Screening of tyrosinase inhibitors by capillary electrophoresis with immobilized enzyme microreactor and molecular docking

A new method for screening tyrosinase inhibitors from traditional Chinese medicines (TCMs) was successfully developed by capillary electrophoresis with reliable online immobilized enzyme microreactor (IMER). In addition, molecular docking study has been used for supporting inhibition interaction between enzyme and inhibitors. The IMER of tyrosinase was constructed at the outlet of the capillary by using glutaraldehyde as cross‐linker. The parameters including enzyme reaction, separation of the substrate and product, and the performance of immobilized tyrosinase were investigated systematically. Because of using short‐end injection procedure, the product and substrate were effectively separated within 2 min. The immobilized tyrosinase could remain 80% active for 30 days at 4°C. The Michaelis–Menten constant of tyrosinase was determined as 1.78 mM. Kojic acid, a known tyrosinase inhibitor, was used as a model compound for the validation of the inhibitors screening method. The half‐maximal inhibitory concentration of kojic acid was 5.55 μM. The method was successfully applied for screening tyrosinase inhibitors from 15 compounds of TCM. Four compounds including quercetin, kaempferol, bavachinin, and bakuchiol were found having inhibitory potentials. The results obtained in this work were supported by molecular docking study.     

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.     

靶向亲和-液相色谱-质谱联用技术快速筛选黄藤总生物碱中乙酰胆碱酯酶抑制剂

采用靶向亲和-液相色谱-质谱联用技术(Target molecule affinity-LC-ESI-MSn)快速筛选黄藤总生物碱中能够抑制乙酰胆碱酯酶活性的成分,共筛选出12种具有潜在抑制乙酰胆碱酯酶的活性成分,并鉴定了6种成分,分别为黄藤素(Palmatine)、小檗碱(Berberine)、药根碱(Jatrorrhizine)、巴马汀红碱(Palmatrubine)、7,8-二氢-8-羟基小檗碱(7,8-Dihydro-8-hydroxyberberine)、Groenlandicine,结合体外酶学实验对这6种化合物进行了活性验证实验.结果表明,黄藤素抑制活性最强,其抑制作用强于阳性对照药盐酸多奈哌齐,说明黄藤素具有开发成抗阿尔茨海默症药物的潜力.本方法简单、快速、准确地从复杂的中药提取物中筛选出具有抑制乙酰胆碱酯酶活性的成分,适用于复杂体系中的高通量筛选.     

Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis–Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half‐maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors.     

Screening of lipase inhibitors in Folium Mori with lipase‐linked magnetic microspheres by high‐performance liquid chromatography and evaluation in diabetic mice

A new method for the screening of compounds with hypoglycemic effect from traditional Chinese medicines employing high‐performance liquid chromatography and lipase‐linked magnetic microspheres has been proposed. We hypothesized that the interaction of traditional Chinese medicine extracts with lipase‐linked magnetic microspheres should decrease the concentration of compounds with hypoglycemic effect. Using this approach, the potential lipase inhibitors in Folium Mori extract were investigated. First, lipase was immobilized on magnetic microspheres by a chemical method. Then, by comparing the chromatograms of samples before and after the interaction with lipase‐linked magnetic microspheres, seven compounds of Folium Mori extract were identified. It was found that protocatechuic acid, chlorogenic acid, protocatechualdehyde, rutin, isoquercitrin, astragalin, and dicaffeoylquinic acid B had evident combination with lipase‐linked magnetic microspheres. Their hypoglycemic effects were verified in streptozocin‐induced diabetic mice. In the present study, astragalin was verified to improve the glucose tolerance and lower the level of glucose in streptozocin‐induced diabetic mice, which indicated that astragalin might be a new highly efficient lipase inhibitor. Based on these significant results, this method could be a convenient approach to screen potential lipase inhibitors from traditional Chinese medicines. Meanwhile, it also could be expanded to screen other active compounds in traditional Chinese medicines.     

