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

Abstract

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.

Prospective, randomized, double‐blind assessment of topical bakuchiol and retinol for facial photoageing

“…This may be attributable to bakuchiol's antioxidant effects, as well as its ability to disrupt melanin synthesis. Interestingly, bakuchiol can interfere with two steps of the melanin synthesis pathway, blocking both α‐melanocyte‐stimulating hormone activation and tyrosinase (the rate‐limiting enzyme in melanin synthesis) . Bakuchiol's suppressive effects on cutaneous melanin production prime the compound for use in both antiageing and antihyperpigmentation cosmeceuticals.…”

Advances in enzyme substrate analysis with capillary electrophoresis

“…The on-line mixing of enzyme, substrate, and inhibitor has been accomplished through electrophoretically mediated microanalysis [58,59,62,63,66,73], transverse diffusion of laminar flow profiles [21,31,41,74,75], and pressure mediated microanalysis [45]. On-line analysis with the use of immobilized enzyme for inhibitor studies have also been developed and high throughput analysis was achieved [18,22,23,56,68,76–78]. …”

“…This is achieved by determining the IC 50 value, which is defined as the inhibitor concentration necessary to obtain 50% of the original response in the absence of inhibitor for a given substrate concentration. The IC 50 is obtained by monitoring the product formation while varying the concentration of inhibitor [13,21–23,29,31,39,46,56,68,71,74,75,81,82]. The lower the IC 50 value is, the more effective the inhibitor.…”

“…The lower the IC 50 value is, the more effective the inhibitor. Alternatively, the percent differences of the product peak area in the absence and presence of inhibitor can also be used to estimate the inhibition efficiency of the inhibitor [13,21,56,68,73–77,82,83]. …”

“…The mechanism used to prevent cancer progression is to reduce the enzyme activity on the pathway that promotes cancer cell growth. In order to find a potent inhibitor, on-line analysis is generally adapted to screen the selected compounds, such as natural extracts [23,56,63,76] or traditional Chinese medicines [18,57,68,73]. In the process of screening the inhibition, the selected inhibitor is evaluated as the relative difference in the product peak area in the presence and in the absence of the inhibitor.…”