Studies of the intestinal absorption of the alkaloids in the Wu-tou decoction combined with different incompatible medicinal herbs in a Caco-2 cell culture system using UPLC-MS/MS

In this study, seven alkaloids were detected in Wu-tou decoction using ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MSn). The aim of this study was to investigate the effect of Fritillariae Cirrhosae Bulbus, Fritillariae Thunbergii Bulbus, Pinelliae Rhizoma in different ratios with Wu-tou decoction (2:1, 1:1, 1:2) by measuring the therapeutic effects in Wu-tou decoction of main seven alkaloids including benzoylaconitine (BA), benzoylmesaconitine (BM), benzoylhypaconitine (BH), hypaconitine (HA), fuziline (FU), niaolin (NE) and deoxyaconitine (DA). The permeability of aconitum alkaloids extract through a Caco-2 cell monolayer was analyzed in the absence and presence of Fritillariae Cirrhosae Bulbus, Fritillariae Thunbergii Bulbus, and Pinelliae Rhizoma, respectively. The results showed that Pinelliae Rhizoma could reduce the absorption of the alkaloids and increase the excretion of the alkaloids, which would attenuate the therapeutic effects of Wu-tou decoction. Therefore, Pinelliae Rhizoma is an incompatible herb of Wu-tou decoction because of the inhibition of the absorption of alkaloids in the intestine. And that Fritillariae Cirrhosae Bulbus and Fritillariae Thunbergii Bulbus showed the effects to improve the permeability of the alkaloids in Wu-tou decoction. These effects of these two herbs were similar, but the former was stronger than the latter, which most likely is due to the fact that the compositions of these two traditional Chinese medicines are similar. The in vitro data suggests that the compounds such as fritillary presented in alkaloids in the formula maybe improve the therapeutic function caused by the increased bioavailability of alkaloids in intestine.     

利用液质联用技术分析干姜对乌头类生物碱在大鼠肠内吸收的影响

为从吸收的角度考察干姜对乌头类双酯型生物碱的解毒机理, 采用外翻肠囊法展开实验. 利用超高液相与三重四极杆质谱联用技术定量检测双酯型生物碱成分, 采用标准曲线法计算乌头碱、中乌头碱、次乌头碱在肠囊内吸收的绝对含量, 采用质谱峰面积直接分析其它双酯型生物碱的相对变化, 结果加入干姜提取液后, 乌头碱、中乌头碱、次乌头碱的单位肠管面积累计吸收量均降低, 10-羟基中乌头碱的的累积峰面积降低; 加入维拉帕米后, 双酯型生物碱的单位肠管面积累计吸收量及累积峰面积均增加; 向含有地高辛的肠营养液中加入干姜提取液后, 地高辛在各实验时间点的单位肠管面积累计吸收量均降低, 根据以上结果推测干姜抑制乌头类双酯型生物碱在大鼠肠囊内吸收的可能机制是通过诱导肠内P-葡糖蛋白, 从而抑制作为P-葡糖蛋白底物的双酯型生物碱的吸收, 最终起到减毒作用.     

中草药中铁锌钙的悬浮液进样-火焰原子吸收光谱法测定

将悬浮液进样技术应用于火焰原子吸收光谱法, 建立了中草药中微量元素的快速分析新方法。将样品粉碎、悬浮在琼脂胶体中制成悬浮液; 取适量样品悬浮液配制成试液, 喷入空气－ 乙炔火焰, 用标准加入法测定。 用该法成功地测定了当归、川芎及川乌中的铁、锌、钙, 测定结果与灰化法一致, 方法简便、快速、准确。     

Rapid quality assessment of Radix Aconiti Preparata using direct analysis in real time mass spectrometry

This study presents a novel and rapid method to identify chemical markers for the quality control of Radix Aconiti Preparata, a world widely used traditional herbal medicine. In the method, the samples with a fast extraction procedure were analyzed using direct analysis in real time mass spectrometry (DART MS) combined with multivariate data analysis. At present, the quality assessment approach of Radix Aconiti Preparata was based on the two processing methods recorded in Chinese Pharmacopoeia for the purpose of reducing the toxicity of Radix Aconiti and ensuring its clinical therapeutic efficacy. In order to ensure the safety and effectivity in clinical use, the processing degree of Radix Aconiti should be well controlled and assessed. In the paper, hierarchical cluster analysis and principal component analysis were performed to evaluate the DART MS data of Radix Aconiti Preparata samples in different processing times. The results showed that the well processed Radix Aconiti Preparata, unqualified processed and the raw Radix Aconiti could be clustered reasonably corresponding to their constituents. The loading plot shows that the main chemical markers having the most influence on the discrimination amongst the qualified and unqualified samples were mainly some monoester diterpenoid aconitines and diester diterpenoid aconitines, i.e. benzoylmesaconine, hypaconitine, mesaconitine, neoline, benzoylhypaconine, benzoylaconine, fuziline, aconitine and 10-OH-mesaconitine. The established DART MS approach in combination with multivariate data analysis provides a very flexible and reliable method for quality assessment of toxic herbal medicine.     