磁珠富集与液相色谱-质谱联用结合筛选枳壳中脂肪酶抑制剂

提出了一种磁珠收集与液相色谱-质谱联用(LC-MS)结合用于快速筛选中药提取物中的脂肪酶抑制剂的方法. 通过制备键合脂肪酶的磁珠, 将其与枳壳总黄酮孵育后, 筛选出枳壳中脂肪酶的配体. 通过LC-MS分析发现, 4个化合物均是潜在的脂肪酶抑制剂, 鉴定其分别为柚皮苷、 新橙皮苷、 橙皮苷和枸橘苷. 对4个化合物进行了体外脂肪酶抑制活性验证. 研究结果表明, 新橙皮苷、 橙皮苷和枸橘苷具有显著的脂肪酶抑制活性, IC₅₀分别为48.04, 52.45和46.18 μg/mL, 其中枸橘苷具有脂肪酶抑制活性为首次报道. 结果表明, 磁珠富集与LC-MS集成技术能够用于快速发现中药活性成分.     

Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography

A simple, rapid, and effective assay based on ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target‐guidance of ultrafiltration combined with the high‐performance liquid chromatography experiment, liquiritin ( 1 ), isoliquiritin ( 2 ), and liquiritigenin ( 3 ) were separated by high‐speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC₅₀ of 8.95 μM. The results of the present study indicated that the combinative method using ultrafiltration, high‐performance liquid chromatography and high‐speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.     

A strategy for screening trypsin inhibitors from traditional Chinese medicine based on a monolithic capillary immobilized enzyme reactor coupled with offline liquid chromatography and mass spectrometry

A novel strategy was successfully developed for screening trypsin inhibitors in traditional Chinese medicines based on monolithic capillary immobilized enzyme reactors combined with liquid chromatography‐tandem mass spectrometry. Organic polymer based monolithic enzyme reactors were firstly prepared by covalently bonding trypsin to a poly(glycidyl methacrylate‐co‐poly (ethylene glycol) diacrylate) monolith by the ring‐opening reaction of epoxy groups. The activity and kinetic parameters of the obtained monolithic trypsin reactors were systematically evaluated using micro‐liquid chromatography. Fourier transform infrared spectroscopy and scanning electron microscopy were also used to characterize the monolithic trypsin reactors. The resulting functional and denatured monolithic trypsin reactors were applied as affinity solid‐phase extraction columns, and offline coupled with a liquid chromatography‐tandem mass spectrometry system to construct a binding affinity screening platform. Subsequently, the proposed platform was applied for screening trypsin binders in a Scutellaria baicalensis Georgi extract. Three compounds, namely scutellarin, baicalin, and wogonoside were identified, and their inhibitory activities were further confirmed via an in vitro enzymatic inhibition assay. Additionally, molecular docking was also performed to study the interactions between trypsin and these three compounds.     

微透析/液相色谱-质谱联用筛选黄芩中α-葡萄糖苷酶抑制剂

应用微透析/液相色谱-质谱联用法筛选黄芩中α-葡萄糖苷酶抑制剂,并对筛选出的具有酶抑制活性的化合物进行结构鉴定。实验采用迪马C18色谱柱(150 mm×4.6 mm,5μm),以0.5%醋酸水-乙腈溶液为流动相,梯度洗脱,流速0.5 mL/min,柱温30℃,以正离子电喷雾质谱模式采集数据,从黄芩中筛选出10种潜在的α-葡萄糖苷酶抑制剂,10种化合物与α-葡萄糖苷酶的结合率为36.18%~52.71%。该方法可快速、有效地筛选黄芩中的α-葡萄糖苷酶的抑制剂,同时可为从其他中药中筛选酶抑制剂提供方法学借鉴。     

Dopamine-polyethyleneimine co-deposition of a capillary for α-glucosidase immobilization and its application in enzyme inhibitor screening

