Phytochemical analysis of an antiviral fraction of Radix astragali using HPLC–DAD–ESI–MS/MS

Radix astragali (Huangqi in Chinese) is a well-known traditional Chinese medicinal herb that has been used clinically in China for centuries to cure various diseases. To profile the antiviral constituents of this herb, a high-performance liquid chromatography–diode array detector–electrospray ionization–tandem mass spectrometry (HPLC–DAD–ESI–MS/MS) analytical method was developed to separate and determinate the active part of the extract of Radix astragali, which showed potent inhibition of several viruses. By comparing their retention time and MS data with those obtained from the authentic compounds and the published data, a total of 18 compounds, comprising 11 flavonoids and 7 saponins, were identified. This study provides an approach to rapidly characterize bioactive constituents in traditional Chinese medicines (TCMs).     

A novel blood plasma analysis technique combining membrane electrophoresis with silver nanoparticle-based SERS spectroscopy for potential applications in noninvasive cancer detection

Combining membrane electrophoresis with silver nanoparticle-based surface-enhanced Raman spectroscopy (SERS), we have developed a novel method for blood plasma analysis for cancer detection applications. In this method, total serum proteins are isolated from blood plasma by membrane electrophoresis and mixed with silver nanoparticles to perform SERS spectral analysis. The obtained SERS spectra present information-rich, fingerprint-type signatures of the biochemical constituents of whole proteins. We evaluated the utility of this method by analyzing blood plasma samples from patients with gastric cancer (n = 31) and healthy volunteers (n = 33). Principal components analysis of the spectra revealed that the data points for the two groups form distinct, completely separated clusters with no overlap. The gastric cancer group can be unambiguously distinguished from the normal group in this initial test—that is, with both diagnostic sensitivity and specificity of 100%. These results are very promising for developing a label-free, noninvasive clinical tool for cancer detection and screening.

From the Clinical Editor

In this paper by Lin et al, a novel nanotechnology-based blood plasma test is presented for sensitive detection of malignant cells. The method utilizes membrane electrophoresis followed by silver-nanoparticle based surface-enhanced Raman spectroscopy to analyze the purified proteins for spectral changes to determine if cancer is present.     

Analysis by surface enhanced Raman spectroscopy on silver hydrosols and silver coated filter papers

Surface enhanced Raman spectrometry (SERS) is an analytical technique with a sensitivity comparable to that of conventional molecular absorption or fluorescence spectroscopy, with the additional major advantage of selectivity inherent in vibrational spectroscopies. The analytical application of flowing silver hydrosols is described. Under the controlled experimental conditions of flow injection analysis, it was possible to detect as low as 30 ng of p-aminobenzoic acid. The linear range was two orders of magnitude (1–100 μg ml⁻¹) with a signal reproducibility of 3.2%. Silver coated filter paper is another SERS active substrate that is simple to prepare and handle. The SERS spectra of several nitrogen-containing molecules were obtained on these substrates. The effects of laser power and paper hydration are described. The relative advantages of both substrates are compared.     

盐酸苯乙双胍与盐酸二甲双胍在纳米银表面共吸附的表面增强拉曼光谱研究

目的：研究盐酸苯乙双胍与同类药物盐酸二甲双胍在不同酸碱度务件下共存时的表面增强拉曼光谱。方法：用便携式拉曼光谱仪对不同酸碱条件下盐酸苯乙双胍和盐酸二甲双胍的表面增强拉曼光谱及它们在不同酸碱度条件下共存时的表面增强拉曼光谱进行检测。结果：研究表明,在酸性和中性条件下,二者的表面增强拉曼光谱均不明显,二者共存体系亦如此;而在偏碱性的条件下,二者均表现出特征性明显的表面增强拉曼光谱信息,二者共存体系在偏碱性的条件下能将二者区分开来。结论：在表面增强拉曼光谱检测时,偏碱性的条件有利于盐酸苯乙双胍与盐酸二甲双胍的同时检出。     

Towards the development of a rapid,portable, surface enhanced Raman spectroscopy based cleaning verification system for the drug nelarabine

