rg3、Rf、Rb1、Rb2、Rb3、Rc、Rd、Rh1 和Rh2).结果表明,应用皂苷化合物(包括人参皂苷、田七皂苷和绞股蓝皂苷)的质谱及裂解规律可在缺少相应对照品的情况下对其进行可靠的鉴定.在此基础上,对田七花样品以加压溶剂萃取法提取,然后以LC-MS/MS分析,从中鉴定出22种皂苷,其中六糖皂苷Ⅰ和Ⅱ、乙酰基Rb1为首次报道,并且定量测定了其中10种皂苷的含量.     

电喷雾串联质谱分析附子炮制中的化学成分变化

利用电喷雾质谱方法(ESI-MS)分析了附子加辅料(甘草)炮制前后水煎液中二萜类生物碱在种类和含量方面的变化,通过加入内标化合物,建立了电喷雾质谱的半定量分析方法。此方法具有快速、准确、灵敏的特点,能够更加全面地反映中药配伍炮制过程中多种化学成分的含量变化,并能根据电喷雾串联质谱的分析结果鉴定配伍后产生的新的化学成分,在共煎液中的次乌头碱、中乌头碱和乌头碱的相对含量分别是单煎液中的5.67%、4.05%和4.88%。通过研究附子与甘草的单煎液、共煎液以及药渣中化学成分的变化,揭示了甘草作为辅料,在炮制过程中对附子减毒作用机理。     

UPLC-Q-TOF/MS分析不同比例丹参配伍藜芦毒性生物碱变化规律

利用超高效液相色谱-飞行时间质谱联用技术研究不同配伍比例的丹参藜芦的化学指纹图谱, 并分析毒性生物碱在不同比例配伍混煎前后溶出量的变化. 同时利用小鼠的急性毒性实验来考察配伍后的毒性变化规律. 结果显示在正离子模式下, 各配伍组(A～H)样本之间的差异能得到明显区分, 并结合Loadings图谱和数据库确定了配伍化学指纹图谱中的17种藜芦生物碱毒性成分, 当固定藜芦用量为LD₅₀且藜芦比例大于丹参时, 藜芦定、伪介芬胺等生物碱溶出高于或与藜芦单煎液相当, 但随着丹参比例增加, 上述生物碱溶出呈逐渐下降趋势, 当丹参比例大于藜芦时, 生物碱溶出低于藜芦单煎液. 不同比例丹参藜芦配伍急性毒性实验毒性变化规律与上述生物碱含量的变化趋势吻合, 表明上述毒性生物碱可能是藜芦与丹参特定比例配伍后毒性增强主要化学标志物.     

非靶向代谢组学用于藜芦妨害人参治疗脾气虚的机制研究

采用超高效液相色谱串联四级杆飞行时间质谱( UPLC/Q-TOF-MS)联用技术,通过非靶向代谢组学方法分析大鼠尿液内源性代谢物的变化,研究藜芦妨害人参发挥药效作用的机制。建立脾气虚大鼠模型,连续给药15天,测定力竭游泳时间及血液中白细胞、红细胞、血红蛋白的含量。结果表明,人参可显著提高脾气虚模型大鼠的力竭游泳时间(p﹤0.01),升高白细胞、红细胞及血红蛋白含量(p﹤0.05,p﹤0.01),藜芦对脾气虚模型大鼠各项指标无明显影响(p>0.05),人参与藜芦配伍后对脾气虚模型大鼠各项指标均无显著影响(p>0.05),表明藜芦妨害了人参发挥药效作用。采用UPLC/Q-TOF-MS技术及非靶向代谢组学的方法分析了空白组、模型组、人参组、藜芦组、参藜组对脾气虚模型大鼠的尿液代谢组差异,其中主成分分析( PCA)得分图显示各组代谢轮廓有显著差别,并通过正交偏最小二乘法-判别分析( OPLS-DA)及数据库检索,鉴定出15种人参干预调节脾气虚模型大鼠的潜在生物标志物,从中找出了7种人参藜芦配伍后减弱人参上述干预作用的潜在生物标志物,并对其涉及的代谢通路进行了系统分析。上述研究结果表明,人参藜芦配伍后妨害了人参对脾气虚模型大鼠的治疗作用,其机理可能是影响人参对体内能量代谢、免疫平衡及氧化还原反应等相关代谢的调节。     

