Application of glabridin raceme

Abstract

The invention discloses application of glabridin raceme, and belongs to the technical field of glabridin. Both the (S)-glabridin and the glabridin raceme have tyrosinase inhibitory activity. The glabridin racemate shows the tyrosinase inhibitory activity superior to that of glabridin with two optical configurations, and can be used in whitening cosmetics to improve the whitening effect of the cosmetics.

Description

Technical Field

[0001] The invention relates to the technical field of glabridin, and in particular to an application of a glabridin racemate. Background Art

[0002] Naturally isolated ( R )-Glycyrrhizin has strong tyrosinase inhibitory activity, but the glabridin on the market is mainly derived from natural plant extracts, and there is no commercial supply of fully synthetic glabridin. There are few synthetic routes for optically pure glabridin and only R The other synthetic routes are all for synthesizing the racemic form of glabridin. Wang Xudong and other researchers proposed in "Progress in the Synthesis of Glabridin" that glabridin, as a natural extract with multiple medical uses, has always attracted the attention of workers in the field of organic synthesis and pharmaceutical synthesis. At present, there are many methods for synthesizing the racemic form of glabridin, and for ( S )-Glycyrrhizin, Kim et al. performed chiral separation of racemic glycyrrhizin by using a semi-preparative chiral column and finally obtained ( S )-Glycyrrhizin, probably due to the difficulty of separation and the small amount of separation, it has not been tested for activity. That is to say, the application of glabridin reported in the prior art only reports on the R configuration, and there are no reports on the application of racemic and S configuration glabridin. It is speculated that its effectiveness is closely related to its configuration. At the same time, there is no effective chiral separation method for the racemic glabridin. Therefore, it is of great significance to study the application of the racemic glabridin. Summary of the invention

[0003] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an application of a glabridin racemate, which can effectively inhibit tyrosinase activity without the need for chiral separation.

[0004] The technical solution of the present invention to solve the above technical problems is as follows: The first object of the present invention is to provide the use of glabridin racemate in inhibiting tyrosinase activity.

[0005] The beneficial effects of the present invention are: R )-Glycyrrhizin has strong tyrosinase inhibitory activity, but the prior art has not reported ( S )-Glycyrrhizin has tyrosinase inhibitory activity. S )-The tyrosinase inhibitory activity of glabridin is significantly lower than that of ( R )-Glycyrrhizin, but by ( R )-Glycyrrhizin and ( S )-Glycyrrhizin formed glabridin racemate, namely ( Rac)-Glycyrrhizin's tyrosinase inhibitory activity showed significantly better than the tyrosinase inhibitory activity of the two optical configurations of Glycyrrhizin, which indicates that ( R )-Glycyrrhizin and ( S )-Glycyrrhizin showed a synergistic effect.

[0006] After molecular docking analysis, ( R )-Glycyrrhizin and ( S )-The binding position of glabridin and tyrosinase is quite different. R )-Glycyrrhizin binds more tightly to tyrosinase inside the molecule, ( S )-Glycyrrhizin rule binds to tyrosinase on the outside, and then speculates that it may be ( R )-Glycyrrhizin and ( S )-Glycyrrhizin has different mechanisms of tyrosinase inhibition activity. The two different inhibition mechanisms lead to a synergistic effect of racemic glycyrrhizin.

[0007] The second object of the present invention is to provide the use of glabridin racemate in cosmetics, which are whitening cosmetics.

[0008] The third object of the present invention is to provide a cosmetic containing glabridin racemate.

[0009] The beneficial effects of the present invention are as follows: the glabridin racemate has the function of inhibiting tyrosinase activity, and can be used in cosmetics to achieve a whitening effect.

[0010] The present invention has the following beneficial effects: The present invention studies the tyrosinase inhibitory activity of the glabridin racemate, and measures and analyzes the tyrosinase inhibitory activity of the glabridin racemate from two aspects: the monophenolase inhibitory activity and the diphenolase inhibitory activity. R )-Glycyrrhizin and ( S )-Glycyrrhizin's tyrosinase inhibitory activity, it was found that the racemic form of glabridin showed significantly better tyrosinase inhibitory activity than the two optical configurations of glabridin. After molecular docking analysis, ( R )-Glycyrrhizin and ( S )-The binding position of glabridin and tyrosinase is quite different. R )-Glycyrrhizin binds more tightly to tyrosinase inside the molecule, ( S )-Glycyrrhizin rule binds to tyrosinase on the outside, and then speculates that it may be ( R )-Glycyrrhizin and ( S )-Glycyrrhizin has different mechanisms of tyrosinase inhibition activity. The two different inhibition mechanisms lead to a synergistic effect of racemic glycyrrhizin. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the activity curve of monophenolase.