Objectives Cleaning verification is a scientific and economic problem for the pharmaceutical industry. A large amount of potential manufacturing time is lost to the process of cleaning verification. This involves the analysis of residues on spoiled manufacturing equipment, with high‐performance liquid chromatography (HPLC) being the predominantly employed analytical technique. The aim of this study was to develop a portable cleaning verification system for nelarabine using surface enhanced Raman spectroscopy (SERS). Methods SERS was conducted using a portable Raman spectrometer and a commercially available SERS substrate to develop a rapid and portable cleaning verification system for nelarabine. Samples of standard solutions and swab extracts were deposited onto the SERS active surfaces, allowed to dry and then subjected to spectroscopic analysis. Key findings Nelarabine was amenable to analysis by SERS and the necessary levels of sensitivity were achievable. It is possible to use this technology for a semi‐quantitative limits test. Replicate precision, however, was poor due to the heterogeneous drying pattern of nelarabine on the SERS active surface. Understanding and improving the drying process in order to produce a consistent SERS signal for quantitative analysis is desirable. Conclusions This work shows the potential application of SERS for cleaning verification analysis. SERS may not replace HPLC as the definitive analytical technique, but it could be used in conjunction with HPLC so that swabbing is only carried out once the portable SERS equipment has demonstrated that the manufacturing equipment is below the threshold contamination level.     

盐酸二甲双胍的表面增强拉曼光谱研究

目的:研究盐酸二甲双胍在不同激发光波长下的固体拉曼光谱以及不同酸碱度条件下的表面增强拉曼光谱,对比固体谱,分析盐酸二甲双胍表面增强拉曼光谱中的分子振动模式.方法:用2种不同的激发光波长对盐酸二甲双胍固体粉末及不同pH 条件下盐酸二甲双胍溶液的表面增强拉曼散射(SERS)进行考察.结果:体系的pH对盐酸二甲双胍的增强效应有较大的影响,在碱性条件下,盐酸二甲双胍的SERS效应最强,而其他两种pH条件下均不明显.另外,激发光波长对盐酸二甲双胍的SERS效应也有较大影响.结论:本方法简单、快速、专属性强,可以作为分析盐酸二甲双胍分子结构信息的方法.     

Effect of Selected Essential Oils and Pure Compounds on Saprolegnia parasitica

Saprolegnia parasitica Coker is the main agent of saprolegniosis, one of the most important causes of economic losses in the fish farming industry, affecting all developmental stages. In this work the activity of some essential oils are compared with the activity of their isolated chemical constituents (alcohols, aldehydes, ketones, phenols, and hydrocarbons) to evaluate molecular classes active against S. parasitica. This suggests potential new products to use against saprolegniosis. Sixteen commercial essential oils and 37 pure constituents were tested against a strain of Saprolegnia parasitica from cutaneous lesions of Perca fluviatilis Linnaeus, using a continuously agitated broth technique that allows both the Minimum Inhibitory Concentration (MIC) and the Minimum Lethal Concentration (MLC) to be evaluated. It was possible to evaluate the efficacy of each essential oil against S. parasitica in relation to its specific composition and the efficacy of its main constituents. Carvacrol (MIC of 50?ppm and a MLC of 100?ppm) and p-cymene (MIC and MLC = 200?ppm) are the main constituents of Origanum vulgare L. and Thymus vulgaris L. (MIC = 100?ppm, MLC = 200?ppm) and are the primarly responsible for the efficacy of these two essential oils against S. parasitica. The structure of the constituents was also evaluated in relation to their activities against S. parasitica. Besides monoterpenic aromatic compounds, the acyclic mono- and sesquiterpenic primary alcohols and aldehydes seem to be the most active compounds. The open chain alcohol 1-decanol [CH₃-(CH₂)₈-CH₂-OH] proved to be the most active agent with a MLC = 50?ppm.     

