Screening the anti-gout traditional herbs from TCM using an in vitro method

The aim of this work was to evaluate the ability of 33 herbal extracts in inhibiting the acute inflammation and xanthine oxidase(XOD) activity.The anti-inflammation effects of the herbal extracts were detected by an in vitro cell model,which was established by stimulating human umbilical vein endothelial cells(HUVEC) using sodium urate(MSU).In this model,the intercellular adhesion molecule-1(ICAM-1) and interleukin-1 beta(IL-1β) were expressed,and the anti-inflammation effects of herbal extracts were evaluated by detecting the content changes of ICAM-1 and IL-1β in cell lysates and cell culture supernates using an enzyme-linked immunosorbent assay(ELISA).Moreover,an ultrahigh performance liquid chromatography and tandem mass spectrometry(UPLC-MS/MS) method was used for the detection of XOD activity and the screening of XOD inhibitors in this research.The amount of uric acid from each analyte was directly detected using the multiple reaction monitoring mode and the uric acid level could be reduced via the addition of an inhibitor.Results indicated that Salviae Miltiorrhizae Radix et Rhizome,Rhei Radix et Rhizoma,Polygoni Cuspidati Rhizoma et Radix,Selaginellae Herba,Paeoniae Radix Rubra,especially Ginkgo Folium seemed to be more effective in anti-inflammation and inhibiting XOD activity.The anti-inflammation and enzyme inhibitory activities of the herbal extracts may be correlated with their bioactive components.And the differences between the herbal extracts were correlated with the amount of flavonoid and anthraquinone components.In our study,we have investigated the potential anti-inflammation bioactivity of 33 herbal extracts in vitro,which could provide a reference for further in vivo research in the prevention and treatment of gout.     

Profiling of secondary metabolites in tissues from Rheum palmatum L. using laser microdissection and liquid chromatography mass spectrometry

Evaluating the quality of herbal medicines by morphological features is a convenient, quick, and practical method compared with other methods that mostly depend on modern instruments. Here, laser microdissection and ultra-performance liquid chromatography are combined with mass spectrometry to map the distribution of secondary metabolites in cells or tissues of a herb itself for correlating its bioactive components and morphological features. The root and rhizome of Rheum palmatum L. were taken as research target, which is the Chinese medicine, Radix et Rhizoma Rhei. According to fluorescent microscopic characteristics, 12 herbal cells or tissues of Radix et Rhizoma Rhei were separated by laser microdissection. Thirty-eight compounds were identified or tentatively characterized in the microdissected tissues. (+)-Catechin, 1-O-galloyl-2-O-cinnamoyl-β-d-glucose, and emodin were found to be the major components in most of the tissues. The brown ergastic substances found in rays of normal and anomalous vascular bundles as well as the parenchymatous cells of rhizome pith and the parenchymatous cells of root xylem contained higher than average amounts of these three components and more kinds of secondary metabolites. Overall, results suggest that Radix et Rhizoma Rhei of larger size and with conspicuous “brocaded patterns” and star spots are of higher quality as they tend to have greater contents of bioactive components. The study provides quantitative and specific criteria by which the quality of Radix et Rhizoma Rhei can be judged. This research also established a new, reliable, and practical method for direct profiling and imaging of secondary metabolites in any herbal tissue.

Structural elucidation and identification of alkaloids in Rhizoma Coptidis by electrospray ionization tandem mass spectrometry

Simple, convenient, sensitive and accurate analytical methods are needed for the structural characterization and identification of alkaloid components in Rhizoma Coptidis in traditional Chinese herbal medicine, which has important bioactivity. In this work, the identification of alkaloid compounds in Rhizoma Coptidis was investigated by obtaining molecular mass information using electrospray ionization mass spectrometry (ESI-MS). Multi-stage tandem mass spectrometric (ESI-MS(n)) data for the alkaloid compounds were used for detailed structural characterization, then structure information was obtained by comparison of the fragmentation mechanisms of both alkaloids in Rhizoma Coptidis and standard samples of berberine, palmatine, coptisine and jatrorrhizine by MS. Based on the results obtained, the structure of a novel compound was elucidated. The results of the experiments demonstrate that ESI-MS(n) is a sensitive, selective and effective tool for the rapid determination of alkaloids in Rhizoma Coptidis.     