[0012] Figure 2 for( R )-Glycyrrhizin's inhibition results on monophenolase activity.

[0013] Figure 3 for( S )-Glycyrrhizin's inhibition results on monophenolase activity.

[0014] Figure 4 for( Rac )-Glycyrrhizin's inhibition results on monophenolase activity.

[0015] Figure 5 The results show the inhibition of monophenolase activity by glycyrrhizin with different configurations.

[0016] Figure 6 This is the activity curve of bisphenolase.

[0017] Figure 7 for( R )-Glycyrrhizin's inhibition results on bisphenolase activity.

[0018] Figure 8 for( S )-Glycyrrhizin's inhibition results on bisphenolase activity.

[0019] Fig. 9 for( Rac )-Glycyrrhizin's inhibition results on bisphenolase activity.

[0020] Fig.10 The results show the inhibition of bisphenolase activity by glycyrrhizin with different configurations.

[0021] Fig.11 for( R )-Glycyrrhizin and ( S )-Glycyrrhizin molecular docking results; where a is ( R )-Glycyrrhizin and ( S )-the best binding position of glabridin; b is the best position for amino acid residue connection; ( R )-Glycyrrhizin and ( S )-Glycyrrhizin are all represented by rod-shaped structures, ( R )-Glycyrrhizin is represented by green sticks, ( S )-Glycyrrhizin is represented by a yellow stick structure. DETAILED DESCRIPTION

[0022] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0023] Tyrosinase, as a bifunctional enzyme, exhibits both monophenolase and diphenolase activities. This bifunctional property enables tyrosinase to participate in a variety of complex biochemical reactions in vivo, including the hydroxylation of monophenols (under the action of monophenolase, tyrosine is hydroxylated to L-DOPA) and the oxidation of diphenols (under the action of diphenolase, L-DOPA is oxidized to dopaquinone, which then undergoes a series of reactions to produce melanin). Therefore, evaluating only its effect on the overall activity of tyrosinase may not fully reveal the interaction mechanism between active ingredients and tyrosinase. Monophenolase activity is the key step in tyrosinase-catalyzed conversion of monophenols to diphenols. Studying the inhibition of active ingredients on monophenolase activity can reveal its effect on this specific reaction stage, thereby gaining a deeper understanding of its specific mechanism of action in vivo. Diphenolase activity is the key step in tyrosinase-catalyzed oxidation of diphenols to quinones. By evaluating the inhibition of active ingredients on diphenolase activity, we can further understand its role in subsequent biochemical reactions and the specific effects it may have on organisms. Therefore, decomposing the activity of tyrosinase into two independent parts, monophenolase and diphenolase, can improve the accuracy and pertinence of experimental design. For example, reference: Gong Shengzhao, Yang Zhuoru, Cheng Jiang. Inhibition of tyrosinase activity by vanillin [J]. Journal of South China University of Technology: Natural Science Edition, 2006, 34(5): 53-57. Therefore, the present invention conducts monophenolase and bisphenolase activity tests on the glabridin racemate to illustrate the inhibitory effect of the glabridin racemate on tyrosinase activity, as follows: Embodiment 1: Glabridin racemate (denoted as ( Rac )-glabridin), which contains an equal amount of ( R )-Glycyrrhizin and ( S )-Glycyrrhizin. This example studies the inhibitory effect of racemic glabridin on tyrosinase activity. Rac )-Glycyrrhizin, ( R )-Glycyrrhizin and ( S )-Glycyrrhizin was used as the research object, and the specific process was as follows: 1. Determination of tyrosinase activity 1. Preparation of standard curve Without adding the sample to be tested, add 100 μL of 9 mmol / L L-tyrosine and 100 μL of 6 mmol / L L-DOPA (dissolved in phosphate buffer solution, pH 6.8) to 70 μL phosphate buffer solution (pH 6.8), then add 10 μL of 2 mg / mL and 1 mg / mL tyrosinase buffer aqueous solution, respectively. Measure the absorbance at 450 nm, and draw a standard curve of enzyme activity with time as the horizontal axis and absorbance as the vertical axis.

