马兜铃酸类成分检测与分析研究进展

马兜铃酸类成分是广泛存在于马兜铃科植物中的硝基菲类化合物,已被证实具有肾毒性、致癌和致基因突变等作用。我国自2003年以来采取一系列风险控制措施,其中马兜铃酸含量高的关木通、广防己和青木香等药材已被禁用。目前,一些马兜铃酸含量低的中药材与中成药仍在使用中,鉴于马兜铃酸成分对人体的严重危害性,有必要进一步加强相关药材与制剂的风险评估。在归纳马兜铃酸类成分结构等基本信息的基础上,对近年来的检测分析方法进展进行了总结,为其风险控制与安全使用提供了科学依据。     

Comparative study of the contents of analogues of aristolochic acid in two kinds of Aristolochiae Fructus by high-performance liquid chromatography

Aristolochiae Fructus (??Madouling??) is derived from the fruits of Aristolochia contorta and A. debilis (Aristolochiaceae). These two species contain potentially nephrotoxic constituents, but are officially used in China. Distinction of constituents and toxicity between these two species remains unclear. A high-performance liquid chromatography method was developed and validated for the simultaneous determination of seven analogues of aristolochic acid (aristolochic acids I, II, IIIa, IVa and VIIa), as well as aristololactams I and II in Aristolochiae Fructus. Chromatographic separation was achieved on a Zorbax SB-C₁₈ column with a gradient mobile phase comprising acetonitrile and 1?% acetic acid?C30?mM triethylamine (20:1, v/v) buffer. Analytes were detected with a diode array detector at 250 and 260?nm. The contents of seven constituents in samples (11 batches of A. contorta fruits, 15 batches of A. debilis fruits and 33 commercial samples of Madouling) were determined. The content of aristolochic acid IVa was higher than that of aristolochic acid VIIa in A. contorta fruits, whereas the opposite was true in A. debilis fruits. This feature can be used to distinguish the two species from each other and identify the resource plant of Madouling. Through a morphological method and a newly found principle based on the ratio AA-IVa/AA-VIIa, we found that the 33 commercial samples collected from 12 provinces in China were all derived from the fruits of A. contorta.     

An extractive nanoelectrospray ionization-mass spectrometry method for Chinese herbal medicine authentication

Herein, we describe a rapid, sensitive, and nondestructive method—extractive nanoelectrospray ionization-mass spectrometry (EnESI-MS)—for traditional Chinese medicine (TCM) authentication. The mass-spectral fingerprints of volatile compounds released from various TCMs can be rapidly acquired using EnESI-MS without sample pretreatment. EnESI-MS was applied to successfully differentiate between two commonly used medicinal herbs, Schisandra chinensis and Schisandra sphenanthera, which are morphologically similar but exhibit different therapeutic effects. Specific volatile compounds of each herb in a ten-component Chinese herbal product, Jia Wei Xiao Yao San, were also identified, and the method was applied to discriminate between the commercial product and a substandard version.     

基于转运体的中药毒性及药物相互作用研究进展

中药-药物的相互作用已成为药物毒性研究的热点。许多中药的活性成分或其提取物在体内的药效和毒性与转运体相关，转运体的表达或活性可明显影响中药在体内的药动学和生物活性。其中，基于转运体的马兜铃酸、雷公藤甲素等中药成分的肝、肾毒性的研究十分广泛，而中药对转运体的作用也会对其他药物的代谢产生影响从而发生药物的相互作用。主要基于转运体角度来概述中药毒性及对其他药物代谢的影响，对中药的毒性研究及临床上的合理用药具有指导意义。     

HPLC analysis,semi-preparative HPLC preparation and identification of three impurities in salidroside bulk drug

