Study on the dynamic metabolic characteristic of main active ingredients in Danggui Buxue Decoction by liquid chromatography-tandem mass spectrometry based on in situ sequential metabolism strategy

Danggui Buxue Decoction is a classic formula for replenishing qi and nourishing blood. Despite its widespread use, its dynamic metabolism involved remains unclear. Based on the sequential metabolism strategy, blood samples from different metabolic sites were obtained via in situ closed intestine ring integrated with a jugular venous continuous blood supply technique. An ultra-high-performance liquid chromatography-linear triple quadruple-Orbitrap-tandem mass spectrometry method was developed for the identification of prototypes and metabolites in rat plasma. The dynamic absorption and metabolic landscape of flavonoids, saponins, and phthalides were characterized. Flavonoids could be deglycosylated, deacetylated, demethylated, dehydroxylated, and glucuronicated in the gut and then absorbed for further metabolism. Jejunum is an important metabolic site for saponins biotransformation. Saponins that are substituted by Acetyl groups tend to lose their acetyl groups and convert to Astragaloside IV in the jejunum. Phthalides could be hydroxylated and glucuronidated in the gut and then absorbed for further metabolism. Seven components serve as crucial joints in the metabolic network and are potential candidates for the quality control of Danggui Buxue Decoction. The sequential metabolism strategy described in this study could be useful for characterizing the metabolic pathways of Chinese medicine and natural products in the digestive system.     

固定化脂质体色谱与动物小肠吸收模型的相关性研究方法及其应用

固定化脂质体色谱(ILC)是一种新颖的筛选经被动吸收药物的技术,建立了ILC与常用的动物小肠吸收模型的相关性研究方法,考察了当归补血汤在ILC上有保留的成分的保留值与其在大鼠在体和离体小肠吸收模型中被吸收值的相关性。结果显示,当归补血汤在ILC上有保留成分的保留值与其通过大鼠在体小肠灌流和离体小肠实验测得的吸收值具有很好的相关性,其相关系数分别为0．9356和0．9350;当归补血汤在ILC上有保留的成分主要以被动扩散的方式被机体吸收。     

透析-高效液相色谱法在当归补血汤药效物质基础研究中的应用

将脂质体作为模拟生物膜,采用平衡透析与液相色谱联用技术,建立了一种研究中药成分与模拟生物具有相互作用的新方法。应用该方法对当归补血汤进行了分析,同时考察了模拟生物膜的浓度、pH值、缓冲系统和胆固醇的加入等因素对当归补血汤与模拟生物膜相互作用的影响。结果表明:当归补血汤中有7个组分与模拟生物膜相互作用明显;模拟生物膜的浓度影响最大,pH值对酸性组分阿魏酸的作用影响较大,其它因素的影响较小。该方法可用于预测药物在体内的吸收情况,进而研究中草药及复方的药效物质基础。     

Simultaneous determination of bioactive constituents in Danggui Buxue Tang for quality control by HPLC coupled with a diode array detector,an evaporative light scattering detector and mass spectrometry

Danggui Buxue Tang (DBT), a classical traditional Chinese formula comprising Radix Angelicae Sinensis (RAS) and Radix Astragali (RA), has been widely used to treat menopausal irregularity in Chinese women for nearly 800 years. In this study, a comprehensive analytical method of simultaneously determining the main types of bioactive constituents, eighteen in all from the formula, involving flavonoids, saponins, organic acid and some volatile compounds, was developed. This method was based on HPLC coupled to a diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) on a common reverse-phase C(18) column. Liquid chromatography coupled with on-line electrospray ionization mass spectrometry (LC-ESI-MS) was also used to further validate and analyze the constituents. It was found that 0.3% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution. This method, which showed good precision and accuracy, was successfully used to quantify the bioactive constituents in six products. As a result, the validated HPLC method, together with the LC-ESI-MS analysis, provided a new basis for assessing the quality of traditional Chinese medicinal compound preparations (TCMCPs) consisting of many bioactive components.     