基于生物可给性和靶器官毒性剂量法的乌梢蛇中铅和砷联合暴露评估

目的 通过考察乌梢蛇Zaocys dhumnades（Cantor）中铅（Pb）和砷（As）的生物可给性,并探索靶器官毒性剂量（TTD）法在评估中药中重金属联合暴露风险中的应用,为限量标准的制定提供参考。方法 通过胃-肠两步模拟消化（in vitro PBET）联合电感耦合等离子体质谱（ICP-MS）法对乌梢蛇中Pb和As的生物可给性进行考察,根据其生物可给性计算其日暴露量。采用危害指数（HI）法对于Pb和As联合暴露产生的健康风险进行初步筛查;进一步针对不同毒理学终点,采用TTD法对Pb和As的累积风险进行更加精确的评估。结果 8批乌梢蛇中Pb和As的合格率为100%。HI法的初步评估结果表明,所有批次乌梢蛇中Pb和As的HI值均＜1。TTD法评估结果表明,作用终点心血管系统、神经系统、肾脏、血液和睾丸,所有批次乌梢蛇的HI值均＜1,健康风险可接受。结论 基于生物可给性,探索乌梢蛇中Pb和As的累积风险评估方法,为中药外源性有害残留物风险评估的方法开发提供新的思路,为制定更加科学的限量标准提供技术支撑。     

外翻肠囊法研究金芪降糖片的肠吸收特性

目的:研究金芪降糖片中主要化学成分的肠吸收特性。方法:采用大鼠离体外翻肠囊模型,收集金芪降糖片3种剂量在肠道不同部位给药后不同时间的肠囊液样品,用HPLC对有效成分进行检测分析,计算各有效成分的累积吸收量(Q)及吸收速率常数(K_a)以分析它们在肠道的吸收特征。结果:可检测到金芪降糖片中的9个成分吸收进入肠囊,分别为新绿原酸、隐绿原酸、绿原酸、咖啡酸、表小檗碱、黄连碱、药根碱、巴马汀、小檗碱。金芪降糖片中的9个成分在不同肠段不同浓度下的吸收均为线性吸收,其回归相关系数(R²)均大于0.9,符合零级吸收,且9个成分的K_a值在不同肠段均随药物剂量的增加而增加(P<0.05),表明其为被动吸收。肠道不同部位的吸收实验表明,以金芪降糖片高剂量中绿原酸和小檗碱在120 min的累积吸收量为例,绿原酸在各肠段的累积吸收量分别为:十二指肠,626.9 μg;空肠,1 116.8 μg;回肠,516.9 μg;结肠,215.8 μg,小檗碱在各肠段的累积吸收量分别为:十二指肠,178.6 μg;空肠,208.6 μg;回肠,97.5 μg;结肠,66.8 μg。说明空肠对各成分的吸收最为明显,其次是回肠,且有机酸类成分的在肠吸收优于生物碱类成分。结论:金芪降糖片中4个有机酸类成分及5个生物碱类成分可吸收进入肠囊,各成分的吸收形式可能为被动吸收。     

A high throughput screening approach to identify isocitrate lyase inhibitors from traditional Chinese medicine sources

Isocitrate lyase (ICL) catalyses the first step of the glyoxylate bypass pathway, which reversibly cleaves isocitrate into succinate and glyoxylate. This pathway occurs in a wide range of pathogens and plays a key role in the pathogenesis of Mycobacterium tuberculosis (MTB) suggesting that it may represent a drug target for the treatment of tuberculosis. ICL was cloned, expressed, and purified, and a high‐throughput screen (HTS) developed to screen active extracts derived from traditional Chinese medicines (TCMs) for inhibition of ICL. A colorimetric assay based on the formation of glyoxylate‐phenylhydrazone was used to measure ICL activity. The assay had signal to noise (S/N) of 12.74 and Z′ factor of 0.72, indicating that the assay was suitable for HTS. Screening of a collection of 465 extracts derived from TCMs resulted in the identification of two extracts from Illicium verum Hook.f (Illiciaceae, XHD‐1) and Zingiber officinale Rosc (Zingiberaceae, XHD‐2), which inhibited ICL with IC₅₀ values of 47.7 ± 16.9 and 18.2 ± 0.9 µg/ml, respectively. Drug Dev. Res. 67:818–823, 2006. © 2007 Wiley‐Liss, Inc.     