Salidroside is a bioactive compound mainly distributed in Rhodiola L. (Crassulaceae). It has been widely used in Chinese traditional medicine. In this paper, three impurities were found during the analysis of salidroside bulk drug. The enrichment of impurities was carried out by ODS column chromatography, using methanol–water (13:87, v/v) as eluent and the purification of impurities was achieved by semi-preparative HPLC, using methanol–water (11:89, v/v) as mobile phase, respectively. Three impurities were characterized as 4-(2-hydroxylethyl)-phenol-1-O-β-d-glucopyranoside, 4-hydroxyphenacyl-d-glucopyranoside and p-acetylphenyl-O-β-d-glucopyranoside by a variety of spectral data (IR, UV, MS, ¹H NMR, ¹³C NMR, DEPT and 2D NMR). The simultaneous quantitative determination of salidroside and its impurities (Imp. 1, 2 and 3) was performed by reverse-phase HPLC method with UV detection. Specificity, linearity, sensitivity, precision and accuracy were evaluated.     

Simultaneous HPLC Determination of Four Active Compounds in Fengshiding Capsules,a Chinese Medicine

A high performance liquid chromatography method was established for simultaneously determining four bioactive components, salicin, liquiritin, paeonolum, and imperatorin in Fengshiding capsule, a widely used traditional Chinese medicine for treating rheumatic disease. The chromatographic separation was performed on a Shimadzu Shim-pack Stable Bond C₁₈ column using gradient elution with methanol and water. The analytical method was validated through precision, repeatability and stability, and the relative standard deviation values were less than 3%, respectively. The recoveries of the four investigated compounds ranged from 95.80 to 101.21% with relative standard deviation values less than 3.2%. Then this proposed method was successfully applied to determine six batches of Fengshiding commercial products of capsule dosage form from two pharmaceutical factories. This study might provide a basis for quality control for this traditional Chinese medicine preparation.     

Simultaneous determination of 11 active components in two well-known traditional Chinese medicines by HPLC coupled with diode array detection for quality control

A simple and sensitive high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) method was investigated for simultaneous determination of 11 components (chlorogenic acid, coptisine, epiberberine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and chrysin) in Qinhuanghouzheng (QHHZ) capsule and Xiaoerqingre (XEQR) tablet, for quality control of these two well-known traditional Chinese medicines (TCMs). The method was established using an Eclipse Plus C₁₈ (150 mm × 4.6 mm i.d., 5 μm) column. The mobile phase comprising methanol (A) 3% phosphoric acid (B) (pH 2.0, adjusted by triethylamine) was used to elute the targets in gradient elution mode. Flow rate and detection wavelength were set at 0.8 mL/min and 270 nm, respectively. All calibration curves showed good linearity with R² > 0.9995. Inter- and intra-day precisions for all investigated components expressed as relative standard deviation (R.S.D.) ranged from 0.26% to 1.77%. Recoveries measured at three concentrations were in the range of 95.0–103.0% with R.S.D. ≤ 3%. The validated method is simple, reliable, and successfully applied to determine the contents of the selected compounds in QHHZ capsule and XEQR tablet for quality evaluation and control. The 11 main active marker compounds measured occur only in 2 or 3 plant species out of 7–10 species comprising the two TCMs. Additional procedures need to be developed for the quality control of plant materials other than Coptis chinensis Franch, Scutellaria baicalensis Georgi and Phellodendron amurense Rupr.     

中药安全性事件的危险因素与防治对策

回顾马兜铃酸、雷公藤、八角莲、槟榔、仙灵骨葆胶囊、鱼腥草注射剂、双黄连注射剂等引发的中药安全性事件,旨在探讨影响中药安全性的危险因素,对中药毒性成分、生产工艺、储藏条件、体质差异、给药剂量、风险管控等方面存在的安全性问题进行探讨,提出加强源头控制以确保质量合格、探明配伍机制以增效减毒、提高生产标准以推进质量统一、完善法规政策以加强管控、健全安全性评价体系以确保质量达标等建议,以期为促进临床合理用药、减少中药安全性事件提供帮助。     

Renal toxic ingredients and their toxicology from traditional Chinese medicine

Introduction: There have been increasing concerns regarding adverse reactions and toxicity incidents caused by traditional Chinese medicines (TCMs), among which the nephrotoxicity is particularly worrying.