An online method based on CE was established to screen α-glucosidase inhibitors from traditional Tibetan medicine extracts. First, the inner wall at the inlet of capillary column was simply and effectively functionalized by dopamine-polyethyleneimine co-deposition method, which combines the adhesion property of dopamine and easy cationization of polyethyleneimine. Then α-glucosidase was rapidly immobilized on the inner wall of the capillary column by electrostatic adsorption. The inter- and intraday repeatability of the peak area of the enzymatic reaction product (p-nitrophenol) in a capillary was evaluated, and RSD% (n = 3) was 0.94% and 1.09%, respectively. Good batch-to-batch reproducibility of the peak area between different capillaries (RSD = 2.1%, n = 5) shows that the preparation method has good reproducibility. The Michaelis–Menten constant of the immobilized α-glucosidase was measured to be 1.18 mM, and the capillary column enzyme reactor retained 85.9% of initial activity after 30 cycles. Finally, it was applied to the screening of enzyme inhibitors in 20 traditional Tibetan medicine extracts. Sixteen medicines with inhibitory activity were screened out, and Rheum australe had the strongest inhibitory effect with an inhibitory rate of 83.3 ± 0.4%. These results showed that this method is effective to find potential enzyme inhibitors.     

Immobilized capillary adenosine deaminase microreactor for inhibitor screening in natural extracts by capillary electrophoresis

A novel strategy for the preparation of in-column adenosine deaminase (ADA) microreactor and rapid screening of enzyme inhibitors in natural extracts was demonstrated. In this approach, ADA was encapsulated in anionic polyelectrolyte alginate that was immobilized on the surface of fused-silica capillary via ionic binding technique with cationic polyelectrolyte polyethylenimine (PEI). On-line enzyme inhibition study was performed by capillary electrophoresis (CE). The substrate and product were baselined separated within 75 s. The enzyme activity was determined by the quantification of peak area of the product. Enzyme inhibition can be read out directly from the reduced peak area of the product in comparison with a reference electropherogram obtained in the absence of any inhibitor. The inhibition percentage was used to evaluate relative activity of ADA microreactor. A known ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) was employed as a model compound for the validation of the inhibitor screening method, and the screening of ADA inhibitor in 19 traditional Chinese herbal medicines was performed.     

Screening of aromatase inhibitors in traditional Chinese medicines by electrophoretically mediated microanalysis in a partially filled capillary

An electrophoretically mediated microanalysis method with partial filling technique was developed for screening aromatase inhibitors in traditional Chinese medicine. The in‐capillary enzymatic reaction was performed in 20 mM sodium phosphate buffer (pH 7.4), and sodium phosphate buffer (20 mM, pH 8.0) was used as a background electrolyte. A long plug of coenzyme reduced β‐nicotinamide adenine dinucleotide 2′‐phosphate hydrate dissolved in the reaction buffer was hydrodynamically injected into a fused silica capillary followed by the injection of reaction buffer, enzyme, and substrate solution. The reaction was initiated with a voltage of 5 kV applied to the capillary for 40 s. The voltage was turned off for 20 min to increase the product amount and again turned on at a constant voltage of 20 kV to separate all the components. Direct detection was performed at 260 nm. The enzyme activity was directly assayed by measuring the peak area of the produced β‐nicotinamide adenine dinucleotide phosphate and the decreased peak area indicated the aromatase inhibition. Using the Lineweaver–Burk equation, the Michaelis–Menten constant was calculated to be 50 ± 4.5 nM. The method was applied to the screening of aromatase inhibitors from 15 natural products. Seven compounds were found to have potent AR inhibitory activity.     

Screening of new non-nucleoside reverse transcriptase inhibitors of HIV-1 based on traditional Chinese medicines database

HIV-1 RT is an important target for the treatment of AIDS.There are two major classes of antiviral agents that inhibit HIV-1 RT have been identified,nucleoside RT inhibitors(NRTIs) and non-nucleoside RT inhibitors(NNRTIs).In this report,a noval class of non-nucleoside compound with potential RT inhibitory activity were found from the traditional Chinese medicines database (TCMD) using a combination of virtual screening,docking,molecular dynamic simulations,where results were ranked by scoring function of...     

