Screening and separation of α‐amylase inhibitors from Solanum nigrum with amylase‐functionalized magnetic graphene oxide combined with high‐speed counter‐current chromatography

A screening method using α‐amylase‐functionalized magnetic graphene oxide combined with high‐speed counter‐current chromatography was proposed and utilized to screen and separate α‐amylase inhibitors from extract of Solanum nigrum . The α‐amylase‐functionalized magnetic graphene oxide was characterized and found to demonstrate satisfactory structure, magnetic response (24.5 emu/g), and reusability (retained 90% of initial activity after five cycles). The conditions for the screening with α‐amylase functionalized magnetic graphene oxide were optimized and set at pH 7.0 and 25°C. As a result, two potent flavonoid compounds, apigenin‐7‐O‐glucuronide ( 1 ) and astragalin ( 2 ), were separated and collected through high‐speed counter‐current chromatography and subjected to high‐performance liquid chromatography analysis with purity higher than 90% (according to HPLC data), which were identified as α‐amylase inhibitors. These results suggested that utilization of α‐amylase functionalized magnetic graphene oxide in the rapid screening and isolation bioactive compounds from complex natural products is a feasible and environmentally friendly method.     

亲和色谱法筛选中药中血管紧张素转化酶抑制剂

以壳聚糖微球为载体、戊二醛（glutaraldehyde,GA）为交联剂对血管紧张素转化酶（Angiotensin converting enzyme,ACE）进行固定化．用固化的ACE作为亲和介质,利用血管紧张素转化酶抑制剂（Angiotensin convertingenzym einhibitor,ACEI）与ACE之间的亲和作用,结合高效液相色谱对亲和前后的体系进行检测,比较两者各组分色谱峰的差异,以此实现快速筛选复杂体系中的ACE抑制剂．应用赖诺普利（Lisinopril）、九肽抑制剂、依那普利（Enalapril）、培哚普利（Perindopril）、卡托普利（Captopril）等已上市的ACEI对方法进行验证,反映方法具有高度选择性．将方法应用于中药地龙及山楂筛选,发现共有5个组分与ACE有亲和作用,并且都能抑制ACE酶活性,它们对酶活性抑制的IC50值在0．45～4．62μg／mL范围．通过对亲和方法重现性考察,6次测定的相对标准偏差小于1％,说明方法可靠．提出的亲和色谱．色谱指纹差异法非常适合于从中药及天然产物等复杂混合物库中快速筛选靶点活性物质．     

Tyrosinase inhibitor screening in traditional Chinese medicines by electrophoretically mediated microanalysis

A capillary‐electrophoresis‐based method for the screening of tyrosinase inhibitors in traditional Chinese medicines was developed. The method integrated electrophoretically mediated microanalysis with sandwich mode injection, partial filling, and rapid polarity switching techniques, and carried out on‐column enzyme reaction and the separation of substrate and product. The conditions were optimized including the background electrolyte, mixing voltage, and the incubation time. Finally, the screening of nine standard natural compounds of traditional Chinese medicines was carried out. The inhibitors can be directly identified from the reduced peak area of the product compared to that obtained without any inhibitor. Chlorogenic acid (100 μM) showed inhibitory activity with the inhibitory percentage of 19.8%, while the other compounds showed no inhibitory activity. This method has great application potential in drug discovery from traditional Chinese medicines.     

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.     