Areas covered: This review summarizes the ingredients with renal toxicity from some TCMs through searching the relevant literature published over the past two decades. Renal toxicity components from TCMs include aristolochic acids (AAS), alkaloids, anthraquinones and others. TCM renal toxicity is most commonly caused by AAS and some alkaloids. AAS mainly come from Aristolochia contorta Bunge, Aristolochia manshuriensis Kom, Clematis Chinensis Osbeck, Aristolochia cathcartii Hook. Some renal toxic alkaloids are derived from Tripterygium regelii Sprague et Takeda, Stephania tetrandra S. Moore, Strychnos nux-vomica Linn. and Aconitum carmichaeli Debx. A few kinds of anthraquinones, flavonoids, and glycosides from TCMs also cause renal toxicity. All of these renal toxicity components and their associated renal toxicity, structures and toxic mechanism are introduced in detail in this review.

Expert opinion: Given the complexity of the toxic components, a lot of work needs to be done to analyze the specific modes of action of toxic components in vivo and in vitro, in particular, to elucidate the molecular mechanism of toxicity, in order to reduce the occurrence of renal toxicity of TCM.     

Hypothesis of potential active components in Angelica sinensis by using biomembrane extraction and high performance liquid chromatography

The screening and analysis of bioactive components in traditional Chinese medicines (TCMs) is very important not only for the quality control of crude drugs but also for elucidating the therapeutic principle. In this study, a method for screening potential active components from TCMs was developed by using biomembrane extraction and high performance liquid chromatography. Based on the methodology, aqueous extract of Angelica sinensis (WEAS) was used, and four compounds were detected by HPLC in the desorption eluate of red cell membrane extraction for WEAS. The compounds were identified as ferulic acid, ligustilide, senkyunolide H and senkyunolide I based on their UV, MS and NMR spectra. Actually, ferulic acid and ligustilide are considered as major active components in Angelica sinensis. Therefore, this method may be applied to predict the potential bioactivities of multiple compounds in TCMs simultaneously.     

Screening of immunomodulatory components in Yu-ping-feng-san using splenocyte binding and HPLC

Yu-ping-feng-san (YPFS) is a widely used immunomodulatory herbal medication used in traditional Chinese medicine, but the active molecules remain obscure. To screen for bioactive components we combined splenocyte binding with high performance liquid chromatography (SB-HPLC). After enrichment by splenocyte binding, two YPFS components (C1 and C2) were analyzed by HPLC. Compound C2 was identified as linoleic acid (LA) based on UV absorption and mass spectrometry. Silica gel chromatography was used to purify compound C1 from Radix Saposhnikoviae, a major constituent of YPFS. This allowed identification of the molecule as panaxynol (PAN) based on EI-MS and NMR spectrometry. Bioassay in vitro demonstrated that PAN significantly inhibited splenocyte proliferation induced by concanavalin A (ConA) in a concentration-dependent manner, whereas LA had no significant effect on splenocyte proliferation. In vivo, PAN was found to attenuate allergic contact dermatitis in a mouse model of delayed-type hypersensitivity (DTH), a pharmacological activity not previously reported for this molecule. It is suggested that PAN contributes to the anti-DTH effects of YPFS. SB-HPLC provides a rapid and efficient method for the identification of potential immunomodulatory components in traditional Chinese medicines.     