高效液相色谱-飞行时间质谱联用法分析人参及人参皂苷与山楂配伍过程中的水解行为

利用高效液相色谱-飞行时间质谱联用的方法,分别对人参配伍山楂前后人参皂苷的变化进行分析,同时对人参皂苷Re、Rg1、Rb1、Rd与山楂配伍的水解规律进行系统研究,并与单独煎煮液、仿山楂配伍pH值煎煮液的水解产物进行比较,结果发现人参与山楂配伍后人参皂苷Rg1、Rb1含量明显减少,而人参皂苷Re、Rd、Rg2、Rg3、F2、Rh1含量明显增加,其中人参皂苷Re与山楂配伍后水解产物为人参皂苷20(R)-Rg2、20(S)-Rg2,仿山楂配伍pH值水解产物为人参皂苷20(R)-Rg2、20(S)-Rg2、Rg4、Rg6;人参皂苷Rg1与山楂配伍后水解产物为20(S)-Rh1、20(R)-Rh1,仿山楂pH值水解产物为20(S)-Rh1、20(R)-Rh1、Rh4、Rk3;人参皂苷Rb1与山楂配伍后水解产物为Rd、20(S)-Rg3,仿山楂pH值水解产物为F2、20(S)-Rg3;人参皂苷Rd与山楂配伍后水解产物为F2、20(S)-Rg3、20(R)-Rg3,仿山楂pH值水解产物为20(S)-Rg3、20(R)-Rg3。研究表明,不同人参皂苷和山楂配伍后与仿山楂pH值的水解产物并不相同,人参与山楂配伍改变了人参皂苷成分的种类及含量。本研究为临床方剂中人参与山楂配伍后成分的变化提供物质基础数据。     

人参中皂苷类化合物的HPLC-ESI MS研究

利用高效液相色谱与电喷雾质谱联用技术分析鉴定了人参粗提物中人参皂苷类化合物。实验采用反相C18色谱柱，二元线性梯度洗脱（0．2％的醋酸水溶液和乙腈），分离并检测了人参中的皂苷类化合物：通过与电喷雾质谱的联用和质谱的源内CID技术获得了其中主要色谱峰相对应化合物的相对分子质量和结构信息。     

HPLC-MS结合多元统计分析区分人参产地及筛选皂苷类标志物

利用高效液相色谱-质谱联用(HPLC-MS)技术结合多元统计分析方法, 区分中国人参主产区5个不同产地的45个人参样本, 筛选出差异性皂苷类标志物. 根据人参总皂苷在反相C₁₈色谱柱中的洗脱顺序, 结合串联质谱分析和标准品比对, 在提取的人参总皂苷中鉴定出15种原人参三醇型、 24种原人参二醇型和2种齐墩果酸型共41种皂苷. 对人参总皂苷的HPLC-MS全扫描数据进行了多元统计分析. 正交偏最小二乘-判别分析(OPLS-DA)结果表明, 所建立的分析模型具有良好的数据描述能力和预测能力. 所有人参样本能够根据产地被区分, 并筛选得到同时区分5个产地的差异性皂苷类组分18种; 能够区分任意2个产地人参样本的差异性组分主要为在人参中含量较高的人参皂苷Rb1, Rg1, Re, Rc, Rd, Ro和m-Rb1等. 分层聚类分析(HCA)结果显示, 黑龙江和吉林两省的样本能够独自聚类, 但是绥化市的样本更接近于吉林省. 初步推断原因为绥化市地理位置较接近吉林省, 两地人参生长环境相似并可能存在种质资源交换.     

Study on intestinal transport of Veratrum alkaloids compatible with Panax ginseng across the Caco-2 cell monolayer model by UPLC-ESI-MS method

The Caco-2 cells have been recognized as effective tools to be applied to imitate the drug absorption in human intestine for the transport of drug. In this study, Caco-2 cell monolayer model was used to study compatibility of the transport of the Veratrum alkaloids in different proportions with Panax ginseng. A specific ultra-high performance liquid chromatographic-electrospray ionization-mass spectrometric (UPLC-ESI-MS) method is developed for the semi-quantitative determination of Veratrum alkaloids on intestinal transport with berberine as internal standard (IS). In the Caco-2 model constructed, three influencing factors are investigated, including time, concentration and recovery rates of the Veratrum alkaloids during the uptake from AP (apical side) to BL (basolateral side). The results suggest that the flux of Veratrum alkaloids is time dependent and concentration dependent. And the absorption of all eight Veratrum alkaloids increase after compatibility with Panax ginseng compared to the single Veratrum nigrum extraction. This research was studied from the perspective of intestinal absorption by the UPLCESI-MSmethod. Thismethod was successfully applied to transport studies of the Veratrum alkaloids and the interaction mechanism between Veratrum nigrum and Panax ginseng.     