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％。     

Studies of the Synergistic Actions of Three Herbs in Tong-Mai Formula

Tong-Mai oral liquid, containing three herbs (Pueraria radix, radix Salviae miltiorrhizae and rhizoma Chuanxiong), is a formula created under the theory of traditional Chinese medicine (TCM) to invigorate the ‘blood’ and dispel ‘blood stasis’, which arises from poor blood circulation. In Tong-Mai formula, Pueraria radix is used for improved blood circulation and to treat cardiovascular diseases and arrhythmia. Radix Salviae miltiorrhizae was widely used in TCMs for treatment of coronary heart disease, blood circulation diseases, and other cardiovascular diseases; rhizoma Chuanxiong is used to treat thrombosis and platelet aggregation. Clinically, combining the use of these herbs was therapeutically valuable, which suggests that they might synergistically prevent thrombosis. In this study, the pharmacokinetic behaviors of puerarin and daidzein in rat plasma were studied after orally administrated to four kinds of extracts. Puerarin and daidzein were set as index components. In the Tong-Mai oral liquid group, puerarin and daidzein showed the maximum plasma concentration and the lowest clearance rate of plasma. The results indicated that the herbal constituents in Tong-Mai formula showed synergism, and it may be helpful to certificate the rationality of the matching in Tong-Mai formula.     

Studies of the Synergistic Actions of Three Herbs in Tong-Mai Formula

Tong-Mai oral liquid, containing three herbs (Pueraria radix, radix Salviae miltiorrhizae and rhizoma Chuanxiong), is a formula created under the theory of traditional Chinese medicine (TCM) to invigorate the ‘blood’ and dispel ‘blood stasis’, which arises from poor blood circulation. In Tong-Mai formula, Pueraria radix is used for improved blood circulation and to treat cardiovascular diseases and arrhythmia. Radix Salviae miltiorrhizae was widely used in TCMs for treatment of coronary heart disease, blood circulation diseases, and other cardiovascular diseases; rhizoma Chuanxiong is used to treat thrombosis and platelet aggregation. Clinically, combining the use of these herbs was therapeutically valuable, which suggests that they might synergistically prevent thrombosis. In this study, the pharmacokinetic behaviors of puerarin and daidzein in rat plasma were studied after orally administrated to four kinds of extracts. Puerarin and daidzein were set as index components. In the Tong-Mai oral liquid group, puerarin and daidzein showed the maximum plasma concentration and the lowest clearance rate of plasma. The results indicated that the herbal constituents in Tong-Mai formula showed synergism, and it may be helpful to certificate the rationality of the matching in Tong-Mai formula.

     

Metabolomics study of the effect of Danggui Buxue Tang on rats with chronic fatigue syndrome

Danggui Buxue Tang (DBT), a traditional Chinese medicine (TCM) formula for ‘invigorating qi and enriching blood’, has been reported to produce a good effect on chronic fatigue syndrome (CFS). However, the related mechanism remains largely undetermined. This study devised a metabolomics approach with GC–MS combined with pattern recognition to estimate the extent to which DBT alleviated CFS induced by food restriction and force swimming in rats. After 4 weeks of treatment, the endurance capability of rats was significantly better, and the motionless time was significantly shorter in the DBT group than in the CFS model group. Moreover, the activities of superoxide dismutase and glutathione peroxidase increased, whereas the levels of malondialdehyde, interleukin 6, and tumor necrosis factor alpha decreased in the DBT treatment group. Fifteen significantly changed metabolites were observed in the serum of rats with CFS, which was reversed markedly by DBT treatment. Metabolic pathway analysis showed that DBT could possibly alleviate CFS in rats by regulating the biosynthesis of phenylalanine, tyrosine, and tryptophan and the metabolism of glycine, serine, threonine, glycerolipid, glyoxylate, dicarboxylate, and tyrosine. It was observed that the metabolism of glycine, serine, and threonine was most closely related to the improvement in CFS by DBT treatment. This study showed that DBT could improve CFS effectively, and metabolomics was a powerful means to gain insights into the TCM formulas against CFS.     