Characterization and determination of the major constituents in Belamcandae Rhizoma by HPLC-DAD-ESI-MS(n)

Belamcandae Rhizoma, derived from the rhizome of Belamcanda chinensis (L.) DC., has been used as traditional Chinese medicine for the treatment of coughing and pharyngitis. However, there have been few studies dealing with the systematic analysis of the bioactive constituents in Belamcandae Rhizoma. In this work, high performance liquid chromatography-diode array detection-electrospray ionization multiple-stage mass spectrometry (HPLC-DAD-ESI-MSⁿ) combined with liquid chromatography-time of flight-mass spectrometry (HPLC-TOF/MS) was established for profiling and characterization of multi-constituent in Belamcandae Rhizoma. The ESI-MSⁿ fragmentation behaviors of the authentic references were proposed for aiding the structural identification of components in the extract. Thirty-five flavonoids, including 30 isoflavones and five xanthones, were identified or tentatively identified by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data. Twelve of the identified compounds (neomangiferin, mangiferin, tectoridin, iristectorin B, iristectorin A, iridin, tectorigenin, iristectorigenin A, irigenin, irisflorentin, irilone and dichtomitin) were determined by HPLC-DAD using a C₁₈ column. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in herbal medicine. This work is expected to provide comprehensive information for the quality evaluation of Belamcandae Rhizoma, which would be a valuable reference for the further study and development of this herb and related medicinal products.     

Identification and determination of the major constituents in Traditional Chinese Medicinal formula Danggui-Shaoyao-San by HPLC–DAD–ESI-MS/MS

Danggui-Shaoyao-San (DSS), a famous traditional Chinese medicine formula consisting of six herbal medicines (Paeonia lactiflora, Angelica sinensis, Ligusticum chuanxiong, Poria cocos, Atractylodis macrocephalae and Rhizoma Alismatis), has been used as a classical gynecological remedy in China for centuries. However, its active substances have remained unknown. In this paper, an HPLC/DAD/ESI-MS/MS method was developed for the qualitative and quantitative analysis of the major constituents in DSS. The ESI-MS/MS fragmentation behavior of the reference compounds was proposed for aiding the structural identification of components in DSS extract. Forty-one compounds including monoterpene glycosides, phenolic acids, phathalides, sesquiterpenoids and triterpenes were identified or tentatively characterized by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data, and 14 of them (gallic acid, albiflorin, paeoniflorin, ferulic acid, benzoic acid, senkyunolide I, coniferyl ferulate, senkyunolide A, 3-butylphthalide, Z-ligustilide, Z-butylidenephthalide, atractylcnolide II, atractylcnolide I and levistolide A) were determined by HPLC–DAD using a C₁₈ column and gradient elution of acetonitrile/water–formic acid (100:0.1, v/v). The linearity, precision, accuracy, LOD and LOQ were validated for the quantification method, which proved sensitive, accurate and reproducible. The study might provide a basis for the quality control of DSS extracts and preparations.     

Identification and determination of the major constituents in Deng's herbal tea granules by rapid resolution liquid chromatography coupled with mass spectrometry

Deng's herbal tea (DHT), a famous traditional Chinese herbal tea consisting of six traditional Chinese medicines (Honeysuckle, Chrysanthemum, Rhizoma imperatae, Folium mori, dandelion and liquorice), is widely used in China for its health benefits. In this paper, a rapid resolution liquid chromatography coupled with mass spectrometry (RRLC-MS) method was developed for the identification and determination of the major constituents in DHT granules. A good RRLC separation was achieved using an Agilent Poroshell 120 SB-C₁₈ column and gradient elution (0.5% formic acid in water/acetonitrile) within 30 min. Twenty-eight compounds were identified or tentatively characterized based on their exact molecular weights and fragmentation patterns. Fifteen major bioactive constituents of those 28 compounds were chosen as the benchmark substances. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple-reaction monitoring mode, and a full quantitative analysis of the 15 major constituents was performed by our developed RRLC-MS/MS method in only 10 min. Of the 16 DHT granule samples tested, the quality of the results was stable, which confirms that the developed method was efficient and robust for the quality control of DHT granules.     

