Screening of bioactive compounds in Rhizoma Polygoni Cuspidati with hepatocyte membranes by HPLC and LC–MS

A new method employing HPLC, LC–MS, and hepatocyte membranes for the screening of bioactive compounds in traditional Chinese medicines (TCMs) has been proposed. We hypothesized that exposure of the TCM extracts to hepatocyte membranes should decrease the concentration of membrane‐permeable compounds in the solution. Using this approach, the permeability of the compounds in Rhizoma Polygoni Cuspidati was investigated. By comparing chromatograms of samples prepared both before and after interaction with hepatocyte membranes, seven permeable compounds of Rhizoma Polygoni Cuspidati were identified. Additionally, it was found that piceid, resveratrol, emodin‐8‐β‐d‐glucoside, physcion‐8‐β‐d‐glucoside, aloe‐emodin, emodin, and physcion combined specifically with hepatocyte membranes, which might indicate a useful approach for revealing the antiatherosclerotic effects of Rhizoma Polygoni Cuspidati. Therefore, the proposed method could be a good approach to predict the potential bioactivities of multiple compounds in TCMs simultaneously. Based on the significance of these results, this method could be a novel approach for identifying potentially bioactive components in other TCMs.     

中药黄芩中神经氨酸酶抑制剂的超滤质谱筛选研究

采用超滤质谱分析技术, 结合体外酶活性实验方法, 从传统中药黄芩提取物中筛选神经氨酸酶抑制剂. 研究结果表明, 中药黄芩提取物具有较强的神经氨酸酶抑制活性, 利用超滤质谱方法从中筛选并鉴定出了六种具有潜在神经氨酸酶抑制活性的化合物, 为开发神经氨酸酶抑制剂提供了实验依据.     

基质固相分散液相色谱法检测辣椒产品中的黄曲霉毒素

建立了中性氧化铝-石墨化碳黑的基质固相分散共柱提取净化前处理和以溴为衍生试剂的液相色谱-柱后在线衍生荧光检测法,并将该方法用于辣椒产品中黄曲霉毒素B1,B2,G1,G2的分析。对固相分散剂及共柱净化剂进行了选择和优化。该方法对B1,B2,G1,G2的平均回收率分别为95.4%,87.3%,91.5%和92.6%;方法对B1,G1的检出限为0.25 ng/g,对B2,G2的检出限为0.10 ng/g;对B1,B2,G1,G2进行测定的相对标准偏差(RSD)分别为3.3%,5.8%,4.7%和6.1%。对基质固相分散法和免疫亲和柱法的净化效果进行了比较,结果表明基质固相分散提取净化可以作为一种有效的方法用于辣椒产品中黄曲霉毒素的测定。     

人参中有机磷农残的基质固相分散-HPLC同时快速分析

建立了基质固相分散-高效液相色谱法(MSPD-HPLC)对中药材人参中的三种有机磷农药甲胺磷、甲基对硫磷和乙基对硫磷残留量进行快速分析:. 将HPLC法应用于有机磷农药残留量的检测, 分别采用中性Al2O3和体积比为8%的乙酸乙酯-正己烷作为MSPD的分散剂和洗脱剂, 对分析方法的质量控制问题进行了详细讨论, 并对不同种类的人参中药材进行了准确的定性定量分析. 该方法的线性范围为0.20～10.00 mg•L－1, 相关系数(R)大于0.99815, 相对标准偏差(RSD)均小于11.54%, 检测限(LOD)低于0.017 mg•L－1, 样品的加标回收率为87.29%～92.43%. 其线性范围、相关系数、准确度、精密度和LOD等指标均满足有机磷农残分析的要求.     