Effects of Danggui Buxue Tang, a traditional Chinese herbal decoction, on high glucose-induced proliferation and expression of extracellular matrix proteins in glomerular mesangial cells

Diabetic nephropathy (DN) is the leading cause of end-stage failure of the kidney, but the efficacy of currently available strategies for the prevention of DN remains unsatisfactory. In this study, we investigated the effects of Danggui Buxue Tang (DBT), a Chinese herbal decoction prepared from Radix Astragali (RA) and Radix Angelicae sinensis (RAS), on high glucose-induced proliferation and expression of laminin, type IV collagen (collagen IV) and fibronectin in glomerular mesangial cells (GMCs). The cell proliferation was determined by MTT assay, and the expression of collagen IV, laminin and fibronectin in GMCs was detected by ELISA assay. It was shown that high glucose clearly induced the proliferation of GMCs and increased the release of collagen IV, laminin and fibronectin. Treatment with RA, RAS and DBT inhibited cell proliferation and the expression of collagen IV, laminin and fibronectin induced by high glucose, with DBT, especially at the highest concentration (DBT20), exhibiting a stronger effect than RA and RAS alone. Thus, it is concluded that DBT inhibits increased cell proliferation and the expression of major extracellular matrix proteins that are induced by high glucose, indicating its value for prophylaxis and therapy of DN at the early stages.     

Multiple approaches to characterize and visualize the chemical composition of Sijunzi Decoction comprehensively

Sijunzi Decoction is composed of Ginseng Radix et Rhizoma, Atractylodes Macrocephalae Rhizoma, Poria, and Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle, and it is a classic formula for treating spleen deficiency syndrome in Chinese medicine. Clarifying the active substances is an effective way to develop Traditional Chinese medicine and innovative medicines. Carbohydrates, proteins, amino acids, saponins, flavonoids, phenolic acids, and inorganic elements in the decoction were analyzed by multiple approaches. A molecular network was also used for visualizing the ingredients in Sijunzi Decoction, and representative components were also quantified. The detected components accounted for 74.544% of the Sijunzi Decoction freeze-dried powder, including 41.751% crude polysaccharides, 17.826% sugars (degree of polymerization 1–2), 8.181% total saponins, 2.427% insoluble precipitates, 2.154% free amino acids, 1.177% total flavonoids, 0.546% total phenolic acids, and 0.483% inorganic elements. Molecular network and quantitative analysis used to characterize the chemical composition of Sijunzi Decoction. The present study systematically characterized the constituents of Sijunzi Decoction, revealed the composition ratio of each type of constituent, and provided a reference for study on the substance basis of other Chinese medicine.     

The analysis of Radix Angelicae Sinensis (Danggui)

Radix Angelicae Sinensis, known as Danggui in China, is one of the most popular traditional Chinese medicines (TCMs), which is contained by more than 80 composite formulae. Modern researches indicate that phthalides, organic acids and their esters, polysaccharides are main chemical components related to the bioactivities and pharmacological properties of Danggui. Some of them, such as Z-ligustilide and ferulic acid, are selected as marker compounds to evaluate the quality of Danggui frequently. Because of the diversity of chemical structures and characters of these components, analytical methods of Danggui are various, including GC-MS, HPLC-DAD-MS, TLC, CE-DAD, and so on. Besides that, the development of analytical technology makes the quality control of Danggui more effective and reliable. Quality evaluation is from single or several components' analysis to fingerprinting, or in combination. Furthermore, bioactive components screening of Danggui has also attracted much attention, which will help us evaluate the selected marker components to some extent. In this paper, the literatures about the major phytoconstituents of Danggui, quality control and bioactive components screening methods have been reviewed. Main attention is given to the different methodologies developed to perform chemical analysis, including separation, detection and identification.     

中药配伍的统一分析方法研究Ⅰ.单味药与复方的分离、特征组分的识别以及指纹对比

 中药配伍是传统中药理论之一 ,其物质分析是中药基础理论现代化研究的重点。中药配伍的特点是物质组分复杂、多变和整体性强 ,因此单一或少量组分的分析结果很难表征药物的性质。采用反相液相色谱模式 ,以黄芪、当归以及自制的“当归补血汤”的分离、特征组分识别以及指纹对比的实例分析 ,介绍了中药配伍分析的多模式多柱色谱系统以及统一分析方法的研究思路。     