High-performance liquid chromatography—Two wavelength detection of triterpenoid acids from the fruits of Ziziphus jujuba containing various cultivars in different regions and classification using chemometric analysis

A simple and sensitive HPLC–DAD method has been developed for the first time to simultaneously determine 10 triterpenoid acids (ceanothic acid, alphitolic acid, zizyberanal acid, zizyberanalic acid, epiceanothic acid, ceanothenic acid, betulinic acid, oleanolic acid, ursonic acid and zizyberenalic acid) in the dried fruit of Ziziphus jujuba (called Dazao) which has been widely used as one of the traditional Chinese medicines (TCMs). This HPLC assay was performed on a reversed-phase C₁₈ column (250 mm × 4.6 mm, 5 μm) with the column temperature at 35 °C. The mobile phase was composed of A (acetonitrile) and B (0.05% aqueous phosphoric acid, v/v). The flow rate was 1.0 ml/min and the detection wave length was set at 205 nm for reference compounds 1–9 and 238 nm for reference compound 10. All calibration curves showed good linear regression (r² > 0.9999) within the test range. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 0.43–1.72% and 0.53–2.45%, respectively, and overall recoveries of 94.98–104.09% for the 10 compounds analyzed. The validated method was successfully applied for the simultaneous determination of the 10 triterpenoid acids in 42 batches of Dazao which contained 36 cultivars from 22 cultivation regions, and were investigated and authenticated as Z. jujuba. Hierarchical clustering analysis (HCA) and principal components analysis (PCA) were performed to differentiate and classify the samples based on the contents of the 10 triterpenoid acids. The presented HPLC–DAD method conjugated with chemometrics approach was demonstrated to be very helpful in using Dazao resources, and was possibly useful in chemotaxonomic characterization.     

基于网络搜索快速鉴定抗风湿类中成药和保健食品中非法添加的非那西丁及小檗碱

目的 鉴定抗风湿类中成药和保健食品中非法添加的2个未知成分。方法 采用高效液相色谱-二极管阵列检测器联用（HPLC-DAD）技术进行抗风湿类中成药和保健食品非法添加筛查时发现2个可疑色谱峰,采用UPLC-MS/MS技术获得其准分子离子和二级质谱图,然后通过网络搜索分析初步确定可疑成分,最后与对照品比较检测,最终确定2个非法添加化合物的结构。结果 在抗风湿类保健食品中检出盐酸小檗碱,中成药中检出非那西丁。结论 这2个化合物不在现有抗风湿类中成药和保健食品检验标准13种目标化合物范围内,容易逃脱标准监管范围。     

Chemical fingerprint analysis of Gentianae Radix et Rhizoma by high-performance liquid chromatography

Gentianae Radix et Rhizoma (also called “Longdan” in Chinese) is commonly used for eliminating damp-heat and quenching the fire of liver and gall bladder in traditional Chinese medicine. In this study, a novel and reliable method using high-performance liquid chromatography (HPLC) was developed both for quantitative analysis of four bioactive compounds (loganic acid, swertiamarin, gentiopicroside and sweroside) and chemical fingerprint analysis of “Longdan”. In quantitative analysis, four compounds showed good regressions (R²>0.9987) within the test ranges and the recovery of the method was in the range 97.61?102.49%. In fingerprint analysis, ten characteristic peaks were selected to evaluate the similarities of the crude drugs, and the HPLC chromatograms of twenty samples from different regions of China showed similar patterns. The results demonstrated that the combination of the quantitative and chromatographic fingerprint analyses offered an efficient way to evaluate the quality consistency of Gentianae Radix et Rhizoma.     

液质联用法检测镇静安神类中药制剂及保健品中非法掺入的化学品

目的: 建立检测镇静安神类中药制剂和保健品中非法掺入化学品的液质联用方法,并对10批市售样品进行检测。方法: 采用液相色谱-离子阱质谱法,选用C₁₈柱,分别以甲醇-乙腈-0.5%甲酸(15:25:60)和甲醇-水(60:40)为流动相,对样品的提取液进行分析。结果: 在10批受试制剂中,1批被检测出非法掺有地西泮。结论: 该法选择性强,灵敏度高,可以用于分析检测中药制剂及保健品中是否掺入相应化学品。