Screening free radical scavengers in Xiexin Tang by HPLC‐ABTS‐DAD‐Q‐TOF/MS

Xiexin Tang (XXT) is a traditional Chinese medicine (TCM) that has been used in herbal clinics for more than 1800 years. Many studies have shown that XXT has therapeutic effects on patients with arteriosclerosis owing to its antioxidant activity. However, there is little information about the relationship between the chemical composition of XXT and its antioxidant activity. In this study, the HPLC‐ABTS‐DAD‐Q‐TOF/MS method, which can simultaneously identify individual components and rapidly screen for antioxidant compounds, was used to screen and identify antioxidant components in XXT. The 15 compounds identified were gluco‐syringic acid, adenine, gallic acid, biflorin, cularine, 6‐C ‐arabinose‐8‐C ‐glucose‐chrysin, 6‐C ‐glucose‐8‐C ‐arabinose–chrysin, baicalin, rhein‐8‐O‐β ‐d ‐glucopyranoside, coptisine, epiberberine, jatrorrhizine, norwogonin, 5,7,2′‐trihydroxy‐6‐ methoxyflavone and baicalein. In addition, the data showed that the antioxidant activity of peaks 4, 6, and 11 was lower in XXT than in its constituent herbs, while the activity of peaks 1, 2, 3, 5, 7, 8, 10, 12, 13, 14 and 15 was higher in XXT. Compound 5 had the strongest antioxidant activity in XXT, while compound 1 showed the strongest antioxidant activity among its constituent herb. The differences between antioxidant activities of major components of XXT and those of its constituent herbs might be due to the interaction of crude drugs that changes the solubility of active components during the decoction process. The results show that the HPLC‐ABTS‐DAD‐Q‐TOF/MS method can successfully combine on‐line mass spectrometry with activity detection system. It is a useful tool for the rapid detection and identification of antioxidants, and for quantitative analysis of individual antioxidants in complex mixtures such as plant extracts. Furthermore, this method does not require extensive extract purification and fraction collection.     

Chinese hamster ovary‐sphingomyelin synthase2 biospecific extraction and liquid chromatography with tandem mass spectrometry analysis for the prediction of bioactive components of Rhizoma Polygoni Cuspidati

A novel strategy for predicting bioactive components in traditional Chinese medicines using Chinese hamster ovary‐sphingomyelin synthase2 (CHO‐SMS₂) cell biospecific extraction and high‐performance liquid chromatography with diode array detection and tandem mass spectrometry analysis was proposed. The hypothesis is that when cells are incubated with the extract of traditional Chinese medicines, the potential bioactive components in the traditional Chinese medicines should selectively combine with the cells, while the cell‐combining components would be detectable in the extract of denatured cells. The identities of the cell‐combining components could be determined by liquid chromatography with tandem mass spectrometry. Using the proposed approach, the potential bioactive components of Rhizoma Polygoni Cuspidati, a commonly used traditional Chinese medicine for atherosclerosis, were detected and identified. Eight compounds in the extract of Rhizoma Polygoni Cuspidati were detected as the components selectively combined with CHO‐SMS₂ cells, which is a stable cell line that highly expresses sphingomyelin synthases, it was found that piceid, resveratrol, emodin‐8‐β‐d‐ glucoside, physcion‐8‐β‐d‐ glucoside, emodin, physcion, 3,5,4‘‐trihydroxystilbene‐3‐O‐(6“‐galloyl)‐glucoside, and emodin‐1‐O‐glucoside combined specifically with CHO‐SMS₂ cells. The results indicate that the proposed approach may be applied to predict the bioactive candidates 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.     

Chemical profiling approach to evaluate the influence of traditional and simplified decoction methods on the holistic quality of Da‐Huang‐Xiao‐Shi decoction using high‐performance liquid chromatography coupled with diode‐array detection and time‐of‐flight mass spectrometry

Da‐Huang‐Xiao‐Shi decoction, consisting of Rheum officinale Baill, Mirabilitum, Phellodendron amurense Rupr. and Gardenia jasminoides Ellis, is a traditional Chinese medicine used for the treatment of jaundice. As described in “Jin Kui Yao Lue”, a traditional multistep decoction of Da‐Huang‐Xiao‐Shi decoction was required while simplified one‐step decoction was used in recent repsorts. To investigate the chemical difference between the decoctions obtained by the traditional and simplified preparations, a sensitive and reliable approach of high‐performance liquid chromatography coupled with diode‐array detection and electrospray ionization time‐of‐flight mass spectrometry was established. As a result, a total of 105 compounds were detected and identified. Analysis of the chromatogram profiles of the two decoctions showed that many compounds in the decoction of simplified preparation had changed obviously compared with those in traditional preparation. The changes of constituents would be bound to cause the differences in the therapeutic effects of the two decoctions. The present study demonstrated that certain preparation methods significantly affect the holistic quality of traditional Chinese medicines and the use of a suitable preparation method is crucial for these medicines to produce special clinical curative effect. This research results elucidated the scientific basis of traditional preparation methods in 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.     

