色谱-质谱联用技术在中药代谢组学研究中的应用

代谢组学是研究生物体被扰动后其代谢产物种类、数量及变化规律的科学,研究理念与中医药理论的整体、动态观念非常一致,目前很多工作已将代谢组学应用于中药药效物质基础、作用机制、复方及配伍规律等研究中,有望推动中医药现代化进程。色谱-质谱联用技术是代谢组学的主要分析技术平台,该文综述了近3年来色谱-质谱联用技术在中药代谢组学研究中的应用,重点介绍不同分离技术的特点及最新进展,并讨论了其存在的问题。     

Comparative pharmacokinetic study of the main components of cortex fraxini after oral administration in normal and hyperuricemic rats

Cortex Fraxini is an important traditional Chinese herbal medicine used for the treatment of gout and hyperuricemia. An efficient and rapid ultra‐performance liquid chromatography mass spectrometry method was developed and validated for simultaneous quantitation of six coumarins (aesculin, fraxin, aesculetin, fraxetin, sopoletin and 7‐hydroxycoumarin) in normal and hyperuricemic rats plasma after oral administration of Cortex Fraxini. The method could successfully be applied for pharmacokinetics studies. The pharmacokinetic behavior of six coumarins in normal and hyperuricemia rats plasma was determined. Results showed that, for some of analytes, the pharmacokinetic parameters (AUC_0–t , AUC_0–∞, C _max, T _max and CL ) were significantly different between normal and hyperuricemic rats. The different pharmacokinetic parameters might result from renal impairment or a change of metabolic enzymes in the pathological state. The pharmacokinetic study in pathological state could provide more useful information to guide the clinical use of traditional Chinese herbal medicine.     

Primary study on the application of Serum Pharmacology in Chinese traditional medicine

In the paper, two main methods, which are Serum Pharmacology and Traditional Pharmacology, were adopted to study Chinese traditional medicine, such as Ginkgo biloba extract (GBE), ginsenosides (GS) and compound GG (GBE + GS), pharmacology in vitro. The results showed that there were evident difference between the results of Serum Pharmacology and that of Traditional Pharmacology. There was no significant difference between the drug effect of crude GS on nitric oxide (NO) production in ECV304 and that of crude GBE, and the drug effect of GG was superior to that of GS and GBE, respectively. But, compared with GBE serum, the GS serum up-regulation of NO production in ECV304 increased significantly, and the GG serum up-regulation of the NO production in ECV304 was inferior to that of GS serum and GBE serum significantly. The results suggested that Serum Pharmacological study should be adopted in the pharmacological investigation on the Chinese traditional medicine and the drug screening of the Chinese traditional medicine.     

荧光光谱法对中药注射液热稳定性的研究

采用荧光光谱法对中药注射液热稳定性进行了初步探讨。发现在恒温（40℃、80℃）条件下、不同中药注射液随时间变化的荧光强度和荧光谱图，呈现出类似规律性的变化，但又有各自的特点。通过对其谱图和荧光强度的分析，能够反映出中药注射液在受热条件下，随时间变化的荧光谱的差异，不同温度对谱峰强度的差异性影响及不同注射剂热稳定性变化曲线的差异，从而提示中药注射液受热变质中组分变化的一些特点。找出了不同温度下不同中药注射液荧光强度减弱10％的时间。     

超声波和微波对中药提取的促进和影响

本文主要讨论超声波、微波在中药有效成分提取中的作用机理,探讨频率、强度、时间、占空比对提取率的影响,介绍现代检测技术与超声波、微波相结合的方法。证明超声波、微波作用于药物提取具有提取率高、可靠性强、操作简便、大大缩短提取时间等优点。     

中药通心络对抑郁-动脉粥样硬化大鼠干预作用的血浆代谢组学研究

采用快速液相色谱串联离子阱飞行时间质谱(UFLC/MS-IT-TOF)技术对通心络干预下的抑郁-动脉粥样硬化大鼠的血浆代谢谱进行研究.结果表明,抑郁-动脉粥样硬化模型大鼠体内的氨基酸代谢、脂肪酸的β-氧化、胆固醇代谢及磷脂代谢发生了异常.中药通心络通过调节模型大鼠体内的色氨酸、苯丙氨酸代谢及某些胆汁酸的代谢,抑制大鼠抑郁-动脉粥样硬化的形成.该研究结果表明代谢组学在中药整体性药效评价、作用机理的阐明等方面具有很好的应用前景.     