固相萃取-超过滤-液相色谱/质谱联用法测定织纹螺中的河豚毒素

建立了液相色谱-质谱法测定织纹螺中河豚毒素的分析方法。匀质后的样品用0.03 mol/L乙酸溶液提取,水浴加热10 min,用Sep-Pak C18固相萃取柱净化,再用截留相对分子质量为3000的超滤管过滤。采用Insertsil ODS-3色谱柱分离,以含有30 mmol/L七氟丁酸的甲酸铵溶液(1 mmol/L)-甲醇(体积比为99∶1)为流动相,采用电喷雾离子源,选择离子监测模式检测。以保留时间和河豚毒素的二级质谱特征碎片离子予以定性确证。结果表明,在此分析条件下,可将河豚毒素及其衍生物分离,在0.01～10.0 mg/L范围内线性关系良好(r2＞0.995),检出限(以3倍信噪比为计)为2 μg/L,平均加标回收率为72.5%～80.4%,相对标准偏差为4.48%～8.87%。将该方法用于实际样品检测,在赤潮后所采集的织纹螺样品中检出了河豚毒素。     

Separation and identification of the organic acids in Angelicae Radix and Ligustici Rhizoma by HPLC and CE

Angelicae Radix (AR) and Ligustici Rhizoma (LR) are both derived from the Umbelliferae plants and contain similar organic acids as their bioactive compounds. Nine of these organic acids, including nicotinic acid, protocatechuic acid, phthalic acid, folinic acid, p-hydroxybenzoic acid, folic acid, vanillic acid, caffeic acid, and ferulic acid were separated by HPLC and CE. Detection at 210 nm with a linear gradient containing 20 mM KH2PO4 (pH 3.5) and H2O-CH3CN in HPLC and with a buffer solution containing 10 mM LTAC, 2 mM Na2HPO4, 9 mM Na2B4O7(pH 9.56), and CH3CN in CE were found to be the most efficient eluents for this separation. The contents of the nine components in crude extracts of either AR or LR could easily be determined within 60 min by LC and within 20 min by CE. The structures of the individual peaks in the LC chromatogram were identified by LC-MS. The effects of buffers on the separation and validation of the two methods were examined.     

Rapid Characterization of the major chemical constituents from Polygoni Multiflori Caulis by liquid chromatography tandem mass spectrometry and comparative analysis with Polygoni Multiflori Radix

Polygoni Multiflori Caulis is a traditional Chinese medicine that has been used for a long time to treat sleep disorders. However, the multiple chemical composition analysis has not been reported. In this study, a simple, rapid and effective ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was established to characterize the components of Polygoni Multiflori Caulis. In addition, a chemical comparative analysis was performed with Polygoni Multiflori Radix, another traditional Chinese medicine from the same plant, through multivariate statistical analysis and semi‐quantitative analysis to screen the difference in chemical ingredients between these two herbal medicines from same medicinal plant. A total of 33 peaks were detected within 25 min, and 28 of them were identified or tentatively characterized by comparing the retention time and mass spectrometry data. Based on the results, 12 characteristic components were screened out by multivariate statistical analysis, and their content change trends were compared by semi‐quantitative analysis.     

Rapid Screening of Lipase Inhibitors from Ophiopogonis Radix Using High-Performance Thin Layer Chromatography by Two Step Gradient Elution Combined with Bioautographic Method

In this study, a high-performance thin layer chromatography (HPTLC) method by two step gradient elution with two mobile phases was developed for the simultaneous analysis of seven constituents in Ophiopogonis Radix. The chromatography was performed on silica gel 60 F₂₅₄ plate with dichloromethane-methanol-ethyl acetate-water (70:25:12:3, v/v/v/v) and dichloromethane-methanol (300:1, v/v) as the mobile phase for two step gradient elution. Then, the HPTLC profiles were observed after derivatization with 10% sulfuric acid in ethanol solution. The obtained HPTLC images were further analyzed by chemometric approaches and the samples could be clustered based on regions and/or growth years, which were two important factors affecting the constituents in Ophiopogonis Radix. Furthermore, five compounds including ophiopogonin D, ophiopojaponin C, ophiopogonin D’, ophiopogonin C’ and methylophiopogonanone B were screened as potential lipase inhibitors from Ophiopogonis Radix by the HPTLC-bioautographic method. The binding modes and interactions between the five compounds and lipase were further explored by molecular docking analysis. The developed HPTLC method could be used for quality control of Ophiopogonis Radix and screening of the potential lipase inhibitors.     