HPLC with quadrupole TOF‐MS and chemometrics analysis for the characterization of Folium Turpiniae from different regions

Folium Turpiniae has been used as a traditional Chinese medicine for the treatment of abscesses, fevers, gastric ulcers, and inflammations. This paper describes a strategy of combining HPLC with photodiode array detection and quadrupole TOF‐MS, as well as phytochemical and chemometrics analysis for the characterization, isolation, and simultaneous quantification of the chemical constituents of Folium Turpiniae. 19 constituents were identified, namely, 11 flavonoids, seven gallic acid derivates, and quinic acid. Among them, 15 compounds were identified in this herbal medicine for the first time; compound 10 appears to be novel and was isolated and confirmed as ellagic acid‐3‐O‐α‐l ‐rhamnopyranoside by NMR spectroscopy and MS. In addition, nine marker compounds, namely gallic acid ( 2 ), ellagic acid‐3‐O‐α‐l ‐rhamnopyranoside ( 10 ), apigenin‐7‐O‐(2′′‐rhamnosyl)gentiobioside ( 11 ), ellagic acid ( 12 ), luteolin‐7‐O‐β‐d ‐neohesperidoside ( 13 ), ligustroflavone ( 14 ), 4′‐O‐methylellagic acid‐3‐O‐α‐l ‐rhamnopyranoside ( 16 ), rhoifolin ( 17 ), and neobudofficide ( 18 ), were quantified simultaneously in ten batches of Folium Turpiniae collected from different regions. Moreover, hierarchical clustering analysis and principal component analysis indicated that both samples from Hubei ( S1 ) and Guangxi ( S3 ) provinces showed apparent differences from the others. Samples from Jiangxi province ( S2 , S4 , and S7–10 ) possessed similar properties and therefore belong to the same group.     

Decyl‐perfluorinated magnetic mesoporous microspheres for extraction and analysis perfluorinated compounds in water using ultrahigh‐performance liquid chromatography–mass spectrometry

In this work, the interior‐walls decyl‐perfluorinated functionalized magnetic mesoporous microspheres (F₁₇–Fe₃O₄@mSiO₂) were synthesized for the first time, and applied as adsorbents to extract and concentrate perfluorinated compounds (PFCs) from water samples. The fluorous functionalized interior pore‐walls contributed to the high‐selective preconcentration of PFCs due to fluorous affinity; and abundant silanol groups on the exterior surface of microspheres contributed to the good dispersibility in water sample. Four kinds of PFCs were selected as model analytes, including perfluorooctanoic acid, perfluorononanoic acid, perfluorododecanoic acid, and perfluorooctane sulphonate. In addition, UHPLC‐ESI/MS/MS was introduced to the fast and sensitive detection of the analytes after sample pretreatment. Important parameters of the extraction procedure were optimized, including salinity, eluting solvent, the amount of F₁₇–Fe₃O₄@mSiO₂ microspheres, and extraction time. The optimized procedure took only 10 min to extract analytes with high recoveries and merely 800‐μL acetonitrile to elute analytes from the magnetic adsorbents. Validation experiments showed good linearity (0.994–0.998), precision (2.6–7.6%), high recovery (93.4–105.7%) of the proposed method, and the limits of detection were from 0.008 to 0.125 μg/L. The F₁₇–Fe₃O₄@mSiO₂ magnetic microspheres have the advantages of great dispersibility in aqueous solution, high specificity of extraction, large surface area, and efficient separation ability. The results showed that the proposed method based on F₁₇–Fe₃O₄@mSiO₂ microspheres is a simple, fast, and sensitive tool for the analysis of PFCs in water sample.     