二维色谱技术及其在中药领域中的应用

二维色谱是近年发展起来的多维色谱分离技术,因其高分辨率、高峰容量、高灵敏度等优点,在复杂样品的分离分析中发挥了巨大作用．中药是一个复杂的化学体系,包含多种类化学成分,因此,分析和阐明中药中的化学成分是中药现代研究的一个关键问题．二维色谱技术在中药等复杂体系的应用中显示了重要的应用价值,也具有广阔的发展前景．本文对二维色谱的分类、主要部件以及在中药领域中的应用进行综述,重点介绍二维气相色谱和二维液相色谱在中药研究中的应用．     

Chemometrics and modernization of traditional Chinese medicine

Development of chromatographic fingerprinting and its related chemometric methods in the research of quality control of traditional Chinese medicines(TCMs) are discussed. The quality control methods for guarantying the authentication and stability of products and semi-products of TCMs are firstly assessed. The technique based on chromatographic fingerprinting is essentially a kind of high-through put and integral tools to explore the complexity of herbal medicines. In order to further control the comprehensive quality of TCMs,confirmation and identification of their important chemical components are necessary. Some new strategies are proposed to trace the chemical changes of chromatographic fingerprints both in product processing and/or after their administration by modern chromatographic techniques and chemometrics. Combined with systems biology and bioinformatics,it seems possible for one to reveal the working mechanism of TCMs and to further control their intrinsic quality comprehensively.     

The metabolomic study on atherosclerosis mice and its application in a traditional Chinese medicine Sishen granule

Although an atherosclerosis (AS) model using low‐density lipoprotein receptor deletion mice has been widely applied, its pathological pathway in metabolite level is still not clear. To further reveal the metabolite profile and identify the potential biomarkers in AS development, a serum metabolomic approach was developed based on reversed‐phase liquid chromatography/quadrupole time‐of‐flight mass spectrometry (LC‐Q‐TOF‐MS). The established metabolomic platform was also used for elucidating the therapeutic mechanism of a traditional Chinese medicine named Sishen granule (SSKL). Twenty‐one potential biomarkers in AS mouse serum were identified. Through functional analysis of these biomarkers, inflammation, proliferation, dysfunction of energy metabolism and amino acid metabolism were considered the most relevant pathological changes in AS. DNA damage products were found for the first time in the metabolomic study of AS. The network established by 20 biomarkers revealed that pyruvate metabolism, citrate cycle, fatty acid metabolism and urea metabolism were seriously disturbed. This metabolomic study not only supplied a systematic view of the progression of AS but also provided a theoretical basis for the treatment of AS. This metabolomic study also demonstrated that SSKL had therapeutic effectiveness for AS through partly reversing the inflammation reaction and amino acid metabolism dysfunction. Copyright © 2015 John Wiley & Sons, Ltd.     

Application of gas chromatography-mass spectrometry in research of traditional Chinese medicine

It is well known that traditional Chinese medicine (TCM) plays a more and more important role in modern pharmaceutical industry. It has been used in the therapy of many diseases for several thousand years because of its high pharmacological activity, low toxicity and rare side effects. In TCM, as an important group of secondary metabolites, essential oils have attracted a great deal of attention in recent years. Gas chromatography-mass spectrometry (GC-MS) is the most commonly used technique for the analysis of liposoluble constituents, especially volatile/semi-volatile compounds, and their metabolites in biological fluids due to its high resolution, selectivity and sensitivity. This review briefly describes the applications of GC-MS for the isolation and characterization of volatile compounds from TCM. In addition, GC-MS methods adopted in the metabolic profiling of volatile compounds in biological matrices are also described.     