复方乌头汤配伍贝母化学成分变化研究

将超高效液相色谱-质谱联用(UPLC-MS) 及UPLC-MSⁿ技术与偏最小二乘法判别分析(PLS-DA)结合, 对乌头汤分别与川贝、 浙贝按照制川乌与贝母生药质量比1: 1配伍前后化学成分的变化情况进行了研究. 在PLS-DA模型中, 乌头汤与川贝、 浙贝配伍前后的煎煮液均可明显区分. 乌头汤与川贝共煎后获得8个化学标志物, 可鉴定出7个化合物, 其中尼奥灵、 附子灵及苯甲酰基新乌头原碱在共煎后含量上升, 塔拉定、 10-OH苯甲酰基乌头原碱、 10-OH苯甲酰基新乌头原碱和苯甲酰基去氧乌头原碱在配伍后含量下降. 乌头汤与浙贝共煎后获得7个化学标志物, 分别为Chuanfumine、 Songorine、 塔拉定、 尼奥灵、 附子灵、 苯甲酰基新乌头原碱和10-OH苯甲酰基新乌头原碱, 所有化学标志物的含量在共煎后均降低.     

半夏淀粉的理化特性

研究了不同产地的4种半夏淀粉的理化特性,包括直链淀粉含量、膨胀度、溶解性、持水性、淀粉粒大小和形貌、结晶类型、热特性和糊化特性等。结果表明,这些半夏淀粉中直链淀粉含量为18.60%～23.91%;膨胀度21.53%～23.09%;溶解度11.5%～32.3%;持水性100.3%～119.0%。淀粉粒单粒球形,卵形或圆半球形,直径2～20μm,复粒由2～3个分粒组成,其结晶类型均为C型,结晶度15.0%～37.9%。用差示扫描量热仪测得的转变温度TO、TP和TC分别为71.58～77.75℃、83.03～83.84℃和89.41～90.99℃,热焓为4.316～5.809 J/g。用快速粘度分析仪测定了4种半夏淀粉的糊化特征值:峰值粘度、热糊粘度、冷糊粘度、稀懈值和回复值分别为149.5～226.2、97.7～127.2、141.8～194.3、50.4～99.0和44.2～67.2 RVU。糊化温度77.8～79.9℃,峰值时间8.3～8.7 min。     

近红外光谱法快速测定附子中6个生物碱的含量

应用近红外光谱(NIRS)技术结合偏最小二乘(PLS)和最小二乘支持向量机(LS-SVM)建立了附子中多指标成分的快速无损检测方法.选取38批样品建立了同时测定附子样品中6种成分含量的高效液相色谱(HPLC)方法;通过采集附子样品的NIRS图,分别采用PLS和LS-SVM建立了各个成分HPLC测定值与NIRS图的定量校...     

基于新型介质阻挡放电离子源的药物快速检测方法研究

本研究将单电极放电技术的DBDI与质谱(MS)联用,快速检测了4种低极性的合成药物,结果表明,4种合成药物主要生成[M+H]+分子离子.此外还利用DBDI-MS对草乌、制草乌切片进行快速分析,在草乌中检测到乌头碱、中乌头碱、脱氧乌头碱的[M+H]+离子,以及[M+H-60]+碎片离子;在制草乌中只检测到乌头碱、中乌头碱、脱氧乌头碱的[M+H-60]+碎片离子.所测草乌中的标志性药效成分主要为双酯类生物碱,制草乌中的标志性药效成分主要为单酯类生物碱.新型DBDI为药物研究提供了一种新的、快速检测方法,具有十分重要的理论和实际应用意义,在药物研究领域具有极大的应用潜力.     

