以人血清白蛋白为固定相的分子生物色谱分析几种中药活性成分的研究

提出用人血清白蛋白分子生物色谱分析中药活性成分的方法,分别比较了当归等4种单味中药的分离结果,考察了提取当归的3种溶剂及提取时间对活性物质提取量的影响,同时建立了当归中阿魏酸与藁本内酯的定量分析方法.     

Evaluation of the Quality of Angelica sinensis by High-Performance Liquid Chromatography

Quality control and consistent quality are prerequisites to guarantee the safety and efficacy of pharmaceutical preparations and dietary supplements. In this paper, the intra- and inter-annual consistency and stability of Angelica sinensis (Oliv.) Diels, cultivated according to Good Agricultural Practices (GAP) in the main production areas of China, were evaluated by high-performance liquid chromatographic fingerprints composed of twenty-four common components. Subsequently, thirteen compounds were identified in the chromatograms using diode array detection and mass spectrometry. Z-Ligustilide, senkyunolide H, senkyunolide I, and ferulic acid were determined to evaluate the distribution of chemical components. In addition, robust principal component analysis was applied to further evaluate the samples, and four discriminatory “markers” among the common twenty-four were pinpointed by the loading plots that were used to distinguish quality differences of Angelica sinensis from different harvest years.     

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％。     

Chemical composition analysis of Angelica sinensis (Oliv.) Diels and its four processed products by ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry combining with nontargeted metabolomics

Angelica sinensis (Oliv.) Diels. has been used for women to enrich the blood, prevent and treat blood deficiency syndrome in Traditional Chinese Medicine for thousands of years. Wine-processed Angelica sinensis, soil-processed Angelica sinensis, oil-processed Angelica sinensis, and charred-processed Angelica sinensis are the most significant four processed products used in Chinese clinic. However, there have been few studies aimed at comparing their chemical differences. Ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry combining with nontargeted metabolomics was applied to investigate the diversity of processed products of Angelica sinensis. A total of 74 compounds with the variable importance in the projection value more than 1.5 and P less than 0.05 in ANOVA were highlighted as the compounds that contribute most to the discrimination of Angelica sinensis and four processed products. The results showed the metabolic changes between Angelica sinensis and its four processed products, there were 19 metabolites, 3 metabolites, 6 metabolites, and 45 metabolites were tentatively assigned in soil-processed Angelica sinensis, wine-processed Angelica sinensis, oil-processed Angelica sinensis, and charred-processed Angelica sinensis, respectively. These results suggested that the proposed metabolomics approach was useful for the quality evaluation and control of processed products of Angelica sinensis.     

Inhibitory effects of tetramethylpyrazine and ferulic acid on spontaneous movement of rat uterus in situ

Tetramethylpyrazine is one of the alkaloids contained in Ligusticum wallichii Franch. Ferulic acid is a phenolic compound contained in Ligusticum wallichii Franch and Angelica sinensis (Oliv.) Diels. The present study was carried out to examine the effect of tetramethylpyrazine and ferulic acid and the combined effect of both compounds on spontaneous uterine contractions in rats in situ. Tetramethylpyrazine and ferulic acid showed an inhibitory effect on uterine movement when given perorally and intravenously, respectively. The combination of both compounds, at doses individually insufficient to inhibit, synergistically inhibited uterine contraction. It was found that tetramethylpyrazine and ferulic acid inhibited uterine contractions and the inhibitory effect induced by the combination of both was due to the potentiation.     

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.     