Determination of bioactive constituents in traditional Chinese medicines by CE with electrochemical detection

This paper reviews the recent advances and the key strategies in capillary electrophoresis (CE) with electrochemical detection (ECD) for separating and detecting a variety of bioactive constituents in traditional Chinese medicines (TCMs). The subjects covered include the separation modes for the CE analysis of the constituents in TCMs, the CE-ECD system, the sample preparations of TCMs, the ECD of TCMs, the applications of CE-ECD in the determination of various bioactive constituents in Chinese medicinal materials and their preparations, the identification and differentiation of TCMs by CE-ECD, and future prospects. It is expected that CE-ECD will become a powerful tool in the herbal medicinal fields and will lead to the creation of truly routine devices for TCM analysis.     

表面增强拉曼光谱法快速鉴别失活白色念珠菌

目的 建立表面增强拉曼光谱（surface-enhanced Raman spectroscopy,SERS）法快速判别失活白色念珠菌。方法 利用热、甲醛、杀菌药（两性霉素B）分别灭活白色念珠菌,然后利用SERS法对失活前后的白色念珠菌进行光谱考察比较。结果 白色念珠菌灭活后的SERS光谱显示出相似的特征峰,且与失活前的光谱有明显差异。结论 该实验方法快速、简便,实现了对失活白色念珠菌的快速检测,有望成为快速鉴别更多病原微生物失活状态的有利工具。     

Novel optical nanosensors for probing and imaging live cells

This review introduces multifunctional optical nanosensors based on surface-enhanced Raman scattering (SERS) and demonstrates their application in live cells. The novel nanosensors have the potential to improve our understanding of cellular processes on the molecular level. The hybrid sensor consists of gold or silver nanoparticles with an attached reporter species. The sensor can be detected and imaged based on the SERS signature of the reporter. This results in several advantages, such as high spectral specificity, multiplex capabilities, improved contrast, and photostability. SERS sensors not only highlight cellular structures, based on enhanced Raman spectra of intrinsic cellular molecules measured in the local optical fields of the gold nanoparticles, they also provide molecular structural information on their cellular environment. Moreover, the SERS signature of the reporter can deliver information on the local pH value inside a cell at subendosomal resolution. SERS sensors are suitable for one- and two-photon excitation.From the Clinical EditorThis review introduces multifunctional optical nanosensors based on surface enhanced Raman scattering (SERS) and demonstrates their application in live cells. These hybrid sensors consist of gold or silver nanoparticles with an attached reporter species. The sensor can be detected and imaged based on the SERS signature of the reporter. SERS sensors highlight cellular structures and provide molecular structural information on their cellular environment. They can also deliver information on the intracellular pH-value at subendosomal resolution.     

延胡索活性部位入血成分的LC-MS/MS研究

为深入研究延胡索活性部位体内作用的物质基础, 本文利用LC-MS/MS提供的色谱及离子碎片信息, 对口服延胡索活性部位后大鼠血浆中的移行成分进行分析鉴定, 通过保留时间比对、离子碎片解析等综合分析, 鉴定了9个入血成分均为原形成分, 同时也发现部分生物碱是以葡糖醛酸结合物的形式存在。入血成分及代谢产物可能为延胡索体内直接作用的药效成分, 延胡索活性部位血清药物化学表征为阐明延胡索的药理作用及其机制提供了依据。     

两种减肥药的表面增强拉曼光谱研究

目的：研究盐酸西布曲明与盐酸芬氟拉明的表面增强拉曼光谱,对比它们的拉曼固体光谱,对表面增强拉曼光谱中的分子振动模式进行识别。方法：使用便携式拉曼光谱仪时盐酸西布曲明与盐酸芬氟拉明的表面增强拉曼光谱与拉曼固体谱进行考察。结果：研究表明,药物分子-银胶体系的pH对盐酸西布曲明与盐酸芬氟拉明的增强效应有较大的影响,这是pH对药物分子-银胶体系的凝聚状态和药物分子存在状态综合影响的结果。结论：本方法可以作为分析不同pH情况下盐酸西布曲明与盐酸芬氟拉明分子在纳米表面吸附情况。     

Online identification of the antioxidant constituents of traditional Chinese medicine formula Chaihu-Shu-Gan-San by LC–LTQ-Orbitrap mass spectrometry and microplate spectrophotometer