Practical method for the rapid screening of xanthine oxidase inhibitors in herbal extracts by high‐performance liquid chromatography based on on‐line precolumn enzymatic reaction

A high‐performance liquid chromatography method with on‐line precolumn enzymatic reaction for the screening of xanthine oxidase inhibitors in natural extracts was developed. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, after which the reaction product, uric acid, was separated and detected by liquid chromatography using ultraviolet absorption at 295 nm. Enzyme inhibition can be read out directly from the reduced peak area of uric acid in comparison to a reference chromatogram obtained in the absence of any inhibitor. In the present study, the availability of on‐line precolumn enzymatic reaction with ultraviolet detection was firstly evaluated by determining the inhibitory mechanism and IC₅₀ values of allopurinol, a commercially available positive drug. Then, the newly developed method was applied to screening of ten natural extracts from traditional Chinese medicine and as a result, the extract of Epimedium sagittatum (Sieb. et Zucc.) Maxim was found to be most positive for xanthine oxidase inhibition. The results obtained were compared with those obtained by offline enzyme assay and the effectiveness of the present method was confirmed. A rapid, low‐cost, and fully automated method for xanthine oxidase inhibitor screening was proposed.     

Screening neuraminidase inhibitors from glycosaminoglycan and natural extract by capillary electrophoresis

An electrophoretically mediated microanalysis (EMMA) method for screening neuraminidase inhibitors in depolymerized glycosaminoglycan and natural extracts is described. In the present method, enzyme and substrate were individually introduced into the capillary as distinct plugs, and then mixed for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract. The measured peak area of the product at 214 nm represents the enzyme activity. The electrophoretic conditions for the enzyme reaction and separation of substrate and product were optimized in this study. Under the optimal conditions, the Michaelis–Menten constant and the inhibitive mechanism of zanamivir were studied, which agreed with the literature data. Furthermore, the inhibitory ratios of enzymatic activity of depolymerized glycosaminoglycan and traditional Chinese drugs were determined. The EMMA method has superiority over traditional assay methods, in not only minimizing the false-positive results but also in simplifying the experimental procedure. Therefore, it could be employed to screen inhibitors from natural sources.     

前体离子扫描法在快速筛查中药及保健食品中非法添加的5型磷酸二酯酶抑制剂及其未知衍生物中的应用

改造有明确疗效的药物,合成新的衍生物以避开法定检验方法是目前化学药物非法添加的趋势之一。本文提出将液相色谱-三重四极杆质谱联用仪的质谱前体离子扫描模式应用于中药及保健食品等复杂体系中非法添加药物衍生物的快速筛查策略,以5型磷酸二酯酶抑制剂为实验对象,通过分析该类化合物的结构和质谱特点将其分类,筛选各类共有的子离子碎片,优化质谱参数,建立了前体离子扫描模式的LC-MS筛查方法,讨论了质谱参数和碎片离子的选择对筛选结果的影响,并应用于实际样品的测定。结果表明,该方法既可以满足已知化合物的测定需要,又可以对复杂体系中未知的同类衍生物进行快速筛查,防止未知衍生物的漏检。该方法灵敏、专属、高效,值得进一步研究。     

Simultaneous determination of six α‑dicarbonyl compounds in traditional Chinese medicines using high-performance liquid chromatography-fluorescence detector with pre-column derivatization

A reliable method for determination of six α-dicarbonyl compounds in traditional Chinese medicines was first developed and validated by high-performance liquid chromatography-fluorescence detector with pre-column derivatization. α-Dicarbonyl compounds in traditional Chinese medicines were extracted and derivatized with 2,3-diaminaphthalene. The derivatization procedure of six α-dicarbonyl compounds was confirmed by high-resolution mass spectrometry. The limits of quantitation for six α-dicarbonyl compounds ranged from 3.70 × 10⁻³ to 2.21 × 10⁻² μM. The established method showed good linearity (regression coefficient > 0.9990), precision (relative standard deviation < 3.37%), and high recovery (97.8%∼113.1%). The developed method was successfully applied to detect the six α-dicarbonyl compounds in traditional Chinese medicines. The result exhibited six α-dicarbonyl compounds was found in the 15 kinds of traditional Chinese medicines, which suggested us that the determination of α-dicarbonyl compounds should be paid more attention in the quality control of traditional Chinese medicines.     

