Fabrication of a Magnetic Porous Organic Polymer for α-Glucosidase Immobilization and Its Application in Inhibitor Screening.

The technology based on immobilized enzymes was employed to screen the constituents inhibiting disease-related enzyme activity from traditional Chinese medicine, which is expected to become an important approach of innovative drug development. Herein, the Fe3O4@POP composite with a core-shell structure was constructed for the first time with Fe3O4 magnetic nanoparticles as the core, 1,3,5-tris (4-aminophenyl) benzene (TAPB) and 2,5-divinylterephthalaldehyde (DVA) as organic monomers, and used as the support for immobilizing α-glucosidase. Fe3O4@POP was characterized by transmission electron microscopy, energy-dispersive spectrometry, Fourier transform infrared, powder X-ray diffraction, X-ray photoelectron spectroscopy, and vibrating sample magnetometry. Fe3O4@POP exhibited a distinct core-shell structure and excellent magnetic response (45.2 emu g-1). α-Glucosidase was covalently immobilized on core-shell Fe3O4@POP magnetic nanoparticles using glutaraldehyde as the cross-linking agent. The immobilized α-glucosidase possessed improved pH stability and thermal stability as well as good storage stability and reusability. More importantly, the immobilized enzyme exhibited a lower Km value and enhanced affinity for the substrate than the free one. The immobilized α-glucosidase was subsequently used for inhibitor screening from 18 traditional Chinese medicines in combination with capillary electrophoresis analysis among which Rhodiola rosea exhibited the highest enzyme inhibitory activity. These positive results demonstrated that such magnetic POP-based core-shell nanoparticles were a promising carrier for enzyme immobilization and the screening strategy based on immobilized enzyme provided an effective way to rapidly explore the targeted active compounds from medicinal plants.

Pancreatic lipase and alpha-glucosidase inhibitors screening from Schisandra chinensis based on spectrum-effect relationship and ultra-high-performance liquid chromatography-tandem mass spectrometry.

As a traditional Chinese medicine, Schisandra chinensis has a potential weight-loss effect by delaying carbohydrate absorption and improving lipid metabolic disorders. However, its active components are still unclear and require in-depth research. In this study, the active components of Schisandra chinensis responsible for pancreatic lipase and alpha-glucosidase inhibitory activity were screened and identified based on a spectrum-effect relationship study in combination with ultra-performance liquid chromatography-tandem mass spectrometry analysis. The ultra-high-performance liquid chromatography fingerprints of 17 batches of Schisandra chinensis were established, and 14 common peaks were specified by similarity analysis. The half-maximal inhibition concentration values for pancreatic lipase and alpha-glucosidase inhibition were separately measured by enzymatic reactions. Using multivariate statistical methods including principal component analysis, partial least square analysis, and grey relational analysis, the correlation models between the peak areas of 14 common peaks and half-maximal inhibition concentration values were constructed, and the chromatographic peaks making a great contribution to efficacy were screened out. Peak1, Peak2, Peak4, Peak6, Peak9, Peak10, Peak11, and Peak13 were responsible for alpha-glucosidase inhibitory activity, while Peak1, Peak4, Peak6, Peak9, Peak10, and Peak11 for pancreatic lipase inhibitory activity. Finally, the 70% ethanol extracts of Schisandra chinensis were characterized by ultra-high-performance liquid chromatography-tandem mass spectrometry analysis, and 14 lignans were identified to further elucidate the active constituents of Schisandra chinensis. The positive results suggested the proposed strategy is simple and effective to screen active components from complex medicinal plants.

MMP3 -1171 5A/6A Promoter Genotype Influences Serum MMP3 Levels and Is Associated with Deep Venous Thrombosis.

BACKGROUND: The aim of this study was to investigate the roles of MMP3 (matrix metalloproteinase-3) gene polymorphism and protein expression in deep venous thrombosis (DVT) among Chinese Han population. METHODS: A total of 280 subjects were included in this study and categorized as case group (144 DVT patients) and control group (136 healthy individuals). Polymerase chain reaction-restriction fragment length polymorphism was used to detect MMP3 promoter -1171 5A>6A genotype and allele frequencies. MMP3 serum levels were measured by enzyme-linked immunosorbent assay. SPSS version 18.0 statistical software was used for data analysis. RESULTS: There was significant difference in genotype frequencies of MMP3 gene -1171 5A>6A between the case group and the control group (all P < 0.05). Furthermore, the 6A allele on MMP3 -1171 5A>6A may be associated with increased risk of DVT (odds ratio 1.961, 95% confidence interval 1.309-2.939, P < 0.01). The MMP3 serum level in DVT patients was markedly higher than the control group (case group: 28.45 ± 10.97 vs. CONTROL GROUP: 18.18 ± 9.03, P < 0.05). Serum MMP3 level in DVT patients carrying 5A/6A and 6A/6A genotypes was higher than the control group (P < 0.05). The bilateral calf circumference difference was significantly higher in DVT patients than the control group among all the genotypes at MMP3 gene -1171 5A>6A (all P < 0.05). CONCLUSION: MMP3 gene -1171 5A>6A polymorphism and upregulated protein expression may be associated with DVT risk in Chinese Han population.

