中药指纹图谱研究的某些进展与展望

在中药现代化的过程中,中药的质量控制标准直接影响到中药的国际化问题。近年来,国内很多学者对中药指纹图谱用于质量控制的问题进行了深入的研究,取得了很多成果。本文着重就近几年来中药指纹图谱的某些进展进行了综述,内容包括气相色谱指纹图谱、液相色谱指纹图谱以及其他指纹图谱。最后就指纹图谱研究的发展提出了几点看法。共引用文献161篇。     

Affinity chromatography with immobilized DNA stationary phase for biological fingerprinting analysis of traditional Chinese medicines

A stationary phase for high performance affinity chromatography with immobilization of DNA onto silica gel was prepared and characterized. The effect of the ionic strength, concentration of Mg2+, EDTA and CH3CN in the mobile phase on the retention of alkaloids were investigated. With this stationary phase, biological fingerprinting analysis of traditional Chinese medicines (TCMs) Coptis chinensis Franch and Rheum palmatum L. was performed with both one-dimensional (1-D) and two-dimensional (2-D) chromatography. The 1-D chromatography was performed with isocratic and gradient elution and 2-D chromatography was developed with immobilized DNA column combined with silica monolithic ODS column. It was found that 7 compounds in Coptis chinensis Franch including berberine, palmatine and jatrorrhizine, 14 compounds in Rheum palmatum L. including aloe-emodin, rhein, emodin, chrysophannol-8-O-glucophranoside and physionl-8-O-glucophranoside were active in binding to the immobilized DNA.     

色谱指纹图谱与中药质量控制

本文综述了近年来色谱指纹图谱研究中多种色谱技术、相关化学计量学方法的发展及其在中药质量控制中的应用,并对中药质量稳定性和有效性的全面评价方法和可行性进行了初步探讨。提出以色谱指纹图谱为核心,依托现代色谱技术、化学计量学和系统生物学方法,建立中药化学成分信息与药效(毒性)信息相关关系的中药质量控制研究策略,深层次揭示中药的内在作用机理,期望真正实现中药质量的可控、安全和有效。     

Qualitative and quantitative analysis in quality control of traditional Chinese medicines

Separation techniques with high efficiency and sensitive detection have been widely used for quality control of traditional Chinese medicines (TCMs). High-performance liquid chromatography, gas chromatography, and capillary electrophoresis are commonly used to separate various components in TCMs. Ultraviolet detection, fluorescence detection, evaporative light-scattering detection, mass spectrometry and nuclear magnetic resonance can be applied to separation techniques for qualitative and quantitative analysis of TCMs. The development of quality control for TCMs based on quantitative and qualitative analysis from 2000 to 2007 are reviewed; the fingerprint technique is also discussed due to its broad application in the quality control of TCMs. Prospects for further research based on our primary results are also discussed.     

Feasibility of sterilizing traditional Chinese medicines by gamma-irradiation

The feasibility of sterilizing traditional Chinese medicine (TCMs) by γ-irradiation has been systematically evaluated by the biological, toxicological and physicochemical tests on irradiated hundreds of TCMs. Those TCMs investigated in general show no significant biological or toxicological changes after irradiation, yet physicochemical changes are detectable in some irradiated TCMs, and water in TCMs enhances the effects. Those results obtained from radiolysis of some major effective components of TCMs in aqueous or ethanolic solutions reveal that the site selection of radiolytically generated radicals follows the example of simple compounds with same function groups. Wholesomeness and chemical clearance present a bright future to sterilizing TCMs by γ irradiation, however, some important measures and steps should be adopted: (1) The producers must strictly execute manufacturing procedure to reduce microbiological contamination thus lower the applied dose for sterilization which is recommended to be controlled under 5, 7 or 10 kGy, 10 kGy for dry herb, 7 kGy for herbal medicine and 5 kGy for some special herbal medicine; (2) Herb to be sterilized by γ-irradiation should exist in possible dry state; (3) Powder TCMs is recommended to mix with honey forming bolus, which can minimize the decomposition of herb.     