Comparative separation of biologically active components inRhizoma chuanxiong by affinity chromatography with α1-acid glycoprotein and human serum albumin as stationary phasesglycoprotein and human serum albumin as stationary phases

Summary Affinity chromatography with α₁-acid glycoprotein (AGP) and human serum albumin (HSA) stationary phases was applied to screen and analyze the biologically active components ofRhizoma chuanxiong. Five major peaks and a number of small peaks were resolved based on their affinity for AGP and HSA, respectively, and three of them, identified as ferulic acid, chuanxiongzine and ligustilide via standard compounds, are regarded as effective components. The effects of acetonitrile concentration, pH, inorganic salt concentration and temperature on the retention behaviors of five major components on the two stationary phases were also investigated. It was observed that hydrophobicity is the major contributor to retention on both stationary phases, and ferulic acid has a weak electrostatic interaction with HSA. It demonstrated that the chromatograms ofRhizoma chuanxiong on the two stationary phases have concise and different fingerprinting characteristics. The amount of ferulic acid, chuanxiongzine and ligustilide inRhizoma chuanxiong determined using the AGP column are as much as 0.064%, 0.021% and 2.00% by weight.     

Fractionation and analysis of Artemisia capillaris Thunb. by affinity chromatography with human serum albumin as stationary phase

A method for the screening and analysis of biologically active compounds in traditional Chinese medicine is proposed. Affinity chromatography using a human serum albumin (HSA) stationary phase was applied to separate and analyze the bioactive compounds from Artemisia capillaris Thunb. Five major peaks and several minor peaks were resolved based on their affinity to HSA, two of them were identified as scoparone (SCO, 6,7-dimethoxycoumarin) and capillarisin (CAP). CAP shows a much higher affinity to HSA than SCO. The effects of acetonitrile concentration, eluent pH, phosphate concentration and temperature on the retention behaviors of several major active components were also investigated, and it was found that hydrophobicity and eluent pH play major roles in changing retention values. The results demonstrate that the affinity chromatography with a HSA stationary phase is an effective way for analyzing and screening biologically active compounds in traditional Chinese medicine.     

Separation and determination of three phenylpropanoids in the traditional Chinese medicine and its preparations by capillary electrophoresis

Three phenylpropanoids (ferulic acid, chlorogenic acid, and caffeic acid) are simultaneously separated and determined within 13 min by a new capillary electrophoresis method using 15 mmol/L sodium borate (pH 8.71) as run buffer. The optimum conditions for the separation as well as the analytical characteristics, such as the calibration graph and limit of detection (LOD) for the determination of these three compounds, are studied. The linear range for the determination of ferulic, chlorogenic, and caffeic acid is 5.0 approximately 70.0, 8.0 approximately 112.0, and 9.0 approximately 64.0 microg/mL, with the LOD as 1.5, 2.25, and 6.0 microg/mL, respectively. The method, which is very simple, rapid, and of requisite sensitivity and reproduction, is satisfactorily used for the separation and determination not only of ferulic, chlorogenic, and caffeic acid in Cimicifuga foelida Li and its preparation (Yin-huang-han-pian), but also of ferulic acid and chlorogenic acid in Ligusticum chuanxiong hort. and Angelica sinensis (Oliv.) Diels.     

Assay of free ferulic acid and total ferulic acid for quality assessment of Angelica sinensis

Activity of Chinese Danggui (DG), the processed root of Angelica sinensis (Oliv.) Diels, is linked to the ferulic acid content but the stability of ferulic acid during extraction for medicinal use is not known. The stabilities of ferulic acid and coniferyl ferulate were evaluated in the extracts of DG using a variety of extraction solvents. These included various combinations and proportions of methanol, water, formic acid, 1 M aqueous hydrochloric acid and 2% sodium hydrogen carbonate (NaHCO3) in water. Coniferyl ferulate was found liable to hydrolyze into ferulic acid in neutral, strongly acidic and basic solvents, where heat and water could facilitate this hydrolysis. However, the hydrolysis was relatively resisted in weakly organic acid. Based on the stability evaluation, two new terms, namely: free ferulic acid and total ferulic acid, were suggested and defined. Free ferulic acid refers to the natural content of ferulic acid in herbs. Total ferulic acid means the sum of free ferulic acid plus the amount of related hydrolyzed components. Meanwhile, the high-performance liquid chromatographic (HPLC) method was developed to assay free ferulic acid and total ferulic acid in DG using methanol-formic acid (95:5) and methanol-2% NaHCO3 in water (95:5) as extraction solvents, respectively. Ten DG samples were investigated on their contents of free and total ferulic acid. The results indicated that the amount variety of free ferulic acid was larger than that of their counterparts, and the ratio of total ferulic acid to free ferulic acid was 4.07 +/- 2.73 (mean +/- SD, n = 10). The chemical assay of DG using total ferulic acid content would be a better choice to assess the herbal quality and was recommended.     

