CHU Pan-pan.Kinetics of Tyrosinase Inhibition Catalyzed by Black Bean Skin Anthocyanins[J].Soybean Science,2021,40(06):780-791.[doi:10.11861/j.issn.1000-9841.2021.06.0780]
黑豆皮花青素抑制酪氨酸酶催化反应动力学研究
- Title:
- Kinetics of Tyrosinase Inhibition Catalyzed by Black Bean Skin Anthocyanins
- Keywords:
- Black bean skin anthocyanins; Tyrosinase; Inhibition rate; Kinetics; Ultraviolet spectrum; Fluorescence spectrum
- 文献标志码:
- A
- 摘要:
- 为研究黑豆皮花青素抑制酪氨酸酶催化反应的动力学机制，在不同条件下研究黑豆皮花青素对酪氨酸酶单酚酶和二酚酶的抑制特性，确定最佳抑制条件、抑制类型和半抑制浓度（IC50），并使用紫外和荧光光谱分析二者的相互作用机制。正交试验分析表明：黑豆皮花青素对酪氨酸酶单酚酶的最佳作用条件为黑豆皮花青素浓度0.6 mg?mL-1、L-酪氨酸浓度6 mmol?L-1、温度37 ℃、时间15 min，此时抑制率高达50.37%；对二酚酶的最佳作用条件为黑豆皮花青素浓度0.8 mg?mL-1、L-多巴浓度6 mmol?L-1、温度35 ℃、时间10 min，此时抑制率52.45%。与VC相比，黑豆皮花青素对酪氨酸酶单、二酚酶的抑制作用更强。动力学试验表明，黑豆皮花青素对酪氨酸酶单、二酚酶活性有抑制作用，IC50分别为1.03和0.82 mg?mL-1，对酪氨酸酶有可逆的混合型抑制作用，其抑制常数Ki为0.61 mg?mL-1。紫外光谱分析表明，黑豆皮花青素使酪氨酸酶发生蓝移，表明此酶构象改变。荧光光谱分析表明，黑豆皮花青素对酪氨酸酶有静态猝灭作用，298，303和308 K时其结合位点数分别为0.76，0.71和0.65，结合常数KA分别为159.66，94.23和54.89 L?mol-1。研究结果说明黑豆皮花青素可以有效抑制酪氨酸酶活性，是天然植物来源的酪氨酸酶抑制剂。
- Abstract:
- In order to study the kinetic mechanism of black bean skin anthocyanins inhibiting tyrosinase catalytic reaction, we studied the inhibitory properties of black bean skin anthocyanins on tyrosinase monophenolase and diphenolase under different conditions, and determined the optimal inhibitory conditions, inhibitory types and the semi-inhibitory concentration(IC50). The UV and fluorescence spectra were used to analyze the interaction mechanism. The orthogonal analysis showed that the optimal conditions of anthocyanins on tyrosinase monophenol were as follows, 0.6 mg?mL-1 anthocyanins concentration, 6 mmol?L-1 L-tyrosine concentration, 37 ℃, 15 min, and the inhibition rate was 50.37%. The optimal action conditions of diphenol enzyme were as follows, 0.8 mg?mL-1 anthocyanin concentration, 6 mmol?L-1 L-dopa concentration, 35 ℃, 10 min, the inhibition rate of diphenolase was 52.45%. Compared with VC, black bean skin anthocyanins had stronger inhibitory effect on tyrosinase monophenolase and diphenolase.The kinetic experiments showed that anthocyanins from black seed could inhibit tyrosinase mono phenolase and diphenolase activities with IC50 of 1.03 and 0.82 mg?mL-1, respectively. It was a reversible mixed inhibition of tyrosinase, and the inhibition constant Ki was 0.61 mg? mL-1. The ultraviolet spectrum analysis showed that the anthocyanins in black bean skin caused the blue shift of tyrosinase, indicating that the conformation of tyrosinase was changed. The fluorescence spectra showed that the binding-site number was 0.76, 0.71 and 0.65 at 298, 303 and 308 K, and the binding constant KA was 159.66, 94.23 and 54.89 L?mol-1, respectively.The result shows that black bean skin anthocyanins can effectively inhibit the activity of tyrosinase as a natural plant origin tyrosinase inhibitor.
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备注/Memo
收稿日期：2021-08-05