Paper
ZHUANG Yuanbei, YE Yongjing , YANG Yueqi , WEI Aihong , ZHANG Shengyuan,
Currently, the methods used for evaluating tyrosinase inhibitory activity in tyrosinase inhibitor screening
are not suitable for the screening of traditional Chinese medicinal plants. Therefore, this study established a novel in vitro
screening method for tyrosinase inhibitors based on HPLC. With the substrate levodopa content as the detection indicator, the
chromatographic and reaction conditions of this method were optimized. The optimized chromatographic conditions were as
follows: XBridge Peptide C18 column(4.6 mm×250 mm, 5 μm), 280 nm of detection wavelength, mobile phase consisting of
0.1％ formic acid-acetonitrile(in a volume ratio of 95 ∶ 5) under isocratic elution, 20 μL of injection volume, 30 ℃
of column temperature, and 0.8 mL/min of flow rate. The optimized enzyme reaction system consisted of 300 μL
of PBS(pH 6.8), 150 μL of test sample, 300 μL of 300 IU/mL tyrosinase solution. The system was mixed and incubated
at 37 ℃ for 10 min, followed by the addition of 150 μL of 10 mmol/L levodopa sdution. After that, the above system was
mixed and reacted at 37 ℃ for 10 min again, and then terminated with 4.2 mL of 0.4 mol/L hydrochloric acid. The tyrosinase
inhibitory activities of the positive controls(vitamin C and kojic acid) were detected by both this method and the microplate
reader method, and the results showed no significant difference in IC50. When applied to screen nine medicinal plant
extracts, the HPLC method detected tyrosinase inhibitory activity in all extracts, whereas the microplate reader method only
detected activity in four extracts, with higher IC50 values compared with the HPLC results. These results indicated that the
screening method based on HPLC outperformed the traditional microplate reader method in terms of specificity, accuracy,
and precision, providing a reliable analytical approach for screening natural tyrosinase inhibitors, particularly suitable for
complex matrix samples.