Concentrating the extract of traditional Chinese medicine by direct contact membrane distillation

This study applies direct contact membrane distillation (DCMD) to concentrating the extract of traditional Chinese medicine (TCM). The trans-membrane flux under various operation conditions was measured in real-time during concentration process. By decoupling the factors affecting the trans-membrane flux decline, it was found that the observed flux decline throughout the process could be attributed to the membrane fouling, the reduction of water vapor pressure and the increase of transport resistance at feed side. Analysis of the combined factors was given to show in detail the mechanism of flux decline. Factors that may affect the flux level, such as feed velocity, feed temperature and pretreatment were experimentally examined. Gas bubbling or sparging was introduced into DCMD system for reducing membrane fouling, and it was found that both gas–liquid two-phase flow at the feed side and gas back-washing within membrane module are effective ways to control membrane fouling.     

Establishing the chromatographic fingerprint of traditional Chinese medicine standard decoction based on quality by design approach: A case study of Licorice

A novel analytical quality by design approach for developing a chromatographic fingerprint was established for analyzing complex traditional Chinese medicine, using a licorice standard decoction as an example. Considering the characteristics of integrity and ambiguity, the resolution of eight common peaks, total peak number, capacity factor distributions, and peak purity were selected as potential critical method attributes for assessing the quality of the chromatographic fingerprint. A central composite design was used to evaluate the relationship between critical method attributes and critical method parameters, including column temperature, wavelength, flow rate, formic‐acid concentration, and gradient parameters. A standard probability method was employed to calculate the design space of the fingerprint analysis parameters and evaluate the robustness of the methodology. The optimized high‐performance liquid chromatography fingerprint conditions were acetonitrile and 0.1% formic acid water gradient elution (0‐5 min, 5–19% A; 5–10 min, 19% A; 10–50 min, 19–42% A; 50–54 min, 42–100% A; 54–60 min, 100% A), column temperature 25±5°C, detection wavelength 265 nm. The design space of fingerprint analytical method based on the analytical quality by design approach not only met the requirements of the fingerprint analysis, but also improved the robustness and applicability of the fingerprint method.     

Rapid analysis of traditional Chinese medicine Pinellia ternata by microchip electrophoresis with electrochemical detection

Traditional Chinese herbal medicine has long enjoyed the reputation of the world's most advanced system of natural medicine. Pinellia ternata is one of the most commonly used herbs in the traditional Chinese medical science. In this study, five representative ingredients of Pinellia ternata guanosine, methionine, glycine, 3,4‐dihydroxybenzaldehyde, and homogentisic acid, were assayed using simple derivatization procedures. Under optimized experimental condition, five analytes in Pinellia ternata were rapidly separated and detected using microchip electrophoresis, affording the benefits of speed, minimal sample requirements, and sensitive on‐the‐chip electrochemical detection, in 5 min with linearity over a concentration of 20–500 μM (R² = 0.994) with nearly complete recovery (95.6–98.5%).     

Application of two‐dimensional liquid chromatography in the separation of traditional Chinese medicine

Traditional Chinese medicines have been widely used to prevent and cure diseases for thousands of years. For the purpose of better understanding the extremely complicated traditional Chinese medicines, powerful separation techniques are essential. Two‐dimensional liquid chromatography has been proven to be more powerful for the separation of complex traditional Chinese medicines due to its enhanced peak capacity and resolution compared with one‐dimensional liquid chromatography. Enormous efforts have been made on the coupling of independent separation mechanisms to improve the resolving power for complex traditional Chinese medicine samples, including the development and introduction of novel stationary phases. This review aims to give an overview on the applications of two‐dimensional liquid chromatography in traditional Chinese medicine research since 2008, including comprehensive two‐dimensional liquid chromatography, heart‐cutting two‐dimensional liquid chromatography both in on‐line, and off‐line mode. Different couplings of separation modes were respectively discussed based on specific studies, with emphasis on the applications of novel stationary phases in the two‐dimensional liquid chromatography.     