有机化学分析与中药现代化

从中药活性物质的阐明、质检方法的建立以及生产工艺的改进等几个方面讨论了有机化学分析在中药现代化研究进程中的作用.     

HPLC-ICP-MS测定中药中砷的形态

报道了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)联用技术测定中药中砷的形态.采用阴离子交换柱,以含0.2 mmol/L乙二胺四乙酸(EDTA)和2 mmol/L NaH2PO4的水溶液为流动相,pH 6.0,流速为1.0 mL/min,成功分离了亚砷酸(AsⅢ)、砷酸(AsⅤ)、甲基砷(MMA)和二甲基砷(DMA).检出限分别为0.67 μg/L (AsⅢ),0.85 μg/L (DMA),0.43 μg/L (MMA),0.70 μg/L (AsⅤ).中药样品经过(1 1)甲醇水溶液超声提取,离心、过滤、氮气吹干甲醇,超纯水定容.样品加标平均萃取回收率分别为: 92.8% (AsⅢ),108% (DMA),104% (MMA),101% (AsⅤ),RSD (n=7)均小于10%.     

Quality evaluation of traditional Chinese medicines based on fingerprinting

The usage of traditional Chinese medicines has expanded globally, but the data about authentication, efficacy, and safety is far from sufficient to meet the criteria supporting their use worldwide due to complexity in the composition. Fingerprinting describes integral characterization and reflects interactive aspects of complex components; therefore, it can offer the possibility of evaluating quality of traditional Chinese medicines following the overall principle. Chemometric techniques introduce multivariate analytical methods into fingerprinting to obtain more information that is useful, which is consistent with the holistic thought and plays an important role in research on the substantial basis. In this review, we will start with three aspects to expound the quality evaluation of traditional Chinese medicines based on fingerprints. The analytical techniques used in developing fingerprints including chromatographic methods, spectroscopic methods, and capillary electrophoresis are introduced. Strategies for fingerprints analysis usually based on chemometric methods including unsupervised and supervised pattern recognition are described. Applications of fingerprints for multi‐component quantification, quality control, screening of bioactive components, and fingerprint‐efficacy relationship study are also outlined. Finally, we propose challenges and future perspectives of fingerprints in quality evaluation to promote the development of modernization and internationalization of traditional Chinese medicines.     

Screening and analysis of bioactive compounds with biofingerprinting chromatogram analysis of traditional Chinese medicines targeting DNA by microdialysis/HPLC

Biofingerprinting chromatogram analysis, which is defined as the comparison of fingerprinting chromatograms of the extract of traditional Chinese medicines (TCMs) before and after the interaction with biological systems (DNA, protein, cell, etc.), was proposed for screening and analysis of the multiple bioactive compounds in TCMs. A method of microdialysis sampling combined with high performance liquid chromatography (HPLC) was applied to the study of DNA-binding property for the extracts of TCMs. Seven compounds were found to bind to calf thymus DNA (ct-DNA) from the TCMs of Coptis chinensis Franch (Coptis), but only three ones from Phellodendron amurense Rupr. (Phellodendron) and none from Sophoraflavescens Ait. (Sophora) to bind to ct-DNA, respectively. Three of them were identified as berberine, palmatine and jatrorrhizine and their association constants (K) to ct-DNA were determined by microdialysis/HPLC. Competitive binding behaviors of them to ct-DNA were also investigated.     