高效液相色谱法同时测定中药材虎掌南星的核苷类成分

建立了高效液相色谱同时测定中药材虎掌南星中核苷类活性成分(腺嘌呤、次黄嘌呤、黄嘌呤、尿苷、胸腺嘧啶、腺苷、鸟苷)含量的方法。以Lichrospher C18色谱柱(150 mm×4.6 mm, 5 μm)分离,以乙腈-水(含0.1%甲酸)为流动相,梯度洗脱,在254 nm下检测,腺嘌呤、次黄嘌呤、黄嘌呤、尿苷及鸟苷分别在1.6～50 mg/L范围内、胸腺嘧啶和腺苷分别在1.2～40 mg/L范围内的线性关系良好,相关系数均大于0.9995,加标回收率为98.9%～101.2%,相对标准偏差均小于3%。方法学考察结果显示符合含量测定要求,并应用于不同产地虎掌南星的测定。该方法操作简便、快速,结果可靠,重现性好,可作为虎掌南星质量评价的参考依据。     

The comparative research on constituents of Radix Aconiti and its processing by HPLC quadrupole TOF-MS

Based upon the regulations stipulated by the State Food and Drug Administration of China, only the processed, detoxified tubers and roots of Aconitum are allowed to be administered orally, used in clinical decoctions and adopted as raw materials for pharmaceutical manufacturing, so the processing principle of preparation of Radix Aconiti is important for ensuring the Radix Aconiti praeparata quality. A simple approach was described for HPLC‐Q‐TOF‐MS screening and identification of many of the aconitine alkaloids present in unprocessed Radix Aconiti and Radix Aconiti praeparata. To compare their fingerprints, the processing principle of preparation of Radix Aconiti was developed. Twenty‐nine compounds and 26 compounds were assigned to aconitine alkaloids and tentatively identified by comparing accurate mass and fragments information with that of the authentic standards or by mass spectrometry analysis and retrieving the reference literature. The nonester alkaloids were almost the same. The diester diterpene alkaloids were decreased, the monoester‐diterpene alkaloids were increased and lipo‐alkaloids decreased obviously in the processing of the preparation. These transformed components could be regarded as potential chemical markers that can be used to distinguish between raw and processed herbs. Copyright © 2012 John Wiley & Sons, Ltd.     

Study on the therapeutic material basis of traditional chinese medicinal preparation Suanzaoren decoction

Methodology for elucidating the therapeutic material basis and quality control indices of Traditional Chinese Medicinal Preparation Suanzaoren decoction was developed. Combination of Suanzaoren decoction was designed and sixteen groups were obtained. Effects on frequency of spontaneous motion, sleeping number and sleeping duration in mice were studied with sixteen combinations of Suanzaoren decoction to get the pharmacological data. RP-HPLC method was developed to analyze the chromatographic profiles of natural components in all combinations. Areas (corrected by weighting amounts) of chromatographic peaks were collected as chemical data. The pharmacological and chemical data were correlated by chemical statistical methods, and then the therapeutic material basis (thirteen chemical constituents with sedative and hypnotic effects among forty-eight chromatographic peaks) of Suanzaoren decoction were elucidated. Spinosin from Semen Ziziphi Spinosae, ferulic acid from Rhizoma Chuanxiong, mangiferin from Rhizoma Anemarrhenae and glycyrrhizic acid from Radix Glycyrrhizae were selected as quality control indices. This paper provided a new methodology for elucidating the therapeutic material basis and quality control indices for T.C.M. It is instructive for modernization of Chinese herbs and its compound preparations.     

基于水溶性浸出物及HPLC指纹图谱评价不同贮存年限半夏的品质

研究不同贮存年限半夏药材的浸出物,建立浸出物的HPLC特征指纹图谱,为半夏药材品质评控提供参考。浸出物测定方法采用药典法;HPLC指纹图谱的色谱条件:采用C_(18)色谱柱(150 mm×4.6 mm,5μm),以水–甲醇为流动相,梯度洗脱,流量为0.8 m L/min,检测波长为260 nm,柱温为25℃,进样体积为50μL。采用相似度评价及聚类分析技术揭示14批样品的相似性及差异性。14批半夏浸出物有12批合格,2批不合格。建立14批半夏浸出物样品的高效液相指纹图谱,确定了3个共有峰,共有峰保留时间的相对标准偏差小于2%,峰面积的相对标准偏差差异较大。1~#~7~#半夏样品有12个共有峰,共有峰保留时间的相对标准偏差小于1.5%,峰面积的相对标准偏差差异较大。各批次药材化学成分组成及含量均存在一定差异。以半夏浸出物数据与其高效液相色谱指纹图谱数据为基础,将指纹图谱相似度评价与聚类分析结合起来,用浸出物含量及评价软件测评结果对半夏品质进行综合评估,可以更精确地对半夏药材进行质量控制。     

