Identification of herb Acanthopanax senticosus (Rupr. Et Maxim.) harms by capillary electrophoresis with electrochemical detection

In this paper, a rapid, high efficient, sensitive and inexpensive approach based on a combination of simple ultrasonic extract and capillary electrophoresis (CE) separation with electrochemical detection (ED), is described to identify herbs by comparing their CE-ED profiles (namely, CE-ED electropherograms). The proposed method takes advantage of ultrasmall sample volume, low consumption of organic solvent, simple sample pretreatment and easy cleanup procedure. It was applied to analyze the CE-ED profiles of stems of herb Acanthopanax senticosus (Rupr. Et Maxim.) Harms from different sources and different parts (roots, rhizomes, stems and leaves) of this herb. By comparing peak number, peak height and peak height ratio, we found that the CE-ED profiles showed big differences for the herbs from the different sources and the different parts of this herb. In addition, the distribution of bioactive compounds (isofraxidin, rutin and chlorogenic acid) in the different parts of this herb and their content variations affected by the source were studied with the CE-ED method. Based on their own unique CE-ED profiles, these herbs from the different sources and the different parts of this herb could be easily distinguished. Therefore, the proposed approach could be used as a rapid, high efficient and sensitive method for the identification of herbal medicines.

Pharmacokinetics of isofraxidin in rat plasma after oral administration of the extract of acanthopanax senticosus using HPLC with solid phase extraction method

High-performance liquid chromatography coupled with solid phase extraction method was developed for determination of isofraxidin in rat plasma after oral administration of Acanthopanax senticosus extract (ASE), and pharmacokinetic parameters of isofraxidin either in ASE or pure compound were measured. The HPLC analysis was performed on a Dikma Diamonsil RP(18) column (4.6 mm x 150 mm, 5 microm) with the isocratic elution of solvent A (acetonitrile) and solvent B (0.1% aqueous phosphoric acid, v/v) (A : B = 22 : 78) and the detection wavelength was set at 343 nm. The calibration curve was linear over the range of 0.156-15.625 microg/ml. The limit of detection was 60 ng/ml. The intra-day precision was 5.8%, and the inter-day precision was 6.0%. The recovery was 87.30+/-1.73%. When the dosage of ASE is equal to pure compound caculated by the amount of isofraxidin, it has been found to have two maximum concentrations in plasma while the pure compound only showed one peak in the plasma concentration-time curve. The determined content of isofraxidin in plasma after oral administration of ASE is the total contents of free isofraxidin and its precursors in ASE in vitro. The pharmacokinetic characteristics of ASE showed the priority of the extract and the properities of traditional Chinese medicine.

HPLC method for preliminary analysis of constituents in rat blood after oral administration of the extract of Acanthopanax senticosus

An HPLC with SPE method has been developed for analysis of constituents in rat blood after oral administration of the extract of Acanthopanax senticosus (ASE). The plasma sample was prepared by SPE method equipped with Oasis HLB cartridge (3cc, 60 mg). The analysis was performed on a Dikma Diamonsil RP(18) column (4.6 mmx150 mm, 5 microm) with the gradient elution of solvent A (ACN) and solvent B (0.1% aqueous phosphoric acid, v/v) and the detection wavelength was set at 270 nm. The calibration curve was linear over the range of 0.156-15.625 microg/mL. The LOD was 60 ng/mL. The intraday precision was less than 5.80%, and the interday precision was less than 6.0%. The recovery was (87.30 +/- 1.73)%. As a result, 19 constituents were detected in rat plasma after oral administration of the ASE, including 11 original compounds in ASE and eight metabolites, and three of the metabolites originated from syringin in ASE. Six constituents were identified by comparing with the corresponding reference compounds.