体外细胞模型和高效液相质谱联用分析预测黄芪中的活性成分

利用体外细胞模型模拟体内细胞对中药有效成分的特异性吸收,结合高效液相色谱/质谱分析筛选中药黄芪中的生物活性成分。将中药黄芪提取液与Caco-2细胞及红细胞分别混合培养,破碎与药材结合后的细胞,使之释放出结合的药材中的成分。运用高效液相色谱/飞行时间质谱(HPLC-ESI/TOFMS)分析中药黄芪提取液与活性细胞有结合的成分,并对其进行结构鉴定。结果显示:黄芪中有10个化合物与Caco-2细胞结合,14个化合物与红细胞结合。本方法可用于预测口服药物在体内的吸收以及与特定靶细胞的结合情况,特异性地筛选中药复杂体系中的药效物质基础。     

Analytical comparison of different parts of Radix Angelicae Sinensis by gas chromatography coupled with mass spectrometry

The three parts of Radix Angelicae Sinensis (Danggui) were reported to have different therapeutic effects. In order to investigate their chemical compositions of different parts of Danggui, gas chromatography-mass spectrometry (GC-MS) combined with chemometrics data analysis was applied to provide a more detailed study. Subwindow factor analysis (SFA) and a modified augmented evolving window orthogonal projection (AEWOP) method were used to resolve the batch GC-MS data sets from the Danggui samples. Then, t-test, Wilcoxon rank sum test, and principal component analysis (PCA) were applied to carry out the comparison job. The results indicate which components are found to have significant differences among the three parts. These findings may be helpful for further research of the pharmacological activities of Danggui.     

Rapid and sensitive screening and characterization of phenolic acids, phthalides, saponins and isoflavonoids in Danggui Buxue Tang by rapid resolution liquid chromatography/diode-array detection coupled with time-of-flight mass spectrometry

A novel rapid resolution liquid chromatography (RRLC) method coupled with diode-array detection (DAD) and time-of-flight mass spectrometry (TOFMS) in both positive and negative modes has been developed for quick and sensitive identification of the major compounds in Danggui Buxue Tang (DBT) preparation. Significant advantages of the use of RRLC with 1.8-microm porous particles include the much higher speed of chromatographic separation and great enhancement in sensitivity, compared with the conventional high-performance liquid chromatography (HPLC). With dynamic adjustment of the key role as fragmentor voltage in TOFMS, an efficient transmission of the ions was achieved to obtain the best sensitivity for providing the molecular formula for each analyte, and abundant fragment ions for structural information. The structural characterization of the major compounds in DBT was elucidated with authentic standards by DAD-TOF/MS, including phenolic acids, phthalides, saponins and isoflavonoids. The targets were rapidly screened from the complicated DBT matrix using a narrow mass window of 0.01 Da to restructure extracted ion chromatograms. By accurate mass measurements within 3 ppm error for each molecular ion and subsequent fragment ions, ten phenolic acids and phthalides including three groups of isomers, thirteen major saponins with a 20,24-epoxy-9,19-cyclolanostane-3,6,16,25-tetrol skeleton, sixteen isoflavonoids, corresponding glycosides, malonylglycosides, and acetylglycosides were identified in DBT preparation. The appropriate fragmentation pathways for them were also proposed based on definite elemental composition of the fragment ions.     

Identification of metabolites of Danggui Buxue Tang in rat urine by liquid chromatography coupled with electrospray ionization time‐of‐flight mass spectrometry

A method coupling liquid chromatography with electrospray ionization time‐of‐flight mass spectrometry (LC/ESI‐TOF/MS) has been developed for rapid and sensitive analysis of rat urinary metabolite profile of Danggui Buxue Tang (DBT), a well‐known Chinese herbal formula. After oral administration of DBT, urine samples were collected during 0–24 h, and then pretreated by solid‐phase extraction. A total of 68 compounds including 13 parent compounds and 55 metabolites were detected in the drug‐containing urines compared with blank urines. The total analytical time was less than 20 min. Metabolites of DBT were identified using dynamic adjustment of the fragmentor voltage to produce structure‐relevant fragment ions. By using this approach, the mass accuracy of precursor and fragment ions was typically within ±5 ppm of the theoretical values, and enabled the identification of 43 metabolites including 27 isoflavanoid and 16 phthalide metabolites. Our results indicated that glucuronidation and sulfation were the major metabolic pathways of isoflavonoids, while glutathione conjugation, glucuronidation and sulfation were the main metabolic pathways of phthalides. No saponin‐related metabolites were detected. The results of the present study provided important structural information relating to the metabolism of DBT. Furthermore, this work demonstrated the potential of the LC/ESI‐TOF/MS approach for identification of metabolites from Chinese herbal medicines in urine. Copyright © 2009 John Wiley & Sons, Ltd.     