Screening prolyl hydroxylase domain 2 inhibitory activity of traditional Chinese medicine by CZE-UV

Prolyl hydroxylase domain 2 (PHD2) is a key enzyme regulating the expression of hypoxia inducible factor (HIF). Its inhibitors can improve the expression of HIF and downstream genes, which can treat hypoxia-related diseases. Therefore, the establishment of a reliable PHD2 inhibitors screening method is of great significance for the drug development of hypoxia-related diseases. In this work, an accurate, rapid, and simple screening method for PHD2 inhibitors was introduced by capillary zone electrophoresis (CZE). In order to improve the detection sensitivity, the derivative reaction of α-ketoglutaric acid (α-OG) and 1,2-diaminobenzene (OPD) was used to enhance the UV absorption of α-OG (the substrate in the enzymatic reaction). The CZE method selected 20 mM Na₂B₄O₇ buffer (pH 9.0) as the separation buffer, +25 kV as the separation voltage, 25°C as the cartridge temperature, and 210 nm as the detection wavelength. Under this condition, the analysis of a single sample can be realized within 9 min. Compared with the existing reported methods, the present work can directly screen the PHD2 inhibitory activity of traditional Chinese medicine (TCM) extracts, which is of significance for the target-purification of bioactive individual compounds from TCMs. Under the optimal conditions, the PHD2 inhibitor screening platform was successfully established, and it was found that 70% methanol/water extracts of Astragali Radix and Codonopsis pilosula had good PHD2 inhibitory activity. Furthermore, the present work provides a novel approach for screening the PHD2 inhibitory activity of TCM extracts and the discovery of anti-hypoxia bioactive compounds.     

中药组方指纹图谱研究方法和思路

中药组方就是将单味中药按照一定的规律配伍成方,因剂量配伍差异会导致组方理化性质的改变,从而导致组方功效强弱、性质方面发生变化,甚至产生新的化学成分。中药组方鉴别与质量控制一直是困扰中药工作者的一个难题。指纹图谱技术具有宏观、准确、稳定等优点,已被广泛用于中药领域。中药组方指纹图谱是解决中药合理组方配伍的最有效手段,其对中药质量控制已经得到普遍关注和重视。由于指纹图谱具有整体定量功能和特征性鉴别作用,对于成分复杂的中药组方能够全面地定性与定量评价。本文就中药组方指纹图谱的研究意义与目的进行了综述,并详细阐述了组方指纹图谱的研究方法与思路,以此为中药及其组方指纹图谱研究提供参考。     

中药质谱分析方法及应用研究

分别从中药成分分析、活性筛选和代谢组学三方面对质谱技术在中药研究中的应用进展进行了全面综述.在中药成分分析方面,重点介绍了寡糖异构体的分析方法,以及质谱指纹图谱技术在中药成分分析及质量控制中的应用;在活性筛选方面,分别介绍了超滤-质谱、细胞膜色谱-质谱、微透析-质谱、亲和色谱-质谱、强度衰减质谱、修饰琼脂糖珠-质谱和直接分析质谱等技术及其应用;在代谢组学研究方面,对中药治疗肝损伤、肾虚、心肌梗死和糖尿病等疾病方面的研究进展进行了阐述.上述内容充分反映了质谱技术在中药创新性研究中的重要性.     

七种无机元素与中药药性的相关性研究

本文研究支持向量机用于中药药性识别的可行性.选择7种无机元素在中药中的含量为指标,运用支持向量机对193种不同药性的中药进行训练,建立平性与非平性中药的预测模型.结果训练集识别正确率95.03%,建立的模型对预测集中平性药的识别正确率为82.14%,对非平性药的识别正确率为70%,总正确率为73.58%.     

代谢组学应用于糖肾方治疗糖尿病肾病的疗效评价

建立了基于超高效液相色谱-飞行时间质谱(UPLC/TOF MS)分析技术的血浆代谢指纹谱,应用多元统计分析方法评价糖尿病肾病患者血浆代谢物变化差异及糖肾方的干预效果。通过研究糖肾方干预糖尿病肾病血浆内源性代谢物的变化,探索与该疾病密切相关的代谢途径,评价糖肾方的治疗效果。结果发现: 经糖肾方治疗后,血浆内源性代谢物发生了明显变化,磷脂代谢、脂肪酸代谢、氨基酸代谢、嘌呤嘧啶代谢、固醇类代谢等多个代谢途径得到纠正。本研究基于UPLC/TOF MS的代谢组学方法,能够从整体水平反映疾病治疗过程中代谢网络的变化趋势,证实糖肾方具有治疗糖尿病肾病的临床疗效并有助于阐释药物作用机理。