Protective effects of various Chinese traditional medicines against experimental cholestasis

In a previous paper, we reported that methanol extracts obtained from 13 Chinese traditional medicines showed remarkable choleretic effects in normal rats. This paper examines the protective effects against experimental cholestasis induced by carbon tetrachloride (CCl4) or alpha-naphthylisothiocyanate (ANIT) in rats. No medicines, including sodium dehydrocholate and 1-phenylpropanol which are used clinically as choleretic drugs, inhibited the decrease of bile flow induced by CCl4. On the other hand, Intinko-to, Saiko-seikan-to and Bohu-tusyo-san revealed marked improvement of the dysfunction in bile secretion induced by ANIT. These three medicines inhibited the decrease of excretion of bile acid or bilirubin in the bile. They also exerted a protective effect against the alterations of serum components induced by ANIT, i.e., of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, alkaline phosphatase and the concentration of serum bilirubin. These results indicate that methanol extracts of Intinko-to, Saiko-seikan-to and Bohu-tusyo-san demonstrate not only choleretic effects but also improvement of cholestasis and liver parenchymal injury in rats.     

Tyrosinase inhibitor screening in traditional Chinese medicines by electrophoretically mediated microanalysis

A capillary‐electrophoresis‐based method for the screening of tyrosinase inhibitors in traditional Chinese medicines was developed. The method integrated electrophoretically mediated microanalysis with sandwich mode injection, partial filling, and rapid polarity switching techniques, and carried out on‐column enzyme reaction and the separation of substrate and product. The conditions were optimized including the background electrolyte, mixing voltage, and the incubation time. Finally, the screening of nine standard natural compounds of traditional Chinese medicines was carried out. The inhibitors can be directly identified from the reduced peak area of the product compared to that obtained without any inhibitor. Chlorogenic acid (100 μM) showed inhibitory activity with the inhibitory percentage of 19.8%, while the other compounds showed no inhibitory activity. This method has great application potential in drug discovery from traditional Chinese medicines.     

共振散射法测定中草药中的微量铬

研究了在H2SO4介质中,Cr(Ⅵ)与碘化物和淀粉形成离子缔合物的共振光散射增强现象,拟定了一种新的测定Cr(Ⅵ)的共振光散射方法,通过实验确定了溶液中Cr(Ⅵ)浓度与散射光强度之间的关系,在λex＝λem＝290 nm处,共振光散射最强,且共振光散射强度与Cr(Ⅵ)浓度呈线性关系,该方法简便快速,线性范围为34～400 μg/L,检出限为6.7 μg/L,可用于中草药样品中铬的测定.     

离子淌度质谱技术在中药化学成分分析中的研究进展

离子淌度质谱(IM-MS)是一种将离子淌度分离与质谱分析相结合的新型分析技术。IM-MS的主要优势不仅是在质谱检测前提供了基于气相离子形状、大小、电荷数等因素的多一维分离,而且能够提供碰撞截面积、漂移时间等质谱信息进而辅助化合物鉴定。近年来,随着IM-MS技术的不断发展,该技术在中药化学成分分析中受到越来越多的关注。首先,IM-MS已成功应用于改善中药复杂成分尤其是同分异构体或等量异位素等成分的分离;其次,IM-MS可通过多重碎裂模式辅助高质量中药小分子质谱信息的获取;此外,IM-MS提供的高维质谱数据信息还可促进中药复杂体系多成分的整合分析。该文在对IM-MS分类和基本原理进行概述的基础上,从分离能力及分离策略、多重碎裂模式、多维质谱数据处理策略3个方面,重点综述了IM-MS在中药化学成分分析中的应用,以期为IM-MS在中药化学成分研究提供参考。     

Inhibition of acrylic acid polymerization by phenothiazine and p-methoxyphenol

The major features of polymerization induction periods for acrylic acid inhibited with phenothiazine and p-methoxyphenol have been characterized at 100°C, including duration of the induction periods, and rates of inhibitor disappearance, molecular oxygen absorption, and peroxide formation. Surprisingly, thermally produced radicals react more rapidly with phenothiazine than with oxygen since there is no detectable oxygen absorption or peroxide for mation during phenothiazine-induced induction periods. Thus, phenothiazine has been used to estimate the thermal rate of radical formation. Phenothiazine's effectiveness as an inhibitor is not directly affected by oxygen, although it does undergo oxygen-promoted, noninhibition-related side reactions. p-Methoxyphenol, on the other hand, depends entirely on the presence of oxygen to function as an inhibitor. Compared with equivalent concentrations of p-methoxyphenol, induction periods obtained with phenothiazine are very long, and the rate of inhibitor disappearance is slower by at least an order of magnitude. The characteristics of p-methoxyphenol inhibition reflect a greater radical flux deriving from the significant rates of oligomeric peroxide formation and decomposition which we measured during p-methoxyphenol-induced induction periods at 100°C. MEHQ is an effective inhibitor at ambient temperatures in part because of the greater stability of the peroxides at these lower temperatures.     