[0024] 2. Determination of monophenolase inhibition activity In a 96-well plate, 140 μL of buffer was added to each well. The remaining wells were divided into three groups. 70 μL of buffer was added to the sample wells. The sample wells were added with concentrations of 0 mg / mL, 0.0005 mg / mL, 0.005 mg / mL, 0.05 mg / mL, and 0.5 mg / mL ( R )-Glycyrrhizin, ( S )-Glycyrrhizin, ( Rac )-Glycyrrhizin DMSO solution 4 μL. Add 100 μL of 9 mmol / L L-tyrosine to each sample well, add 10 μL of 2 mg / mL monophenolase solution to each well, and measure the absorbance at 450 nm.

[0025] 3. Determination of bisphenolase inhibition activity In a 96-well plate, 140 μL of buffer was added to each well. The remaining wells were divided into three groups. 70 μL of buffer was added to the sample wells. The sample wells were added with concentrations of 0 mg / mL, 0.0005 mg / mL, 0.005 mg / mL, 0.05 mg / mL, and 0.5 mg / mL ( R )-Glycyrrhizin, ( S )-Glycyrrhizin, ( Rac )-Glycyrrhizin DMSO solution 4 μL. Add 100 μL of 6 mmol / L L-DOPA to each sample well, and add 10 μL of 1 mg / mL bisphenolase solution to each well, and measure the absorbance at 450 nm.

[0026] 2. Tyrosinase Activity Inhibition Results 1. Results of monophenolase inhibition ( R )-Glycyrrhizin, ( S )-Glycyrrhizin and ( Rac )-Glycyrrhizin inhibits the activity of monophenolase. Figure 1-Figure 5 .

[0027] Depend on Figure 1It can be seen that the inhibition process of glabridin on monophenolase first went through a delay period of about 45 minutes, and then after a linear reaction of 20 minutes, the substrate depletion rose slowly, indicating that its reaction speed was fastest within 45-65 minutes. Figure 2-Figure 4 It can be seen that by adding 0 mg / mL, 0.0005 mg / mL, 0.005 mg / mL, 0.05 mg / mL, 0.5 mg / mL of ( R )-Glycyrrhizin, ( S )-Glycyrrhizin, ( Rac )-Glycyrrhizin, all of which have inhibitory activity on tyrosinase, and as the dosage concentration increases, the monophenolase inhibitory activity also increases. Samples with a significant difference concentration of 0.005 mg / mL were selected for comparison of tyrosinase inhibitory activity. Figure 5 As shown, ( R )-Glycyrrhizin has a much greater inhibitory effect on the activity of monophenolase than ( S )-glabridin inhibits the activity of monophenolase, but the inhibition of optically pure glabridin on the activity of monophenolase is weaker than that of ( Rac )-Glycyrrhizin, Description( R )-Glycyrrhizin and ( S )-Glycyrrhizin combination synergistically improves ( Rac )-Inhibition of monophenolase activity by glabridin.

[0028] 2. Bisphenolase inhibition results ( R )-Glycyrrhizin, ( S )-Glycyrrhizin and ( Rac The results of inhibition of bisphenolase activity by )-glabridin are shown in Figure 6-Figure 10 .

[0029] Depend on Figure 6 It can be seen that the inhibition process of glabridin on polyphenolase first goes through a delay period of about 25 minutes, and then after a linear reaction of 50 minutes, the substrate depletion rises slowly, indicating that its reaction speed is fastest within 25-75 minutes. Figure 7-Figure 9 It can be seen that by adding 0 mg / mL, 0.0005 mg / mL, 0.005 mg / mL, 0.05 mg / mL, 0.5 mg / mL of ( R )-Glycyrrhizin, ( S )-Glycyrrhizin, ( Rac )-Glycyrrhizin, all of which have inhibitory activity on bisphenolase, and as the dosage concentration increases, the bisphenolase inhibitory activity also increases. Samples with a significant difference concentration of 0.05 mg / mL were selected for comparison of bisphenolase inhibitory activity. Fig.10 As shown, ( R)-Glycyrrhizin has a much greater inhibitory effect on bisphenolase activity than ( S )-glabridin inhibits the activity of bisphenolase, but the inhibition of optically pure glabridin on bisphenolase activity is weaker than that of ( Rac )-Glycyrrhizin, Description( R )-Glycyrrhizin and ( S )-Glycyrrhizin combination synergistically improves ( Rac )-Inhibition of bisphenolase activity by glabridin.