Studies on Intestinal Transport of Ginsenoside Compatibility with Veratrum nigrum via Caco-2 Cell Monolayer Model Coupled with UPLC-ESI-MS Method

The Caco-2 cells have been recognized as effective tools to be applied to imitating the drug absorption in human intestine for the transport of drug. Herein, Caco-2 cell monolayer model was used to study the transport of the ginsenoside compatibility with Veratrum nigrum in different proportions. A specific high performance liquid chromatography-electrospray ionization-mass spectrometry(HPLC-ESI-MS) method was developed for the semiquantitative determination of ginsenoside in intestinal transport with Dioscin as an internal standard. For the Caco-2 model constructed, two influencing factors were investigated, including time and concentration. The results suggest that the absorption of ginsenoside Re, Rg1, Rb1, Rc, Rb2 and Rd are time- and concentration-dependent and the excretions of Rb1, Rc, Rb2 and Rd have a relatronship with some transport proteins. The bioavailability of the ginsenosides has reduced compared to the single Panax ginseng extract when compatibility with a certain amount of Veratrum nigrum.     

Determination of Seven Major Ginsenosides in Different Parts of Panax quinquefolius L.（American Ginseng） with Different Ages

Ginsenosides Rgl, Re, Rb1, Rc, Rb2, Rb3, and Rd in different parts of the American ginseng plant were investigated. The extraction process was a pressurized microwave-assisted extraction（PMAE）. The seven ginsenosides were separated and determined by high-performance liquid chromatography（HPLC） with a ultraviolet（UV） detector, at 203 nm. The experiment results showed significant variations in the individual ginsenoside contents of the American ginseng in different parts and ages of the plant. The results demonstrated that the leaves, root hairs, and rhizomes of Panax quinquefolius L. contained higher ginsenoside contents, followed by the main roots and stems. The leaves contained dramatically higher levels of ginsenoside Rg1 Rb3, and Rd than the other four parts. Higher contents of Rb1 and Re were present in the main roots, root hairs, and rhizomes. The amount of ginsenoside content in the stems was the lowest. The total content of the seven ginsenosides in main roots, root hairs and rhizomes increased with the age of the plant. In contrast, the ginsenoside contents in the leaves and stems decreased with a year of growth.     

Brain Concentration of Ginsenosides and Pharmacokinetics after Oral Administration of Mountain‐cultivated Ginseng

Mountain‐cultivated ginseng (MCG ) can be considered a mimic of wild ginseng, whose seeds are sowed artificially, cultivated in the natural environment, and returned to the wild state before being used clinically. Cultivated ginseng (CG ) and MCG are mainly used as the commercial material for clinical applications. However, MCG is much more expensive but effective than CG . The aim of this study is to explore the differences in the pharmacokinetics and brain concentration of Rg1, Re, Rb1, and Rd after oral administration of MCG , CG, and pure ginsenosides. An ultra‐performance liquid chromatography‐tandem mass spectrometry method was developed and validated to determine the concentration of the four ginsenosides in rat plasma and brain tissue. Compared with the pure group, the area under the curve (AUC) of all the four ginsenosides for CG and MCG increased. The mean brain concentrations of the four ginsenosides were found to be 10‐ to 15‐fold lower than the corresponding contents in the plasma, and the poor permeability of ginsenosides into blood–brain barrier was indicated by the low concentrations of the four ginsenosides.     

Liquid chromatography/mass spectrometry of malonyl-ginsenosides in the authentication of ginseng

Different negative ion electrospray (ES) source conditions are required to concentrate the ion current in [M-H](-) for malonylated and non-malonylated ginsenosides. However, both can be ionised optimally in a single liquid chromatography/mass spectrometry (LC/MS) analysis by employing switchable voltages in the post-source ion optics of a quadrupole ion trap mass spectrometer. Coupled with automatic MS/MS scanning and post-acquisition neutral loss data analysis, this method provides a means of profiling the malonylated and acetylated ginsenosides in ginseng extracts. Analyses revealed numerous malonylated ginsenosides that could be partially characterised by serial MS/MS experiments. The ratio of mRb(1) to other isomeric forms present and to mRb(2) and mRc appears to show consistent differences among Panax ginseng (Asian ginseng), P. quinquefolius (American ginseng) and P. notoginseng (Sanchi ginseng). The ratio of malonylated to non-malonylated ginsenosides is reduced in the red form of Asian ginseng compared with the white form and there is a concomitant increase in the levels of the corresponding acetylated ginsenosides. The ability to analyse malonylated ginsenosides is an important contribution to the range of chemical characteristics that can be used to authenticate the different species of ginseng and will assist in quality control and standardisation.