分子生物色谱用于中药活性成分筛选及质量控制方法的研究

报道了近期工作进展,首先阐述了分子生物色谱的基本原理及特点,然后介绍了分子生物色谱对多种中药、不同产地的同种中药活性成分谱图模式的比较,结合已有的工作对活性成分筛选方法、相互作用研究、质量控制方法发展做了细致的说明,并讨论了其发展方向及前景。     

高效液相色谱与质谱联用分析测定川芎中有效成分阿魏酸与藁本内酯

采用HPLC-MS系统地研究了中药川芎有效成分阿魏酸(ferykuc acid)和藁本内酯(liguatilide)的定量分析方法。利用制备色谱制备了藁本内酯对照品,并对其进行了紫外光谱、质谱、红外光谱等结构鉴定。分别考察了水、甲醇、乙醇、95％乙醇4种溶剂以及提取时间对川芎中阿魏酸和藁本内酯提取量的影响。结果表明：水是阿魏酸的最佳提取溶剂,提取时间45min为宜;乙醇是适合提取藁本内酯的溶剂,提取时间75min为宜。以外标法对市售川芎中的阿魏酸与藁本内酯进行了定量分析,二者含量分别是0．15％(m／m)和0．82％(m/m)。     

Fractionation and analysis of Artemisia capillaris Thunb. by affinity chromatography with human serum albumin as stationary phase

A method for the screening and analysis of biologically active compounds in traditional Chinese medicine is proposed. Affinity chromatography using a human serum albumin (HSA) stationary phase was applied to separate and analyze the bioactive compounds from Artemisia capillaris Thunb. Five major peaks and several minor peaks were resolved based on their affinity to HSA, two of them were identified as scoparone (SCO, 6,7-dimethoxycoumarin) and capillarisin (CAP). CAP shows a much higher affinity to HSA than SCO. The effects of acetonitrile concentration, eluent pH, phosphate concentration and temperature on the retention behaviors of several major active components were also investigated, and it was found that hydrophobicity and eluent pH play major roles in changing retention values. The results demonstrate that the affinity chromatography with a HSA stationary phase is an effective way for analyzing and screening biologically active compounds in traditional Chinese medicine.     

Quantification of ligustilides in the roots of Angelica sinensis and related umbelliferous medicinal plants by high-performance liquid chromatography and liquid chromatography-mass spectrometry

A reversed phase high-performance liquid chromatographic method was developed for quantifying E-ligustilide (1) and Z-ligustilide (3) in the roots of Angelica sinensis (Oliv.) Diels with confirmation using UV, atmospheric pressure chemical ionisation (APCI) MS and APCI-MS-MS techniques. Based on the UV spectra of compounds 1, E-butylidenephthalide (2), 3 and Z-butylidenephthalide (4), the absorption at 350 nm was chosen as measuring wavelength in which baseline separation of compounds 1 and 3 could be obtained but avoided the interference of compounds 2 and 4. The identity of compounds 1 and 3 in samples were unambiguously determined by the respective quasi-molecular ions ([M+H]+) in APCI-MS. According to the stability data, acetonitrile was chosen for the preparation of standard solutions in order to minimize the isomerization of compound 3. Compounds 1 and 3 were qualitatively and quantitatively analyzed in seven samples of the roots of Angelica sinensis (Oliv.) Diels, Angelica acutiloba Kitagawa, Angelica acutiloba Kitagawa var. sugiyamae Hikino and the rhizome of Ligusticum chuanxiong Hort. Analysis of an extract from a sample root of Angelica gigas Nakai using LC-MS for the first time could not detect the presence of ligustilide in this herb. The overall analytical procedure is rapid and reproducible which is considered suitable for quantitative analysis of large number of samples.     