Characterization and identification of multiple constituents in Yinhuang granules by high-performance liquid chromatography with diode-array and time-of-flight mass spectrometry detection

A fast high-performance liquid chromatography (HPLC) method with diode-array detection (DAD) and time-of-flight mass spectrometry (TOF/MS) has been developed for the analysis of multi-constituent in Yinhuang granules, a well-known combined herbal remedy prepared from the extract mixtures of Flos Lonicerae and Radix Scutellariae. The fast HPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6×50 mm, 1.8 μm) and 0.2% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution in 17 min, which is five times faster than the performance of conventional columns packed with 5.0 μm particles. With various fragmentor voltages in TOF/MS, accurate mass measurements (<5 ppm error) for molecular ions and characteristic fragment ions represented reliable identification criteria for different constituents. A total of 28 compounds, including nine phenolic acids, three iridoid glycosides and nine saponins from Flos Lonicerae and seven flavonoids from Radix Scutellariae, were identified or tentatively characterized in the extract of Yinhuang granules. The established fast HPLC-DAD-TOF/MS method turns out to be useful and efficient for quality control 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。     

Chemical composition differentiation of Shen‐Shuai‐Ning granule between combined decoction and separated decoction using HPLC‐DAD‐ESI‐QTOF‐MS

Shen‐Shuai‐Ning (SSN) granule, a traditional Chinese medicine formula, is widely used in clinical practice for treating chronic renal failure. However, its detailed chemical profile is unknown. Here, HPLC‐ESI‐QTOF‐MS was employed for the systematic chemical analysis of SSN. A total of 52 compounds were identified and the characteristic ions of the compounds were described. Furthermore, chemical consistency between the combined decoction and the separated decoction of SSN was evaluated using HPLC‐DAD. A chemical comparison between two preparations of SSN granule (combined decoction and separated decoction of Coptides Rhizoma) indicated a significant difference in the content of many compounds, including salvianolic acid A, salvianolic acid B, berberine, palmatine and epiberberine. As a result, separated decoction of Coptides Rhizoma would lead to a significantly decrease in depsides in Salviae Miltiorrhizae Radix et Rhizoma and an increase in alkaloids in Coptidis Rhizoma.     

苦黄注射液中大黄蒽醌含量测定方法改进

按苦黄注射液原质量标准（草案）中测定方法,大黄蒽醌用氯仿提取,阴性试验结果为苦黄注射液大黄蒽酯标示量的１６．５０％（ｎ＝５）,结果偏高;改用石油醚提取,降为６．１５％,重复性试验的ＲＳＤ为１．４４％（ｎ＝５）,加标回收率为１０１．８％（ｎ＝５）,显色在１－４ｈ内稳定,表明该法是可行的。     

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).     

Determination of paeoniflorin, ferulic acid and baicalin in the traditional Chinese medicinal preparation Dang-Guei-San by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of paeoniflorin, ferulic acid and baicalin in the traditional Chinese medicinal preparation Dang-Guei-San, which contains Paeoniae Radix, Swertiae Herba, Cnidii Rhizoma and Scutellariae Radix, was established. The samples were separated with a Cosmosil 5C₁₈-AR column by gradient elution with 0.03% (v/v) phosphoric acid- acetonitrile (0 min 96:4, 5 min 84:16, 7 min 82:18, 14–30 min 78:22) as the mobile phase at a flow-rate of 1.0 ml/min, with detection at 245 nm. Methylparaben was used as the internal standard and three equations were derived showing linear relationships between the peak- area ratios of marker components (paeoniflorin, ferulic acid and baicalin) to methylparaben and concentration. The recoveries of paeoniflorin, ferulic acid and baicalin were 27.86, 33.89 and 49.31%, respectively. The repeatability (relative standard deviation) was generally less than 5% (n = 5). The effects of various processes such as concentration by reduced-pressure evaporation, freeze-drying and spray-drying were studied and commercial concentrated herbal preparations containing Paeoniae Radix, Swertiae Herba, Cnidii Rhizoma and Scutellariae Radix were also analysed.     

Temperature-assisted ionic liquid dispersive liquid-liquid microextraction combined with high performance liquid chromatography for the determination of anthraquinones in Radix et Rhizoma Rhei samples