Chaihu-Shu-Gan-San (CSGS), a traditional Chinese medicine (TCM) formula containing seven herbal medicines, has been used in treatment of gastritis, peptic ulcer, irritable bowel syndrome and depression clinically. However, the chemical constituents in CSGS had not been studied so far. To quickly identify the chemical constituents of CSGS and to understand the chemical profiles related to antioxidant activity of CSGS, liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC–LTQ-Orbitrap) mass spectrometry has been applied for online identification of chemical constituents in complex system, meanwhile, antioxidant profile of CSGS was investigated by the fraction collecting and microplate reading system. As a result, 33 chemical constituents in CSGS were identified. Among them, 13 components could be detected both in positive and in negative ion modes, 20 constituents were determined only in positive ion mode and 2 components were only detected in negative ion mode. Meanwhile, the potential antioxidant profile of CSGS was also characterized by combination of 96-well plate collection of elutes from HPLC analysis and microplate spectrophotometer, in which the scavenging activities of free radical produced by DPPH of each fraction could be directly investigated by the analysis of microplate reader. This study quickly screened the contribution of CSGS fractions to the antioxidant activity and online identified the corresponding active constituents. The results indicated that the combination of LC–MSⁿ and 96-well plate assay system established in this paper would be a useful strategy for correlating the chemical profile of TCMs with their bioactivities without isolation and purification.     

Typical toxic components in traditional Chinese medicine

Introduction: Traditional Chinese medicine (TCM) is widely used around the world. However, with its wide use has been the identification of a number of toxicological issues that have severely restricted its use in clinical treatment. The identification of these toxic substances within TCM has become somewhat of a hot topic in recent years.

Areas covered: This article reviews literature published on professional authoritative journals in the last 10 years on the toxic constituents and toxicology of TCM, including chemical structures, absorption and metabolism. The literature search for this article was based, but not limited to, toxic constituents including: alkaloids, glycosides, toxic proteins, polypeptide, amino acids, phenols or organic acids, terpenes and lactones. The authors discuss the toxic substances referring to their toxicity on organs, tissues and systems.

Expert opinion: More and more toxic constituents from different TCMs have been identified, in addition to information on how they act in the body at a molecular level. However, the toxicology of TCMs is very complex, and although some progress has been made, a lot work is still needed in order to put an end to toxic incidents.     

Berberine impairs embryonic development in vitro and in vivo through oxidative stress‐mediated apoptotic processes

Berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines, has been shown to suppress growth and induce apoptosis in some tumor cell lines. However, berberine has also been reported to attenuate H₂O₂‐induced oxidative injury and apoptosis. The basis for these ambiguous effects of berberine—triggering or preventing apoptosis—has not been well characterized to date. In the current investigation, we examined whether berberine exerts cytotoxic effects on mouse embryos at the blastocyst stage and affects subsequent embryonic development in vitro and in vivo. Treatment of blastocysts with berberine (2.5‐10 μM) induced a significant increase in apoptosis and a corresponding decrease in trophectoderm cell number. Moreover, the implantation success rate of blastocysts pretreated with berberine was lower than that of their control counterparts. Pretreatment with berberine was also associated with increased resorption of postimplantation embryos and decreased fetal weight. In an animal model, intravenous injection of berberine (2, 4, or 6 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst cells and early embryonic developmental injury. Berberine‐induced injury of mouse blastocysts appeared to be attributable to oxidative stress‐triggered intrinsic apoptotic signaling processes that impaired preimplantation and postimplantation embryonic development. Taken together, our results clearly demonstrate that berberine induces apoptosis and retards early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.     

Spatial distribution of theobromine – a low MW drug – in tissues via matrix‐free NALDI‐MS imaging

The ability of nano‐assisted laser desorption‐ionization mass spectrometry imaging (NALDI‐IMS) to provide selective chemical monitoring with appropriate spatial distribution of a low molecular drug in a biological tissue was investigated. NALDI‐IMS is a matrix‐free laser desorption ionization (LDI) protocol based on imprinting of tissue constituents on a nanostructured surface. Using the accumulation of theobromine in rat kidney as a model, NALDI‐IMS was found to provide well‐resolved images of the special distribution of this low molecular weight (MW) drug in tissue. Copyright © 2014 John Wiley & Sons, Ltd.