The difference between blood‐associated and water‐associated herbs of Danggui‐Shaoyao San in theory of TCM,based on serum pharmacochemistry

Danggui‐Shaoyao San (DSS) is a famous Chinese formula for activating blood circulation and promoting urination. This study was to investigate the difference of material basis between a blood‐associated herbs group and a water‐associated herbs group. According to the theory of traditional Chinese medicine, the formula can be divided into a blood‐associated herbs group (Angelica sinensis, Paeonia lactiflora and Ligusticum chuanxiong) and a water‐associated herbs group (Atractylodes macrocephala, Alisma orientale and Poria cocos). The HPLC fingerprint of the formula was established for quality control. Serum samples from rats, orally administrated DSS, and the decomposed recipes of DSS, were analyzed by HPLC‐DAD and the transitional blood components of DSS were identified. Twenty‐one common peaks were identified in the fingerprint of DSS. Contents of paeoniflorin, albiflorin, ferulic acid and alisol B 23‐acetate in co‐decoction were significantly higher than those in individual decoction. Eleven peaks belonged to the blood‐associated herbs group (four metabolites and seven prototype components; paeoniflorin and ferulic acid appeared in prototype components), whereas six peaks belonged to the water‐associated herbs group (three metabolites and three prototype components). It was concluded that the serum pharmacochemistry is a meaningful approach for clarifying the difference between blood‐associated and water‐associated herbs in chemical composition. Copyright © 2015 John Wiley & Sons, Ltd.     

Studies on pharmacokinetics,body distribution,plasma protein binding rate,and excretion of 1-methyl hydantoin in rats in vivo

In this paper, plasma concentration, plasma protein binding rate, body distribution, and excretion of both oral and intravenous administration of rats were determined by high performance liquid chromatography (HPLC) combining with UV detector. The blood drug concentration of oral and intravenous administration was summarized. The bioavailability of T_1/2 was approximately 0.75?hr. At the meanwhile, the bioavailability was about 18.84?±?2.21%. The plasma protein binding rate of 1-methyl hydantoin was about 24.36?±?0.93%, belonging to low protein binding drug. The result shows that 1-methyl hydantoin can be rapidly distributed in various organs and tissues and quickly eliminated within 6?hr without accumulation in the organs. Its discharge from the urine and feces was 16.58?±?4.48% and 3.37?±?0.71%, respectively. All of the results showed that the recovery rate, liner relationship, specificity, stability, and precision of the method were good. The study also proved that 1-methyl hydantoin has been eliminated quite faster in rats.     

Validated method for the determination of platinum from a liposomal source (SPI-77) in human plasma using graphite furnace Zeeman atomic absorption spectrometry

A sensitive analytical method based on flameless atomic absorption spectrometry with Zeeman correction has been validated for the quantitative determination in human plasma of platinum originating from cisplatin in a liposomal source, SPI-77. The performance of the method was acceptable over a sample concentration range of 0. 125-1.25 micromol platinum/L and the lower limit of quantification was determined to be 1.25 micromol platinum/L in undiluted clinical samples. The performance data of the assay were investigated using both a calibration curve with carboplatin in plasma ultrafiltrate and diluted human plasma samples spiked with SPI-77. The recoveries, between-day and the within-day precisions of both methods of calibration were not significantly different allowing carboplatin ultrafiltrate calibration standards to be used to quantify platinum derived from SPI-77 in human plasma. Apparently, the liposomal formulation had no significant influence on the determination of platinum. The usefulness of the presented method was demonstrated in a phase I clinical and pharmacokinetic study. In addition, in vitro experiments were carried out to determine the distribution of SPI-77 in blood. The results indicated that platinum from SPI-77 mainly concentrates in plasma and that binding to and/or endocytosis in red blood cells is negligible.