In this article, a novel method termed as temperature-assisted ionic liquid dispersive liquid-liquid microextraction (TA IL-DLLME) combining high performance liquid chromatography with diode array detection (HPLC-DAD) was developed for the determination of anthraquinones in Radix et Rhizoma Rhei samples. The ionic liquid (1-hexyl-3-methylimidazolium hexafluorophosphate) was used to replace volatile organic solvent as an extraction solvent for the extraction of anthraquinones (aloe-emodin, rhein, emodin, chrysophanol and physcion) from Radix et Rhizoma Rhei. Several important parameters influencing the extraction efficiency of TA IL-DLLME such as the type and volume of extraction solvent and disperser solvent, sample pH, extraction time, extraction temperature, centrifugation time as well as salting-out effects were optimized. Under the optimal conditions, the spiked recovery for each analyte was in the range of 95.2-108.5%. The precisions of the proposed method were varied from 1.1% to 4.4% (RSD). All the analytes exhibited good linearity with correlation coefficients (r²) ranging from 0.9986 to 0.9996. The limits of detection for all target analytes were ranged from 0.50 to 2.02 μg L⁻¹ (S/N = 3). The experimental results indicated that the proposed method was successfully applied to the analysis of anthraquinones in Radix et Rhizoma Rhei.     

Rapid identification of traditional Chinese herbal medicine by direct analysis in real time (DART) mass spectrometry

Direct analysis in real time-mass spectrometry (DART-MS) was employed as a novel fast method to identify traditional Chinese herbal medicine (TCHM). In order to obtain high quality mass spectra, the ionization temperature was optimized for every kind of sample. With minimal or no sample pretreatment, major TCHM components, including alkaloids, flavonoids and some ginsenosides, were directly detected within several seconds, while thirteen ginsenosides need derivatization to get good mass spectra. Pseudoginsenoside F11, compound K, protopanaxatriol (PPT) and protopanaxadiol (PPD), for the first time were detected without derivatization. Among five of eight tested Chinese herbal medicines, Rhizoma Corydalis, Bulbus Fritillariae Thunbergii, Arecae Semen, Ramulus Uncariae Cum Uncis and Scutellariae Radix, were first time identified by DART-MS. In addition, the ionization mechanisms of major herbal components, alkaloids, flavonoids and ginsenosides, were discussed in detail. Our results demonstrated that DART-MS could provide a rapid, reliable and environmental friendly method for the rapid identification of TCHM, and may be applicable to other plants.     

微波消解-原子吸收光谱法测定槐花和大黄中微量元素

采用微波消解-火焰原子吸收光谱法和石墨炉原子吸收光谱法测定了中药槐花和大黄中铜、铁、锌、镉、铬和铅。研究了槐花和大黄的微波消解试剂和消解条件。在原子吸收光谱测定的最佳条件下测定槐花和大黄中铜、铁、锌、镉、铬和铅。方法的回收率在89．2％～112．0％之间。     

Qualitative and Quantitative Analysis for the Quality Control of Rhizoma Coptidis by HPLC-DAD and HPLC-ESI-MS

For quality control purpose, an approach of fingerprinting and simultaneous quantification of five major bioactive constituents of Rhizoma Coptidis was established via a high-performance liquid chromatograph coupled with a photodiode array UV detector(HPLC-DAD) and an electrospray ionization mass spectrometer(HPLC-ESI/MS). The compounds were identified on the basis of the comparison of their mass spectra with literature data and those of standard samples and quantified by the HPLC-DAD method. Baseline separation was achieved on an XTerra C 18 column(5 μm, 250 mm×4.6 mm i. d.) with linear gradient elution of formate buffer(consisting of 0.5% formic acid, adjusted to pH=4.5 with ammonia) and acetonitrile(consisting of 0.2% formic acid and 0.2% triethylamine). The me-thod was validated for linearity(r 2 0.9995), repeatability(RSD3.1%), intra-and inter-day precision(RSD1.8%) with recovery(99.9%―105.1%), limits of detection(0.15―0.35 μg/mL), and limits of quantification(0.53―0.82 μg/mL). The similarities of 32 batches of Rhizoma Coptidis and their classification according to their manufacturers were based on the retention time and peak areas of the characteristic compounds. The five compounds were selected for quality assessment of Rhizoma coptidis via partial least squares analysis(PLS).     

Deciphering chemical interactions between Glycyrrhizae Radix and Coptidis Rhizoma by liquid chromatography with transformed multiple reaction monitoring mass spectrometry

In this study, we propose an integrated strategy for the efficient identification and quantification of herbal constituents using liquid chromatography with mass spectrometry. First, liquid chromatography with quadrupole time‐of‐flight mass spectrometry was employed for the chemical profiling of herbs, where a targeted following nontargeted approach was developed to detect trace constituents by using structural correlations and extracted ion chromatograms. Next, ion pairs and parameters of MS² of quadrupole time‐of‐flight mass spectrometry were selected to design multiple reaction monitoring transitions for the identified compounds on liquid chromatography with triple quadrupole mass spectrometry. The relative concentration of each constituent was then calculated using a semiquantitative calibration curve. The proposed strategy was applied in a study of chemical interactions between Glycyrrhizae Radix and Coptidis Rhizoma. A total of 140 compounds were identified or tentatively characterized from the herbs, 132 of which were relatively quantified. The visualized quantitative results clearly showed codecoction produced significant constituent concentration variations especially for those with a low polarity. The case study also indicated that the present methodology could provide a reliable, accurate, and labor‐saving solution for chemical studies of herbal medicines.     