Comprehensive characterization and quantification of multiple components in Dan‐Huang‐Qu‐Yu capsule using a multivariate data processing approach based on microwave‐assisted extraction with UHPLC and Q Exactive quadrupole‐orbitrap high‐resolution mass spectrometry

Dan‐Huang‐Qu‐Yu capsule, a Chinese herbal medicine compound preparation, is widely used for chronic pelvic inflammatory disease. In this study, a rapid, selective, and sensitive microwave‐assisted extraction ultra‐high‐performance liquid chromatography‐Q Exactive quadrupole‐orbitrap high‐resolution mass spectrometry method was developed for analyzing its chemical compositions. A total of 85 compounds, including 22 flavonoids, 8 terpenoids, 5 quinones, 5 phthaleolactone, 23 organic acids, and 22 other compounds were identified from Dan‐Huang‐Qu‐Yu capsule. Among them, 35 major compounds were unambiguously detected by comparing them with reference standards and selected as quality control markers, which were simultaneously determined in Dan‐Huang‐Qu‐Yu capsule. The established method was successfully validated and applied for simultaneous determination of 35 bioactive compounds in Dan‐Huang‐Qu‐Yu capsule from ten sample batches. The quantitative data of the analytes were analyzed by principal component analysis for quality assessment of Dan‐Huang‐Qu‐Yu capsule. Six compounds (e.g., astragaloside IV, salvianolic acid B, ellagic acid, chlorogenic acid, N‐butylidenephthalide, and luteolin) were screened out and regarded as chemical markers for quality control of Dan‐Huang‐Qu‐Yu capsule. The established method has been proved to be a novel and useful tool for rapid research of Dan‐Huang‐Qu‐Yu capsule. This research will provide reference for the scientific research of traditional Chinese medicines.     

Development of an enzyme‐linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti‐glycyrrhizic acid monoclonal antibody

In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti‐glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid–bovine serum albumin conjugate with the hypoxanthine–aminopterin–thymidine‐sensitive mouse myeloma cell line (Sp2/0‐Ag14). Subsequently, an indirect, competitive enzyme‐linked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12–2500 ng/mL. Both intra‐assay and inter‐assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine.     

Bioactivity‐integrated ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry for the identification of nuclear factor‐κB inhibitors and β2 adrenergic receptor agonists in Chinese medicinal preparation Chuanbeipipa dropping pills

A simple and dual‐target method based on ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry combined with dual‐bioactive [nuclear factor‐κB (NF‐κB) and β₂‐adrenergic receptor] luciferase reporter assay systems was developed to rapidly characterize the chemical structure of various bioactive compounds of TCM preparations. Chuanbeipipa dropping pills, a traditional Chinese medicine preparation used for the clinical therapy of chronic obstructive lung disease and cough caused by bronchial catarrh, was analyzed with this method. Potential anti‐inflammatory and spasmolytic constituents were screened using NF‐κB and β₂‐adrenergic receptor activity luciferase reporter assay systems and simultaneously identified according to the time‐of‐flight mass spectrometry data. One β₂‐adrenergic receptor agonist (ephedrine) and two structural types of NF‐κB inhibitors (platycosides derivatives and ursolic acid derivatives) were characterized. Platycodin D3 and E were considered new NF‐κB inhibitors. Further cytokine and chemokine detection confirmed the anti‐inflammatory effects of the potential NF‐κB inhibitors. Compared with conventional fingerprints, activity‐integrated fingerprints that contain both chemical and bioactive details offer a more comprehensive understanding of the chemical makeup of plant materials. This strategy clearly demonstrated that multiple bioactivity‐integrated fingerprinting is a powerful tool for the improved screening and identification of potential multi‐target lead compounds in complex herbal medicines. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparative analysis of the main active components and hypoglycemic effects after the compatibility of Scutellariae Radix and Coptidis Rhizoma

In this study, a rapid and highly sensitive ultra high performance liquid chromatography with triple quadrupole mass spectrometry method with the mobile phase of acetonitrile and 0.1% aqueous formic acid was established and successfully applied to comparatively analyze main active components after their compatibility. Besides, the effects of Scutellariae Radix, Coptidis Rhizoma and combined extracts on type 2 diabetic rats induced by high‐fat diet along with low dose of streptozocin were investigated. Under the optimized chromatographic conditions, good separation of seven target components was achieved within 12 min. All calibration curves exhibited good linearity (R² ≥ 0.999). The relative standard deviation of precision, repeatability and stability varied from 0.69 to 2.23, 0.98 to 2.56, and 0.92 to 2.57%, respectively. The recovery ranged from 91.11 to 105.35%. The contents of seven active components were notably reduced after compatibility; however, the hypoglycemic effect of combined extracts was stronger than single drug by decreasing the activities of fructose‐1,6‐bisphosphatase, glucose 6‐phosphatase, phosphoenolpyruvate carboxykinase and increasing the activities of glucokinase, phosphofructokinase, pyruvate kinase. Accordingly, the established analytical method was accurate and sensitive enough for quantitative evaluation of seven investigated compounds. Moreover, the combined extract had definite effects on type 2 diabetes through multiple components against multiple targets.     