[0030] Embodiment 2: To further understand ( R )-Glycyrrhizin and ( S )-The interaction between glabridin and tyrosinase, the present invention uses molecular docking for further analysis, the specific process is: using Autodock Vina 1.2.4 software, the binding posture of glabridin and tyrosinase protein is predicted and analyzed. The search volume (53 × 53 × 53 Å3, Spacing is set to 1.00Å) covers the entire cavity of the tyrosinase protein model. The center of the search space is located at X: -35.624; Y: 6.628; Z: -20.323. According to the docking binding energy, the docking simulation results are estimated. The 3D model of tyrosinase protein is derived from ProteinData Bank (PDB ID: 5M8M, http: / / www.rcsb.org / ), the structure diagram is drawn using PyMOL 2.5.2, and the tyrosinase protein is set to a blue cartoon structure. ( R )-Glycyrrhizin is a green rod-shaped structure, ( S )-Glycyrrhizin is a yellow rod-like structure. Fig.11 .

[0031] Depend on Fig.11 It can be seen that the ligand has good coordination on the active site. R )-Glycyrrhizin and ( S )-Glycyrrhizin binds to the same binding pocket, but ( R )-Glycyrrhizin binds to the inside of the molecule, while ( S )-Glycyrrhizin rule binds to the outside of tyrosinase. In the AutoDock Vina docking results, ( R )-Glycyrrhizin binding energy is -9.9 kcal / mol, ( S The binding energy of (R)-glabridin is -8.3 kcal / mol, which indicates that (R)-glabridin binds more tightly to tyrosinase than (S)-glabridin.

[0032] In summary, R )-configuration, ( S)-configuration, and racemic glycyrrhizin were tested for tyrosinase activity, and the results showed that racemic glycyrrhizin had better tyrosinase inhibition activity than natural and non-natural glycyrrhizin. According to the molecular docking results, ( R )-Glycyrrhizin and ( S )-The binding position of glabridin and tyrosinase is quite different. R )-Glycyrrhizin binds more tightly to tyrosinase inside the molecule, ( S )-Glycyrrhizin rule binds to tyrosinase on the outside, and it is speculated that ( R )-Glycyrrhizin and ( S )-Glycyrrhizin has different tyrosinase inhibitory activity mechanisms. The two different inhibitory mechanisms lead to a synergistic effect of racemic glabridin, showing tyrosinase inhibitory activity that is superior to the two optical configurations of glabridin.

[0033] In addition, the main factor for skin darkening is the increase in the content and distribution of melanin. The depth of skin color is closely related to the production and reduction of melanin. Therefore, by inhibiting the production of melanin, a whitening effect can be achieved. Since the formation of melanin mainly occurs in melanocytes, studies on the formation mechanism of melanin in melanocytes have shown that the formation of melanin is mainly the result of the separate or synergistic action of four enzymes in melanocytes: tyrosinase, dopa isomerase (TRP-2), peroxidase, 5,6-dihydroxyindole-2-carboxylic acid, or DHICA oxidase (TRP-1), and currently the relevant intracellular inhibition is mainly focused on the inhibition of tyrosinase (reference: Jia Aiqun, Sun Yang, Wang Jianxin. The current development status of whitening agents and the research progress of their melanin inhibition mechanism [J]. 2001, 1 (31): 41-44.). The glabridin racemate studied in this application has a good inhibitory effect on tyrosinase activity. Therefore, the glabridin racemate can be used in cosmetics, especially whitening cosmetics, to achieve a whitening effect by inhibiting tyrosinase.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of glabridin racemate in inhibiting tyrosinase activity.

2. Application of glabridin racemate in cosmetics.

3. The use according to claim 2, It is characterized in that The cosmetics are whitening cosmetics.

4. A cosmetic product, It is characterized in that include: Glabridin racemate.

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