Formation and conformation of baicalin-berberine and wogonoside-berberine complexes

It is well-known that baicalin-berberine complex (1) precipitates in the water decoction of numerous Chinese Medicinal formulae containing Radix Scutellariae and Rhizoma Coptidis or Cortex Phellodendri. In the current study, ionic interaction between the carboxylate ion of baicalin and the quaternary ammonium ion of berberine was revealed to be responsible for the formation of 1 and wogonoside-berberine (2) by using FAB-MS and NMR titration experiments. In addition, nuclear Overhauser effect spectroscopy (NOESY) correlations observed in 1 and 2 suggested quite different conformation of the two complexes, which was further supported by the fact that the [α](D) of the canadine obtained by reduction of 1 is of an opposite sign to that obtained from 2. Partition coefficients (n-octanol/water) determination demonstrated 12-20 times larger partition coefficient of each complex (1, 2) than that of each single compound (baicalin, wogonoside, and berberine), indicating the significant role of the formation of the complex in the bioavailability enhancement of these pharmacologically active constituents.     

溶剂浮选法分离富集大黄中的有效成分

采用溶剂浮选法对大黄提取液中的芦荟大黄素、大黄素、大黄酚以及大黄素甲醚进行了分离富集,并用高效液相色谱法分别测定了其含量。考察了料液浓度、浮选溶剂、浮选时间、浮选液pH值、氮气流速和电解质NaCl浓度对浮选效率的影响,并与泡沫浮选法和溶剂萃取法进行了比较。结果表明：溶剂萃取效果最差,泡沫浮选次之,溶剂浮选法分离富集效果最好。当料液浓度为6．4mg／mL,浮选时间为30min,浮选液pH一1～2,氮气流速为20mL／min,电解质NaCl浓度为0．4mol／L时,溶剂浮选效率最佳。     

Quality assessment of a formulated Chinese herbal decoction, Kaixinsan, by using rapid resolution liquid chromatography coupled with mass spectrometry: A chemical evaluation of different historical formulae

Kaixinsan is an ancient Chinese herbal decoction mainly prescribed for patients suffering from mental depression. This decoction was created by Sun Si-miao of Tang Dynasty (A.D. 600) in ancient China, and was composed of four herbs: Radix and Rhizome Ginseng, Radix Polygalae, Rhizoma Acori Tatarinowii and Poria. Historically, this decoction has three different formulations, each recorded at a different point in time. In this study, the chemical compositions of all three Kaixinsan formulae were analyzed. By using rapid resolution LC coupled with a diode-array detector and an ESI triple quadrupole tandem MS (QQQ-MS/MS), the Radix and Rhizome Ginseng-derived ginsenosides including Rb(1), Rd, Re, Rg(1), the Radix Polygalae-derived 3,6'-disinapoyl sucrose, the Rhizoma Acori Tatarinowii-derived α- and β-asarone and the Poria-derived pachymic acid were compared among the three different formulations. The results showed variations in the solubility of different chemicals between one formula and the others. This systematic method developed could be used for the quality assessment of this herbal decoction.     

Characterization of protoberberine alkaloids in Coptidis Rhizoma (Huanglian) by HPLC with ESI‐MS/MS

This study aims to qualitatively analyze protoberberine alkaloids in crude extract of Coptidis Rhizoma using HPLC with ESI‐MS/MS. Possible specific molecular weights of protoberberine alkaloids were firstly deduced according to literatures and were adopted to screen the alkaloids in the HPLC with ESI‐MS of crude extract of Coptidis Rhizoma. As a result, 21 protoberberine alkaloids were found, including compounds of very low concentration and compounds coeluted in one peak. Among these, two compounds were positively identified and verified by comparison with standards. Ten of these compounds were first reported in this study for Coptidis Rhizoma. In addition, chromatographic retention parameters a and c of all compounds were obtained using their retention times under five gradient conditions and were applied to confirm the deduction about the structures of protoberberine alkaloids by tandem mass data.