HPLC法测定鳗鱼、猪肉和对虾中的痢特灵残留

在传统法的基础上，常量分析与微量分析技术有效地相结合，研究了测定动物性食品中痢特灵残留的快速ＨＰＬＣ法，用二氯甲烷提取样品中的痢特灵，浓缩提取液至干，８０％乙腈水溶液溶解残渣，在５ｍＬ离心管中用正已烷萃取、分离杂质，清液供ＨＰＬＣ分析用，方法回收率大于８０％，相对标准编差（ｎ＝６）小于８％。     

Identification and Simultaneous Determination of Mangiferin,Neomangiferin, Timosaponin A-III,and Timosaponin C in Rhizoma Anemarrhenae by Rapid Resolution Liquid Chromatography Coupled with Triple Quadrupole Mass Spectrometry

A new and rapid method was developed for simultaneous determination of mangiferin, neomangiferin, timosaponin A-III, and C in Rhizoma Anemarrhenae using rapid resolution liquid chromatography coupled with triple quadrupole mass spectrometry. The analysis was performed on an Eclipse Plus C₁₈ column (I.D. 4.6 × 100 mm, 3.5 μm). Electrospray ionization–tandem interface in the negative mode was employed prior to mass spectrometric detection. Quantitation was based on multiple reaction monitoring (MRM) for determination. Limits of detection (LODs) ranged from 0.7 to 3 pg. The average recoveries ranged from 98.16 to 100.7% with RSDs ≤ 2.03%. The established method was validated, sensitive, and reliable.     

A Ferrocene‐Tagged Amyloid‐β Fragment for Rapid Screening of Aggregation Inhibitors from Natural Compounds by HPLC‐Electrochemical Detection

Aggregates of amyloid beta (Aβ) peptides are believed to be responsible for the neuropathology of Alzheimer’s disease. In this work, ferrocene (Fc) is attached to the aggregating core of the Aβ peptides, KLVFFAE. Inhibition of Fc‐KLVFFAE aggregation by curcumin, a natural compound, was monitored by HPLC‐electrochemical detection (HPLC‐EC). The Fc oxidation current is dependent on the incubation condition and curcumin can retain Fc‐KLVFFAE in its monomeric form. We demonstrate that tagging Fc to KLVFFAE affords a cost‐effective and electroactive mimicry of Aβ(1? 42) and HPLC‐EC is suitable for sensitive, reproducible, and facile screening of drugs for inhibiting the aggregation of Aβ peptides.     

Xanthine Oxidase Inhibitors from Filipendula ulmaria (L.) Maxim. and Their Efficient Detections by HPTLC and HPLC Analyses

Filipendula ulmaria is a plant commonly used for the treatment of several pathologies, such as diarrhoea, ulcers, pain, stomach aches, fevers, and gout. Our study focused on the use of F. ulmaria for the treatment of gout disease. We first studied the chemical composition of a methanolic extract of the aerial parts and demonstrated its xanthine oxidase (XO) inhibitory activity. Then, we performed a fractionation and evaluated the most XO inhibitory active fractions by UV measurement. Purification of some fractions allowed the determination of the inhibitory activity of pure compounds. We demonstrated that spiraeoside, a glycosylated flavonoid, possesses an activity around 25 times higher than allopurinol, used as a reference in the treatment of gout disease. In order to easily and quickly identify potent inhibitors in complex matrix, we developed a complementary strategy based on an HPLC method and an Effect Directed Assay (EDA) method combining HPTLC and biochemical assays. The HPLC method, capable of determining compounds exhibiting interactions with the enzyme, could be an efficient strategy for evaluating potent enzyme inhibitors in a complex mixture. This strategy could be applied for quantitative assays using LC/MS experiments.     