Headspace Solid-phase Microextraction–Gas Chromatographic–Mass Spectrometric Analysis of the Essential Oils of Two Traditional Chinese Medicines, Angelica pubescens and Angelica sinensis

Angelica pubescens and Angelica sinensis belong to the Umbelliferae family and both are used as traditional Chinese medicines. In the present study, headspace solid-phase microextraction (HS-SPME) with gas chromatography-mass spectrometry (GC-MS) was used for the analysis of the volatile constituents present in their roots. Eighty-seven compounds in Angelica pubescens and thirty-six compounds in Angelica sinensis were identified by GC-MS. Their relative contents were calculated by the peak area ratio. HS-SPME was compared to steam distillation (SD) by analyzing the volatile constituents of Angelica sinensis root. A good agreement between results obtained with both techniques was found. As a conclusion, HS-SPME is a powerful tool for determining the volatile constituents present in the TCMs.     

Analysis of Chemical Incredients of Angelica Sinnesis——Analysis of Non-Volatile Constituents of Roots

In the present investigation,the non-volatile constituents of Danguei (Tang-Kuei) roots of Gansu origin [Angelica sinensis (Oliv.) Diels] were analyzed and thirty seven organic constituin s were also identified,fifteen of which have not been recorded in literature before,including a new optically active compound named as Angelicide and an antibiotie,Brereldin A.By plasma emission spectrometry,thirteen minor elements were also detecled     

Antiinflammatory effect of tetramethylpyrazine and ferulic acid.

Tetramethylpyrazine (TMP) is one of the alkaloids contained in Ligusticum wallichii Franch (L. wallichii). Ferulic acid (FA) is a phenolic compound contained in L. wallichii and Angelica sinensis (Oliv.) Diels (A. sinensis). The present study was carried out to examine the antiinflammatory effect and to elucidate the mode of the effect of TMP and FA. Both compounds significantly inhibited the edema induced by carrageenin, the increase of the dye leakage induced by acetic acid and the granuloma formation induced by cotton pellet. And also, TMP and FA inhibited the number of writhes induced by acetic acid. From these results, it is suggested that both compounds have the antiinflammatory effect and the analgesic effect, and both compounds exert an antiinflammatory effect at the early and the late stages of processes in the inflammatory pathology.     

当归化学成份分析研究——毛细管气相色谱-质谱法鉴定当归根挥发油成份

当归具有活血化瘀、镇静、解痉等作用.分析其化学成份对研究其药理作用具有极重要的意义.自1924年开始,即有这方面的研究报导^[1-11].     

当归化学成份分析研究——根部非挥发性成份分析

本文报导了甘肃岷县当归根部非挥发性成份的分析结果。共鉴定出37个有机成份,其中15种未见报导。用等离子体发射光谱检出当归中13种微量元素。     

Characterization of interaction property of multicomponents in Chinese Herb with protein by microdialysis combined with HPLC

Interaction of traditional Chinese Herb Rhizoma Chuanxiong and protein was studied by microdialysis coupled with high performance liquid chromatography. Compounds in Rhizoma Chuanxiong, such as ferulic acid, senkyunolide A and 3-butylphthalide, were identified by HPLC, HPLC-MS and UV-vis. Microdialysis recoveries and binding degrees of compounds in Rhizoma Chuanxiong with human serum albumin (HSA) and other human plasma protein were determined: recoveries of microdialysis sampling ranged from 36.7 to 98.4% with R.S.D. below 3.1%; while binding to HSA ranged from 0 to 91.5% (0.3 mM HSA) and from 0 to 93.5% (0.6 mM HSA), respectively. Compared with HSA, most of compounds bound to human blood serum more extensively and the results showed that binding of these compounds in Rhizoma Chuanxiong was influenced by pH. Two compounds were found to bind to HSA and human blood serum, their binding degrees were consistent with ferulic acid and 3-butylphthalide, the active compounds in Rhizoma Chuangxiong.