A method for screening tyrosinase inhibitors

Abstract

The invention discloses a method for screening tyrosinase inhibitors, comprising: adding chopped Agaricus bisporus into a pre-cooled phosphate buffer solution with pH=6.8; homogenizing the phosphate buffer solution, Then filter with nylon gauze to obtain the crude extract of tyrosinase; add the crude extract of tyrosinase in each hole of the 96-well plate, put the 96 plate into the refrigerator for adsorption; prepare levorotatory polysaccharide Base substrate solution and take the tyrosinase inhibitor solution to be screened; take the 96-well plate obtained in step 3, add the substrate solution on the 96-well plate and add tyrosinase in different substrate solutions Inhibitor solution was carried out as a control culture; compare the color depth of the tyrosinase inhibitor and the control group solution to judge the inhibitory effect of kojic acid on tyrosinase. The invention obtains comparatively pure immobilized tyrosinase by adsorbing the crude extract of tyrosinase, which greatly simplifies the purification steps of tyrosinase.

Description

technical field [0001] The invention relates to a method for screening tyrosinase inhibitors. Background technique [0002] Tyrosinase (TYR), a copper-containing enzyme derived from embryonic nerve sheath cells, is a key enzyme in melanin metabolism and catecholamines. Tyrosinase inhibitors can inhibit tyrosinase and can effectively inhibit melanin metabolism and catecholamines, so they have attracted everyone's attention. In the prior art, the main test of screening tyrosinase inhibitors comprises: constructing the solution system of substrate, tyrosinase and tyrosinase inhibitor (or blank), by testing substrate The color depth or absorbance value of the oxidized product can be used to judge the inhibitory effect of the inhibitor. [0003] And there are several deficiencies in the following methods in the existing method for screening tyrosinase inhibitors: [0004] 1. The current commercialized tyrosinase is only mushroom tyrosinase, and it is expensive. If you extract ...

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Problems solved by technology

[0004] 1. The current commercialized tyrosinase is only mushroom tyrosinase, and it is expensive. If you extract and purify tyrosinase yourself, the steps are cumbersome and time-consuming;

[0005] 2. The traditional method for screening tyrosinase inhibitors is a homogeneous reaction system. After the test, the solution can only be discarded, and tyrosinase cannot be reused, resulting in a serious waste of materials;

[0006] 3. In the traditional method of screening tyrosinase inhibitors, the experimental steps for judging the inhibition mechanism (reversible or irreversible process) of tyrosinase inhibitors are cumbersome, the amount of experiments is large, and the results are not intuitive

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