Preparation of Magnetic Microsphere‐Gold Nanoparticle‐Immobilized Enzyme Batch Reactor and Its Application to Enzyme Inhibitor Screening in Natural Extracts by Capillary Electrophoresis

Abstract

A novel strategy for screening the enzyme inhibitors from the natural products by capillary electrophoresis (CE) with a pre column enzyme batch reactor prepared by magnetic microsphere (MB)-gold nanoparticles (AuNPs) is reported. The enzyme batch reactor was prepared by immobilizing the enzymes on the MB conjugated AuNPs (MB-AuNPs). To demonstrate this strategy, xanthine oxidase (XOD) was employed as a model for the activity of the enzyme, inhibition study, and inhibitor screening. With the developed CE method, the enzyme activity was determined by the quantification of the peak area of the product. Enzyme inhibition can be read out directly from the reduced peak area of product in comparison to a reference electropherogram obtained in the absence of any inhibitor. A statistical parameter Z' factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be 0.7, implying a good accuracy. The screening of two natural extracts from Cortex Phellodendri and Rhizoma Galangae showed that they were positive for XOD inhibition by the present method. Using this immobilized enzyme technology combined with CE separation not only provides the advantages such as convenience, rapidity and low cost, but also provides a new platform for discovering enzyme-inhibitor drug lead compounds.

Capillary electrophoresis in phytochemical analysis (2014–2017)

“…This approach not only realized rapid separation of enzymes from the reaction mixture by magnetism, but also can improve the stability of enzymes. Similar study was also reported, XOD was immobilized on magnetic microsphere ‐gold NPs (AuNPs) for evaluating the XOD inhibitory effects of extracts from Cortex Phellodendri and Rhizoma Galangae .…”

“…CE in screening of bioactive compounds derived from natural products from 2007 to 2017 was well reviewed by Ma et al . Herein, we would only focus on the recent applications of CE in the screening of enzyme or receptor inhibitors, such as tyrosinase (TYR) inhibitors , protein kinase inhibitors (PKIs) of protein kinase A (PKA) , neuraminidase inhibitors , mammalian target of rapamycin (mTOR) inhibitors , epidermal growth factor receptor (EGFR) inhibitors , α‐glucosidase inhibitors , xanthine oxidase (XOD) inhibitors , histone deacetylase 1 (HDAC1) inhibitors , break point cluster region Abelson tyrosine kinase (BCR‐ABL) inhibitors , and thrombin (THR) inhibitors .…”

Overview of in‐capillary enzymatic reactions using capillary electrophoresis

“…This was shown by Liu et al for lipase [55] and Wang et al on separate studies. The latter group grafted xanthine oxidase onto magnetic microspheregold nanoparticles [83].…”

Advances in screening enzyme inhibitors by capillary electrophoresis

“…The developed method has been employed to screen α‐Glu inhibitors from 18 TCMs. Based on magnetic microsphere (MB)‐gold nanoparticles (AuNPs), xanthine oxidase (XOD) was immobilized and used for screening XOD inhibitors from the natural products . With the proposed method, the screening of two natural extracts from Cortex phellodendri and Rhizoma galangae was taken and the results indicated its positive effect for XOD inhibition.…”