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Talanta. 2019 Apr 1;195:127-136. doi: 10.1016/j.talanta.2018.11.009. Epub 2018 Nov 6.

Enzyme immobilized on polyamidoamine-coated magnetic microspheres for α-glucosidase inhibitors screening from Radix Paeoniae Rubra extracts accompanied with molecular modeling.

Author information

1
Fudan University Affiliated Pudong Medical Center & Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203, China.
2
Jing'an District Central Hospital, Fudan University, 259 Xikang Road, Shanghai 200040, China.
3
Department of Biochemical Drugs and Biological Products, Shanghai Institute for Food and Drug Control, No.1500 Zhangheng Road, Pudong New District, Shanghai 201203, China.
4
Fudan University Affiliated Pudong Medical Center & Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203, China. Electronic address: yanli@fudan.edu.cn.
5
Fudan University Affiliated Pudong Medical Center & Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203, China. Electronic address: glduan@shmu.edu.cn.

Abstract

In this study, a method for direct screening and identification of α-glucosidase inhibitors (AGIs) from extracts of natural products was established based on polyamidoamine (PAMAM) coated magnetic microspheres. A facile route to synthesize the magnetic PAMAM was employed and α-glucosidase was successfully covalently attached to its surface through cross linking of glutaraldehyde. Using the enzyme-loaded magnetic microspheres, potential inhibitors were fished out from crude extracts directly, followed by structure confirmation. The inhibitory activities of the screened components were further investigated by the enzyme-loaded magnetic microspheres. The Fe3O4 @PAMAM@α-Glu microspheres displayed favorable dispersibility, fast magnetic separation, large enzyme binding amount (42.9 μg•mg-1) and high enzyme activity. Moreover, the α-glucosidase on the surface of PAMAM coating maintained high storage stability and remarkable reusability. Taking advantage of specific interaction of the α-glucosidase with AGIs, the materials could selectively capture a known AGI (+)-catechin under the interference of an inactive compound salicylic acid, with a binding capacity as high as 15.4%. Additionally, using the Fe3O4 @PAMAM@α-Glu microspheres in the inhibition assay, the enzymatic reaction could be stopped by magnetic separation instead of the traditional addition of Na2CO3 solution, which not only eliminated the disturbance of termination reagent to the results, but also reused the immobilized α-glucosidase. The screening and inhibitory activity verification of potential ligands in Radix Paeoniae Rubra ("Chi-shao" in Chinese) extracts were achieved by using Fe3O4 @PAMAM@α-Glu microspheres, demonstrating practical applicability of our method. Therefore, the magnetic PAMAM-based screening approach could be a feasible and alternative strategy for discovering enzyme inhibitors from natural product extracts.

KEYWORDS:

Immobilized enzyme; Magnetic microspheres; Polyamidoamine; Radix Paeoniae Rubra; α-glucosidase inhibitor

PMID:
30625522
DOI:
10.1016/j.talanta.2018.11.009
[Indexed for MEDLINE]

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