Screening out Potential Cardio-Toxic Components of Chinese Herb Radix Aconiti Lateralis in Rat Dosed Plasma by High Performance Liquid Chromatography/Electrospray Ionization Quadrupole Time-Of-Flight Mass Spectrometry

This study established a new, rapid, and accurate method for bioactive compound screening by the combination of serum pharmacochemistry and serum pharmacology based on a spectrum-effect relationship model. HPLC/ESI-Q-TOF-MS was used for the chemical profiling of samples of dosed plasma, control plasma, and Radix Aconiti Lateralis decoction. A comparison was made of the resulting chemical profiles, which totaled 20 compounds including 16 prototype compounds and 4 metabolites derived from SF decoction that were detected as potential bioactive components. Using MS/MS analysis and accurate molecular weight assessments of TOF, 17 components in 20 potential bioactive compounds were structurally identified. Moreover, the pretreated plasma samples were injected into venous sinus of isolated toad hearts to investigate the cardio-toxicity according to heart ratio (HR) situation and electrocardiograph (ECG) parameters, including PR interval, QRS interval, and QT interval. Finally, 4 compounds in these 20 potential bioactive components that had closest correlation with 4 cardio-toxic experimental indexes respectively were investigated by spectrum-effect relationship model for the first time. The 4 compounds were considered as the real cardio-toxic components. This study proposed a series of potential bioactive components in Radix Aconiti Lateralis and provided a general research pattern to identify the potential bioactivity compounds in Chinese herbs or natural pharmacy.     

Fast profiling of chemical constituents in Yiqing Capsule by ultra-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry

An ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC-ESI-MS(n)) has been developed for structural characterization and identification of multi-constituents in Yiqing Capsule, a well-known combined herbal remedy prepared from the extract mixtures of Rhizoma Coptidis, Radix et Rhizoma Rhei and Radix Scutellariae. The UPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6 mm×50 mm, 1.8 μm) and gradient elution of 0.1% formic acid solution and acetonitrile in 16 min. Based on their retention times and mass spectra in comparison with the data from standards or references, a total of 29 compounds including 3 phenolic acids and 4 anthraquinones from Radix et Rhizoma Rhei, 8 alkaloids from Rhizoma Coptidis and 14 flavonoids from Radix Scutellariae were unambiguously identified or tentatively characterized in the complex system. The MS data and fragmentation information of two isomers of feruloylquinic acid were first reported in Radix et Rhizoma Rhei and in Yiqing Capsules. This study is expected to be accepted as an effective and reliable pattern for comprehensive and systematic characterization of this commonly used Chinese herbal preparation.     

色谱指纹谱用于中药大黄抗肿瘤活性成分的筛选

采用液相色谱-质谱联用方法分析了中药大黄经过SD大鼠肝匀浆体外代谢前后的指纹谱中色谱峰面积、保留值的差异。指出5种游离型蒽醌化合物在SD大鼠肝匀浆体外代谢体系中只有大黄酚发生代谢反应转化为芦荟大黄素。考察了体外代谢条件下,肝匀浆浓度与代谢时间对大黄酚转化及其代谢产物的影响。SD大鼠体外抗肿瘤试验表明,大黄代谢物对于人宫颈癌(HeLa)细胞的抑制活性略高于其提取物。通过比较芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚的活性,并结合大黄酚的体外代谢反应的考察,解释了大黄代谢物对肿瘤细胞活性的抑制率的提高是由大黄酚的代谢产物芦荟大黄素浓度的增加引起的。     

Screening epidermal growth factor receptor antagonists from Radix et Rhizoma Asari by two‐dimensional liquid chromatography

Radix et Rhizoma Asari is a traditional Chinese medicine, and has many pharmacological effects, such as calming, analgesia, anti‐inflammation, antiarrhythmic, antihypertensive, antivirus, etc. But few studies have screened the active compounds from extracts of Radix et Rhizoma Asari for tumor therapy. In this study, a two‐dimensional liquid chromatography system was built to screen active compounds acting on epidermal growth factor receptor (EGFR) from Radix et Rhizoma Asari. The screening result showed that asarinin from Radix et Rhizoma Asari was the targeted component that could act on EGFR specificity. The competitive binding assay and molecular docking assay results showed asarinin binding with EGFR in similar manner as with gefitinib, which was used as a positive control drug. Then the antitumor effect of asarinin was studied through cell growth assay in vitro. The results showed that gefitinib and asarinin could inhibit highly expressed EGFR cell growth in a dose‐dependent manner in the range of dose from 0.10 to 102.4 μM. This two‐dimensional liquid chromatography system will be a useful method in drug discovery from natural medicinal herbs for searching potential antitumor candidates.     