Systematic study on metabolism and activity evaluation of Radix Scutellaria extract in rat plasma using UHPLC with quadrupole time‐of‐flight mass spectrometry and microdialysis intensity‐fading mass spectrometry

Radix Scutellaria is a widely used traditional Chinese medicine in the treatment of various diseases. However, the activities of the absorbed components and metabolites of its main flavones in rat plasma need further investigation. In this study, a systematic method based on ultra‐high performance liquid chromatography with quadruple time‐of‐flight mass spectrometry was developed to speculate the absorbed components and metabolites of the main flavonoids in Radix Scutellaria extract in rat plasma sample after oral administration of the extract. Twelve compounds, including four prototype components and eight metabolites, were confirmed in drug‐containing plasma. In these metabolites, five were originally detected in rat plasma. The possible metabolic pathways of these polyhydroxy flavones in vivo were described and clarified. Microdialysis with intensity‐fading mass spectrometry was originally employed to investigate the binding affinities of the absorbed components and metabolites with α‐glucosidase. The order of their binding affinities was P4 > P3 > P2 > P1≥M5 > M3 > M1. The research result is helpful to deepen the understanding of the absorbed components and metabolic pathways of main flavones from Radix Scutellaria, and provide a new approach to screen potential inhibitors from in vivo components originated from Chinese herb.     

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.     

Rapid screening and identification of α-glucosidase inhibitors from mulberry leaves using enzyme-immobilized magnetic beads coupled with HPLC/MS and NMR

α-Glucosidase plays important roles in the digestion and absorption of carbohydrates in the small intestine. The inhibition of α-glucosidase is regarded as a potential way to treat diabetes. We established an approach to screening α-glucosidase inhibitors from medicinal plants using enzyme-coated magnetic bead. Using 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide as reaction reagents, α-glucosidase was immobilized on the magnetic beads by covalent linkage. The conjugation of α-glucosidase to the magnetic beads was characterized using scanning electron microscope and X-ray diffractometer. The proposed approach was applied in fishing potential α-glucosidase inhibitors from extract of Morus alba, a Chinese medicinal plant. The structures of potential active compounds were identified via liquid chromatography-mass spectrometry and nuclear magnetic resonance. The results demonstrated that two flavonoids (isoquercitrin and astragalin) could bind to α-glucosidase, which was confirmed via conventional α-glucosidase inhibitory assay. Our findings suggested that enzyme-coated magnetic beads may be suitable for discovering active compounds from medicinal plants.     

Covalent immobilization of albumin on micron‐sized magnetic poly(methyl methacrylate‐divinylbenzene‐glycidyl methacrylate) microspheres prepared by modified suspension polymerization

Micron‐sized magnetic poly(methyl methacrylate‐divinylbenzene‐glycidyl methacrylate) microspheres were prepared by a modified suspension polymerization in the presence of oleic acid‐coated magnetite nanoparticles. The magnetic microspheres were functionalized by reacting the epoxy groups with ammonia solution to provide amino groups. After activated with glutaraldehyde (GA), bovine serum albumin was covalently immobilized on these magnetic microspheres. The influence of initial protein concentration, pH and ionic strength of the protein solution on covalent immobilization was studied. Scanning electron micrographs showed that the magnetic microspheres had an average size of 6.4 µm and relative narrow size distribution. Magnetic measurement revealed the magnetic microspheres were superparamagetic with saturation magnetization of 7.32 emu/g. The successful amination of the magnetic microspheres was confirmed by Fourier transform infrared spectroscopy (FT‐IR). Copyright © 2005 John Wiley & Sons, Ltd.