Characterization of triterpenoidic saponin mixture in crude extracts from leaves of Acanthopanax senticosus Harms by saponin structural correlation and mass spectrometry

Eighteen triterpenoidic saponins in crude extracts from leaves of Acanthopanax senticous Harms have been investigated by electrospray ionization multi-stage tandem mass spectrometry and high-resolution mass spectrometry. In ESI-MS spectra, predominant [M + Na](+) ions in the positive ion mode have been observed for molecular mass information. Meanwhile, specific structural correlations between these ions are firstly found. The 18 peaks (ions) can be classified into three groups (group D, E, and F with mass increase) with each group including six peaks. There is a mass difference of 132 Da between group D and E for each corresponding peak in turn (for example peak 1 to peak 7), indicating one more pentose residue was attached to saponins in group E than those corresponding in group D. The mass difference of 146 Da between group E and F implies one more deoxy-hexose attached to saponins in group F than those corresponding in group E. The structural correlations of the corresponding ions are confirmed by tandem mass spectrometry and high-re solution mass spectrometry. These structural features can not only facilitate the rapid characterization of the native known saponins in crude plant extracts, thus avoiding tedious derivation and separation of saponins, but also help find novel compounds of the same type in a specific medicinal plant.

Analysis of flavonoids from leaves of Acanthopanax Senticosus Harms

Using electrospray tandem mass spectrometry (ESI-MSn), the flavonoids obtained from leaves in Acanthopanax Senticosus Harms were analyzed. The typical colorimetric method and the ultroviolet spectrophotometry were also utilized for the determination of the content of total flavonoids. The analytical results showed that there was quercetin as well as its derivatives in leaves of acanthopanax senticosus harms and their content was as high as 37.25%.

Identification of the flavonoid constituents from leaves of Acanthopanax Senticosus Harms

Four flavonoids from leaves of Acanthopanax Senticosus Harms were observed in negative ion mode in the electrospray mass spectra. Two of them were further isolated and identified as quercitrin (quercetin-3-O-alpha-L-rhamnoside) and hyperin (quercetin-3-O-beta-D-galactoside) on the basis of MS' and NMR data. The other two compounds in the mixtures were tentatively established as quercetin and rutin (quercetin-3-O-rutinoside) in terms of their electrospray tandem mass spectrometry (ESI-MSn) data. Three of the four flavonoids (excluding hyperin) haven't been reported in this plant before.

Analysis of flavonoid constituents from leaves of Acanthopanax senticosus Harms by electrospray tandem mass spectrometry

Three known flavonoids, quercetin, quercitrin (quercetin-3-0-rhamnoside) and rutin (quercetin-3-0-rutinoside), have been identified for the first time in the leaves of Acanthopanax senticosus Harms by using electrospray tandem mass spectrometry techniques (ESI-MSn). The flavonoid hyperin (quercetin-3-0-beta-galactoside), already known to be present, was also investigated. The diagnostic fragment ions of the aglycone quercetin were obtained in the ESI-MSn experiments, and a fragmentation mechanism proposed.

Multi-stage mass spectrometric studies of triterpenoid saponins in crude extracts from Acanthopanax senticosus harms

Three triterpenoid saponins in crude extracts from Acanthopanax senticosus Harms have been investigated by use of multi-stage mass spectrometry (MSn) combined with electrospray ionization (ESI), MSn spectra were applied to direct structure elucidation of these saponins in crude plant extracts, in positive and negative ion mode. The characteristic fragmentations of triterpenoid saponins are discussed. The method provides a means of rapid initial screening of crude plant extracts. Copyright (C) 1999 John Wiley & Sons, Ltd.

A rapid and sensitive UPLC-ESI MS method for analysis of isofraxidin, a natural antistress compound, and its metabolites in rat plasma

Isofraxidin is one of the main bioactive constituents in the root of Acanthopanax senticosus, which has antifatigue, antistress, and immuno-accommondating effects. In this study, an ultraperformance LC (UPLC)-ESI MS method was developed for analyzing isofraxidin and its metabolites in rat plasma. The analysis was performed on a UPLC coupled with ESI MS (quadropole MS tandem TOF MS). The lower LOD (LLOD) for isofraxidin was 0.25 ng/mL, the intraday precision was less than 10%, the interday precision was less than 10%, and the extraction recovery was more than 80%. Isofraxidin and two metabolites (M1 and M2) were detected in rat plasma after oral administration of isofraxidin, and the molecular polarities of M1 and M2 were both increased compared to isofraxidin. The metabolites were identified as 5,6-dihydroxyl-7-methoxycoumarin and 5-hydroxyl-6,7-dimethoxycoumarin when subjected to parent ion spectra, product ion spectra, and extract mass and element composition analyses.