Surface-initiated enzymatic polymerization of DNA

We describe a technique to synthesize DNA homopolymers on a surface using surface-initiated enzymatic polymerization (SIEP) with terminal deoxynucleotidyl transferase (TdTase), an enzyme that repetitively adds mononucleotides to the 3'-end of oligonucleotides. The thickness of the synthesized DNA layer was found to depend on the deoxymononucleotide monomer, in the order of dATP > dTTP > dGTP approximately dCTP. In addition, the composition and the surface density of oligonucleotide initiators were also important in controlling the extent of DNA polymerization. The extension of single-stranded DNA chains by SIEP was further verified by their binding to antibodies specific to oligonucleotides. TdTase-mediated SIEP can also be used to grow spatially defined three-dimensional DNA structures by soft lithography and is a new tool for bioinspired fabrication at the micro- and nanoscale.     

基于定量指纹图谱技术的中药质量控制

定量指纹图谱技术是中药指纹图谱技术与多指标成分定量分析相结合的中药质量控制模式。定量指纹图谱技术的发展包括定量组分的制备、过程控制的指纹图谱技术和产品含量测定3个主要部分。本文以丹参为例,通过水提、醇沉、过膜、大孔树脂分离和工业色谱分离5个工艺流程制备了丹参定量组分,对各个工艺步骤以指纹图谱技术考察其稳定性和重复性。对丹参定量组分中的原儿茶醛、迷迭香酸、丹酚酸B 3个成分进行含量测定。3个成分的含量总和大于50%。定量组分的制备以现有的活性成分为目标,经过去粗存精的工艺过程,其质量标准得到了有效的提高。     

Multivariate statistical treatment of PIXE analysis of some traditional Chinese medicines

Elements in two kinds of 30 traditional Chinese medicines were analyzed by PIXE method, and the data were treated by multivariate statistical methods. The results show that these two kinds of traditional Chinese medicines are almost separable according to their elemental contents. The results are congruous with the traditional Chinese medicine practice.     

在线二维液相色谱技术在中药研究中的应用进展

中药的组成复杂,其化学成分的表征和识别一直是中药研究的基础和关键。在线二维液相色谱是基于两种分离模式构建的色谱分析技术,主要包括中心切割二维液相色谱和全二维液相色谱两种模式,因二者具有更高的峰容量而在中药研究中备受青睐。该文对在线二维液相色谱技术的概念和特点进行了讨论,并对二维液相色谱在中药研究中的应用进行了综述,以期为该技术在中药质量控制、物质基础表征、活性成分筛选等研究方面提供一定参考。     

Recent developments in sample preparation techniques for chromatography analysis of traditional Chinese medicines

Traditional Chinese medicines (TCMs) have a long history dating back thousands of years. Recently, there has been increasing interest worldwide in the use of TCMs for the prevention and treatment of various illnesses. In China, a large number of analytical tools, especially chromatographic techniques have been used to analyze the constituents of TCMs in order to control their quality and discover new bioactive compounds. In this paper, recent developments in sample preparation techniques for the extraction, clean-up, and concentration of analytes from TCMs are compared. These techniques include headspace solid-phase microextraction (HS-SPME), headspace liquid-phase microextraction (HS-LPME), microwave-assisted extraction (MAE), supercritical-fluid extraction (SFE), pressurized-liquid extraction (PLE), and microwave distillation (MD).