A platform for fast screening potential anti‐breast cancer compounds in traditional Chinese medicines

Several Chinese herbs, namely, Pu‐Gong‐Ying, Gan‐Cao, Chai‐Hu, Mu‐Xiang, Gua‐Lou and Huang‐Yao‐Zi, are frequently used in complex traditional Chinese medicing formulas for breast hyperplasia and breast tumor therapy. The pharmacological effects of these Chinese herbs are all described as ‘clearing heat‐toxin and resolving masses’ in traditional use. However, the chemical profiles of anti‐breast cancer constituents in these herbs has not been investigated so far. In this study, a bioactivity‐oriented screening platform, which was based on a human breast cancer MCF‐7 cellular model, semi‐preparative high performance liquid chromatography coupled with ultraviolet spectrophotometry and ultraperformance liquid chromatography coupled to quadrupole‐time‐of‐flight mass spectrometer, was developed to rapidly screen the six Chinese herbs. Two potential anti‐breast cancer compounds, which were costunolide (Cos) and dehydrocostus lactone (Dehy), were identified in Mu‐Xiang. Combination of the two compounds showed a synergism on inhibiting the proliferation of MCF‐7 cells in vitro, which exhibits a potential application prospect for breast cancer therapy. This bioactivity‐oriented screening strategy is rapid, economical, reliable and specific for screening potential anti‐breast cancer compounds in traditional Chinese medicines. Copyright © 2013 John Wiley & Sons, Ltd.     

Exploration of Antimicrobial Ingredients in Psoralea corylifolia L. Seed and Related Mechanism against Methicillin-Resistant Staphylococcus aureus

With the abuse of antibiotics, bacterial antibiotic resistance is becoming a major public healthcare issue. Natural plants, especially traditional Chinese herbal medicines, which have antibacterial activity, are important sources for discovering potential bacteriostatic agents. This study aimed to develop a fast and reliable method for screening out antimicrobial compounds targeting the MRSA membrane from Psoralea corylifolia Linn. seed. A UPLC-MS/MS method was applied to identify the prenylated flavonoids in major fractions from the extracts of Psoralea corylifolia Linn. seed. The broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of different fractions and compounds. The morphological and ultrastructural changes of MRSA were determined by scanning electron microscopy (SEM). The membrane-targeting mechanism of the active ingredients was explored by membrane integrity assays, membrane fluidity assays, membrane potential assays, ATP, and ROS determination. We identified eight prenylated flavonoids in Psoralea corylifolia Linn. seed. The antibacterial activity and mechanism studies showed that this type of compound has a unique destructive effect on MRSA cell membranes and does not result in drug resistance. The results revealed that prenylated flavonoids in Psoralea corylifolia Linn. seeds are promising candidates for the development of novel antibiotic agents to combat MRSA-associated infections.     

Pseudotargeted screening and determination of constituents in Qishen granule based on compound biosynthetic correlation using UHPLC coupled with high‐resolution MS

Detection and determination of many known/unknown compounds in traditional Chinese medicines have always been challenging. To comprehensively identify compounds in Qishen granule, which is a widely prescribed herbal formula for treating chronic heart failure, a pseudotargeted screening method was proposed based on compound biosynthetic correlation using ultra high‐performance liquid chromatography coupled with high‐resolution mass spectrometry. Firstly, all possible compounds of Qishen granule were classified into nine types according to their core skeletons, and potential analogue molecular formulas were predicted according to core compound‐related biosynthetic correlations, such as methylation, hydroxylation, and glucosidation. Secondly, nine pseudocompound databases consisting of core compounds, deduced biosynthetic correlations, and predicted analogue molecular formulas were established. Then, compounds of interest were directly located by pseudotargeted screening of high resolution mass spectrometry data and further verified by target tandem mass spectrometry. As a result, 213 constituents were identified and 21 of them were determined as potential new compounds. This demonstrated that pseudotargeted screening based on compound biosynthetic correlations significantly facilitated the processing of extremely large information data and improved the efficiency of compound identification. This research provided essential data for exploration of effective substances in Qishen granule and enriched the methodology for comprehensive characterization of constituents in complex traditional Chinese medicines.