刺五加活性成分及其相关非共价复合物的质谱研究

中药化学成分，尤其是活性成分，是中药发挥药效作用的物质基础，它的深入研究是中药现代化的关键和核心。而中药成分的复杂性和药效的整体性，一般的分析及研究方法无法简捷快速地解决中药有效成分鉴定的难题，直接影响到中药研究开发的进程。质谱技术，尤其是软电离质谱及其联用技术，由于其具有灵敏、快速、分辨率高的优点，使得质谱日益成为研究复杂生物分子和中药体系中的应用问题的强有力手段。本论文以刺五加新药的开发为背景，采用有效部分的研究方法，运用先进的质谱技术，以药理活性筛选为指导，全面地研究了刺五加的活性成分。首先，采用电喷雾多极串联质谱技术，研究了正负离子条件下传统中药刺五加中的有效成分刺五加营B和刺五加营E的特征质谱行为，并提出了质谱碎裂机理，可以此鉴定传统刺五加有效成分，对传统刺五加药材及相关药品的快速鉴别和质量控制具有重要意义。以人参皂营R1为例，定量地研究四种碱金属离子浓度对皂普结构分析的影响，为合理添加碱金属离子以提高皂着分析的灵敏度提供了依据。在此基础上，用电喷雾多极串联质谱技术简便而又快捷地区分了两对生理活性差别较大的人参皂营的同分异构体：Rg1和PF11，Rg2和Rg3，为深入研究人参皂普的药效成分和作用机制奠定了一定的基础。系统地研究了β-香树”脂醇型的三种五环三菇皂营在不同模式下的多级串联质谱特征，为从多角度阐明皂昔类化合物的结构提供了新的思路。结构相近的皂普类化合物在电喷雾多级串联质谱中都表现出了与其自身结构相符的特征质谱行为，为不同皂营混合物的快速鉴定提供了一种快速有效的分析方法。在此基础上，研究了两种典型的刺五加仆卜皂营的质谱特征，并用多种质谱手段从刺五加叶总皂普中检测到18种皂普成分，其中有8种皂营成分在刺五加中而未见有文献报道，并用质谱首次发现了刺五加叶皂营的结构和其在植物体内代谢的相关性，这一结果对研究刺五加皂营的体内代谢有着十分重要的意义。以药理目标活性成分为指标，采用对比实验和一系列正交实验，对刺五加活性成分的提取方法，提取工艺和纯化工艺进行了优化，首次采用联合树脂法一次性分离纯化刺五加中两类有效成分总皂昔和总黄酮，经工艺优化最终得到总有效成分达85％以上的刺五加制剂，为刺五加新药的开发提供了大量的工艺参数。基于环糊精包合物（CDs）在中药剂型改造及提高某些药物稳定性和生物利用度方面广阔的应用前景和刺五加新药开发中的实际需要，系统到研究了三种环糊精（a-CD，β-CD和γ-CD）与刺五加皂昔元齐墩果酸（OA）形成的复合物。纬果表明，三种环糊精都能与OA形成稳定的l:1复合物，而β-CD和γ-CD还能与以形成2:1“三明治”状的复合物。复合物气液两相性质具有良好的相关性，为质谱快速筛选和表征这类非共价复合物提供了必要的条件。采用同样的方法对三种环糊精与刺五加中黄酮类化合物（芦丁和懈皮素）的复合物进行了系统研究，结果发观，三种环糊精都与芦丁（R）和棚皮素（Q）形成1:1复合物。但复合物稳定性有差异，对环糊精与芦丁的1:1复合物，以β-CD复合物最稳定，而对于环糊精与槲皮素1:1复合物则以α-CD复合物最稳定，这与芦丁含有糖链有关。刺五加活性成分黄酮类化合物生物利用度和活性的另一有效途径是制备黄酮-金属络合物。黄酮-金属络合物作为潜在的药物开发正日益受到重视，本文利用电喷雾质谱系统地研究了第四周期五种二价金属离子与棚皮素形成的络合物，并采用多级串联质谱部分表征了这些络合物的结构和稳定性之间的关系，为建立黄酮金属络合物筛选体系和表征提供了一种较好的方法，为这类新药的研制开发提供了一定的理论基础。