川乌与半夏、瓜蒌、贝母、白蔹、白芨配伍禁忌的化学研究

运用高效液相色谱方法,定量分析了川乌单煎液及其与生半夏、法半夏、全瓜蒌、瓜蒌皮、瓜蒌籽、浙贝母、川贝母、白蔹、白芨共煎液的双酯型生物碱含量.并利用电喷雾质谱通过加入内标的半定量分析方法研究生川乌配伍前后生物碱成分和含量的变化.结果显示生川乌与生半夏、瓜蒌籽、全瓜蒌、瓜蒌皮、浙贝母、白芨的共煎液中双酯型生物碱含量高于生川乌单煎液而生川乌与法半夏、川贝母、白蔹的共煎液双酯型生物碱含量变化微弱或有所减少.电喷雾质谱半定量分析方法与高效液相色谱方法的分析结果一致,并且与上述药对的LD50值结果也基本一致.而毒性成分的变化趋势与共煎前后溶液的pH变化相关.     

Application of SiO2 hollow fibers for sorptive microextraction and gas chromatography–mass spectrometry determination of organochlorine pesticides in herbal matrices

A method involving simultaneous extraction and sample clean-up procedure: hollow fiber sorptive microextraction, coupled with gas chromatography–mass spectrometric detection for quantification of seven organochlorine pesticides in Radix et Rhizoma Rhei is described. SiO₂ hollow fiber with porous structure was synthesized for the first time. The internal diameter of SiO₂ hollow fiber is 380 μm and average wall thickness is 100 μm. Aggregated SiO₂ particles deposited on the surface of the hollow fiber in a regular array lead to porous structure. SiO₂ hollow fiber was applied to the determination of organochlorine pesticides in Radix et Rhizoma Rhei to avoid sample clean-up and minimize the matrix effects. Extraction solvent, extraction temperature and equilibration time were optimized. Fiber to fiber repeatability over the concentration ranges were less than 10%. Recoveries were satisfactory (between 63% and 115%) for most of organochlorine pesticides at spiking levels. Furthermore, the proposed method was also applied to determine seven organochlorine pesticides in 43 commercial Radix et Rhizoma Rhei samples, in which the selected pesticides were found in eight samples. The results have been further confirmed by solvent extraction methods according to China Pharmacopoeia (2005).     

野生与栽培羌活药材挥发油含量及组分的比较分析

分析野生和栽培羌活药材挥发油含量及其组分的变化,为羌活药材的引种栽培及进一步的开发利用提供了依据。采用水蒸气蒸馏法提取羌活药材中的挥发油,通过GC-MS对挥发油成分进行分析鉴定;利用聚类分析对测定结果进行分类;通过相关分析研究海拔对羌活药材挥发油含量的影响。6份药材样品挥发油含量范围为1.60～7.98 mL/100g,6份羌活药材挥发油成分经GC-MS分析,共鉴定出57个化合物,共有成分12个;基于挥发油成分种类及相对含量的聚类分析显示了羌活药材挥发性成分种类及含量的种间差异;相关分析结果表明,海拔高度与挥发油含量存在显著正相关。野生和栽培羌活药材挥发油含量存在差异,总体上野生羌活药材的挥发油含量高于栽培种;羌活药材挥发油含量随海拔升高而增加。     

Differentiation of rhizoma et radix polygoni cuspidati from closely related herbs by HPLC fingerprinting

An HPLC-DAD fingerprinting profile of Rhizoma Et Radix Polygoni Cuspidati was established basing on the consistent chromatographic features of 24 authentic herb samples. The major types of chemical constituents, stilbenes and anthraquinones, were analyzed and included in the fingerprint. Eight common peaks of Polygonum Cuspidatum were identified by using HPLC-MS. The developed fingerprint was applied to differentiate Rhizoma Et Radix Polygoni Cuspidati from Radix Polygoni Multiflori and Radix Et Rhizoma Rhei. Although the three herbs belong to the family of Polygonaceae, the results indicated that these could be differentiated by using the established method.