Screening and identification of α‐glucosidase inhibitors from Shenqi Jiangtang Granule by ultrafiltration liquid chromatography and mass spectrometry

Shenqi Jiangtang Granule, a well‐known traditional Chinese herbal preparation, has been widely used for the treatment of type II diabetes mellitus. In this work, an ultrafiltration liquid chromatography with quadrupole time‐of‐flight mass spectrometry method was proposed for the rapid identification of bioactive ingredients from Shenqi Jiangtang Granule using α‐glucosidase as an example. First, the chemical profile of this preparation was clarified, including 37 saponins, 17 flavonoids, 37 lignans, and seven other compounds. After incubation with α‐glucosidase in vitro, the methanol extract with an IC₅₀ value of 0.19 mg/mL exhibited significant inhibitory activity. Then, 18 specific binding peaks were screened, and 15 peaks were identified. Among these, ten compounds were reported to have potential α‐glucosidase inhibitory activity for the first time. Subsequently, the inhibitory activities of these active compounds were evaluated by ultraviolet spectrophotometry with p‐nitrophenyl α‐d‐ glucopyranoside as a substrate. As a result, gomisin J and gomisin D exhibited stronger α‐glucosidase inhibitory activities than other active compounds with IC₅₀ values of 77.69 and 133.85 μM, respectively. The results demonstrated that the integrated ultrafiltration liquid chromatography with mass spectrometry method was an effective and powerful tool for the discovery of active ingredients in Shenqi Jiangtang Granule.     

Screening and isolation of cyclooxygenase‐2 inhibitors from the stem bark of Phellodendron amurense Ruprecht by ultrafiltration with liquid chromatography and tandem mass spectrometry,and complex chromatography

Nonsteroidal anti‐inflammatory drugs appear to reduce the risk of developing cancer. One mechanism through which nonsteroidal anti‐inflammatory drugs act to prevent carcinogenesis is inhibition of the activity of the enzyme cyclooxygenase‐2. The cyclooxygenase‐2 inhibitors are widely used to reduce the risk of developing cancer. Natural products are considered to be a promising source of several novel cyclooxygenase‐2 inhibitors. Ultrafiltration with liquid chromatography and mass spectrometry is an efficient method that can be applied to rapidly screen and identify the ligands from the barks of Phellodendron amurense Ruprecht. A continuous online method comprised of pressurized liquid extraction, countercurrent chromatography, and semi‐preparative liquid chromatography was developed for the efficient scaled‐up production of eight compounds with high purities. The bioactivities of the separated compounds were assessed by an in vitro enzyme inhibition assay. The use of bioactivity screening method combined with preparation method of bioactive compounds and an in vitro enzyme inhibition assay facilitated the efficient screening and isolation of the cyclooxygenase‐2 inhibitors from complex samples. This could be used as an efficient method for the large‐scale production of functional ingredients.     

Rapid screening,identification, and purification of neuraminidase inhibitors from Lithospermum erythrorhizon Sieb.et Zucc. by ultrafiltration with HPLC–ESI‐TOF‐MS combined with semipreparative HPLC

We put forward an efficient strategy based on bioassay guidance for the rapid screening, identification, and purification of the neuraminidase inhibitors from traditional Chinese medicines, and apply to the discovery of anti‐influenza components from Lithospermiun erythrorhizon Sieb.et Zucc. Ultrafiltration with high‐performance liquid chromatography and electrospray ionization time‐of‐flight mass spectrometry was employed for the rapid screening and preliminarily identification of anti‐influenza components from Zicao. Semipreparative high‐performance liquid chromatography was used for the rapid separation and purification of the target compounds. NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. Five compounds were found to have neuraminidase inhibitory activity by this method. Subsequently, the five compounds were separated by semipreparative high‐performance liquid chromatography with the purity over 98% for all of them by high‐performance liquid chromatography test. Combined with the NMR spectroscopy, mass spectrometry, and UV spectroscopy data, they were identified as alkannin, acetylalkannin, isobutyrylalkannin, β,β‐dimethylacryloylalkannin and isovalerylalkannin. The in vitro assay showed that all five compounds had good neuraminidase inhibitory activities. These results suggested that the method is highly efficient, and it can provide platform and methodology supports for the rapid discovery of anti‐influenza active ingredients from complex Chinese herbal medicines.