Catechol-tetraethylenepentamine co-deposition modified cellulose filter paper for α-glucosidase immobilization and inhibitor screening from traditional Chinese medicine.

Cellulose filter paper (CFP) is expected to be an ideal carrier for enzyme immobilization due to its sustainability and biocompatibility. However, the interaction between the carrier and enzyme might change the spatial conformation of the enzyme and its microenvironment, and thus the flexibility of the enzyme molecule or the transport of the substrate to the active site would be hampered. In this work, a two-component system of catechol and tetraethylene pentamine was used to replace dopamine, and a polydopamine-like composite layer was deposited on the surface of CFP to introduce amino groups, which was similar to the self-polymerization-adhesion behavior of dopamine. Using polyethylene glycol diglycidyl ether with flexible spacer arms as the cross-linking agent, α-glucosidase was covalently bonded to amino-modified CFP through an epoxy ring-opening reaction. The immobilized α-glucosidase exhibited greater tolerance to pH and high temperature. After 10 repeated uses, the immobilized α-glucosidase maintained relatively high enzyme activity. Its kinetic behavior was investigated to illustrate the reliability for enzyme inhibitor screening. Finally, a screening method combining an immobilized enzyme and capillary electrophoresis analysis was proposed and applied to screening inhibitors from 11 kinds of traditional Chinese medicines, among which Chebulae Fructus, Phyllanthi Fructus and Terminaliae Relliricae Fructus exhibited strong enzyme inhibitory activities.

Screening and identification of α-glucosidase inhibitors from Cyclocarya paliurus leaves by ultrafiltration coupled with liquid chromatography-mass spectrometry and molecular docking.

Cyclocarya paliurus, as an important edible and medicinal product, has shown a good prospect in the prevention of diabetes mellitus (DM). However, it is unclear which active compounds derived from C. paliurus play a significant role in inhibiting α-glucosidase activity. In present study, affinity-based screening assay was developed to screen and identify potential α-glucosidase inhibitors from C. paliurus leaves based on affinity ultrafiltration coupled with ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) and molecular docking. After being enriched by D-101 macroporous resin, five eluent fractions with different polarity were obtained and their inhibitory activities on α-glucosidase were evaluated by an enzyme inhibition assay in vitro. The result showed that 70% ethanol fraction of C. paliurus leaves exhibited remarkable α-glucosidase inhibitory activity with the IC50 value of 17.81 µg/mL. The 70% ethanol fraction was incubated with α-glucosidase and then active compounds would form enzyme-inhibitor complexes. The complexes could be separated from inactive components by the interception ability of ultrafiltration membrane under centrifugation. A total of 36 active compounds were screened from C. paliurus leaves and the chemical structures were further characterized by UPLC-QTOF-MS/MS. Furthermore, molecular docking was performed to investigate possible inhibitory mechanisms between active compounds and α-glucosidase. The docking result showed that cyclocarioside I, pterocaryoside B, arjunolic acid, cyclocarioside Z5, cypaliuruside D and cyclocarioside N could be embedded well into the active pocket of α-glucosidase, and had significant affinity interactions with critical amino acid residues by forming hydrogen bonds, hydrophobic interactions and van der Waals, and affinity energies ranged from -9.3 to -6.7 kJ/mol. The results indicated that the developed method is rapid and effective for high throughput screening of potential α-glucosidase inhibitors from complex mixtures. Moreover, C. paliurus exhibited a remarkable inhibitory activity on α-glucosidase, making it a promising candidate for the prevention of DM.

α-glucosidase immobilization on magnetic core-shell metal-organic frameworks for inhibitor screening from traditional Chinese medicines.

In this work, a simple and rapid screening strategy was developed combining capillary electrophoresis analysis with enzymatic assay based on immobilized α-glucosidase. For α-glucosidase immobilization, magnetic core-shell metal-organic frameworks composite (Fe3O4@CS@ZIF-8) was fabricated by a step-by-step assembly method, and α-glucosidase was in situ encapsulated in crystal lattice of ZIF-8. The composite was characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction and vibrating sample magnetometer. After immobilization, α-glucosidase exhibited enhanced tolerance to temperature and pH, and its reusability was greatly improved with 74 % of initial enzyme activity after being recycled 10 times. The Michaelis-Menten constant of immobilized enzyme was calculated to be 0.47 mM and its inhibition constant and IC50 for acarbose were 0.57 µM and 0.18 µM, respectively. The immobilized enzyme was subsequently applied to inhibitor screening from 14 TCMs, and Rhei Radix et Rhizoma was screened out. Among the commercially available 10 components presented in Rhei Radix et Rhizoma, gallic acid, (+)-catechin and epicatechin exhibited the strongest inhibitory effect on α-glucosidase. Their binding sites and modes with α-glucosidase were simulated via molecular docking to further verify the inhibition screening assay results. The positive results indicated that the Fe3O4@CS@ZIF-8-based screening strategy may provide a new avenue for discovering enzyme inhibitors from TCMs.