Preparation method and application of tyrosinase inhibitor

Abstract

The invention relates to the technical field of medicines, in particular to a preparation method and application of a tyrosinase inhibitor. According to the present invention, the ratio of the main components 3 ', 5-dimethyl hesperidin to 3'-methyl hesperidin is 0.8: 1-1.25: 1, the tyrosinase activity influence experiment determines that the 3 '-methyl hesperidin and the 3', 5-dimethyl hesperidin can achieve the synergistic addition effect under the ratio condition, and the combination of the 3 '-methyl hesperidin and the 3', 5-dimethyl hesperidin in the ratio shows the better tyrosinase inhibition effect than the single use of the 3 ', 5-dimethyl hesperidin.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a preparation method and application of a tyrosinase inhibitor. Background Art

[0002] Flavonoids play an important role in human health care. The most abundant flavonoid in citrus peel is hesperidin, which has biological and pharmacological properties such as anti-inflammatory, lipid-lowering, antioxidant and anti-cancer. Because hesperidin contains rutinoside, the structure of this glycoside will reduce the liposolubility of flavonoids, making it difficult for them to penetrate biological cell membranes to play physiological roles. And due to the suitable spatial configuration of hesperidin and the presence of a large Π-bond conjugated system, chemical modification methods are commonly used to improve its solubility and bioavailability.

[0003] There is a published literature (Chen Jianqiu, Yang Ke, Hu Zhijun. Study on the derivatization modification and free radical scavenging of hesperidin [J]. Journal of Anhui Agricultural Sciences, 2009, 37(30): 14544-14546) showing that the methylation modification of hesperidin generally involves dissolving hesperidin in sodium hydroxide solution to form hesperidin chalcone, and then obtaining it through methylation with dimethyl carbonate. Under alkaline conditions, the γ-pyrone ring in the hesperidin molecule opens to form hesperidin chalcone, which has unstable properties and forms hesperidin again after acidification. Therefore, the methylation product of hesperidin is a mixture composed of various flavanone and chalcone compounds, and their methylation degrees are also different. In the previous research of the inventors, various flavanone and chalcone derivatives of hesperidin modification were isolated from this mixture.

[0004] Tyrosinase (tyrosinase, EC1.14.18.1) is a key enzyme in the biosynthesis of melanin in animals and plants. Its expression activity determines the speed and quantity of melanin production. Only by inhibiting the activity of tyrosinase can the formation of melanin be reduced to achieve the whitening effect. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and application of a tyrosinase inhibitor. The method provided by the present invention can simply prepare a tyrosinase inhibitor product with a fixed proportion range of main components (the ratio of 3’,5-dimethylhesperidin to 3’-methylhesperidin is in the range of 0.8:1 to 1.25:1), and under the condition of this component ratio range, the two components can have a synergistic effect, and the product can exhibit a tyrosinase inhibitory effect significantly superior to that of any one of its monomer components.

[0006] The following technical scheme is adopted:

[0007] An application of 3’,5-dimethylhesperidin for preparing a preparation for preventing or treating skin hyperpigmentation.

[0008] Specifically, the types of the preparations include, but are not limited to, drugs, health products, functional foods, cosmetics, etc.

[0009] The structural formula of 3’,5-dimethylhesperidin is as follows:

[0010]

[0011] Preferably, it is used for preparing a preparation for inhibiting melanogenesis.

[0012] Preferably, it is used for preparing a preparation for inhibiting tyrosinase

[0013] A preparation containing the above-mentioned 3’,5-dimethylhesperidin includes the 3’,5-dimethylhesperidin and 3’-methylhesperidin.

[0014] The structural formula of 3’-methylhesperidin is as follows:

[0015]

[0016] The inventor found that by applying the compositions of each hesperidin modified derivative and their combinations to tyrosinase, among them, both 3’,5-dimethylhesperidin and 3’-methylhesperidin have a certain tyrosinase inhibitory effect, and the two can have a synergistic effect when used in combination. At the same time, the effect of the combination of the two changes with the change of the ratio of the two in the mixture. When the ratio of 3’,5-dimethylhesperidin to 3’-methylhesperidin is in the range of 0.8:1 to 1.25:1, it has a better tyrosinase inhibitory effect than the single use of the monomer. On this basis, a preparation method for preparing a tyrosinase inhibitor product conforming to the above ratio range from a hesperidin derivatization reaction system was developed.

[0017] Preferably, the molar ratio of the 3’,5-dimethylhesperidin and the 3’-methylhesperidin includes 0.8 to 1.25:1.

[0018] A preparation method of the above-mentioned preparation includes the following steps:

[0019] S1. Using the hesperidin raw material as the initial raw material, adding a methylation reagent and heating for reaction to obtain a methylation product;

[0020] S2. Adding the methylation product to an organic solvent-water binary mixed solution, and after mixing, formulating it into a raw material sample solution;

[0021] S3. Adding the raw material sample solution to a resin material for sample loading treatment to obtain a sample-loaded solution;

[0022] S4. Load the sample solution onto the column, first perform a primary elution with purified water, and then perform a secondary elution with an organic solvent - water binary mixed solution. Monitor the elution endpoint by the ultraviolet rapid detection method, collect the eluate of the secondary elution and dry it to obtain the preparation.

[0023] Preferably, in the organic solvent - water binary mixed solution, the volume ratio of the organic solvent includes 5% - 25%; the organic solvent includes one or more of methanol, ethanol, and acetone; the concentration of the raw material sample solution includes 10 - 15 mg / mL; the resin material includes one or more of the macroporous resins HPD100, AB - 8, and D101.

[0024] Preferably, in the organic solvent - water binary mixed solution, the volume ratio of the organic solvent includes 10% - 20%; the operation of step S1 includes: dissolving the initial raw material with a sodium hydroxide solution, adding dimethyl carbonate and zinc chloride, and reacting in a thermostatic heating and stirring collector, the reaction temperature is 70 degrees Celsius, and the reaction time is 3 hours; in step S2, the mixing treatment includes ultrasonic treatment; in step S3, the resin material is pretreated before use; the sample loading treatment includes oscillation treatment.

[0025] Preferably, in step S2, the operation of the ultrasonic treatment includes: ultrasonic treatment at 300 W for 5 - 10 minutes; in step S3, the operation of the pretreatment includes: weighing the resin material in an amount 5 - 6 times the volume of the raw material sample solution, adding 95% ethanol to completely immerse the resin material, soaking for more than 10 hours, removing the ethanol by suction filtration, slowly adding purified water in an amount 3 - 4 times the volume of the ethanol to wash the resin material, and then drying it in an electrothermal constant temperature blast drying oven for 3 - 4 hours, the drying temperature is 110 - 120 degrees Celsius; the operation of the oscillation treatment includes: constant temperature oscillation in a water bath, the water bath temperature is 30 - 40 degrees Celsius, the oscillation speed is 180 - 220 revolutions per minute, and the time is 1.5 - 2 hours; in step S4, the operation of the primary elution includes: eluting with 2 - 3 times the column volume of purified water; the polarity of the organic solvent - water binary mixed solution in step S4 is not less than that in step S2.

[0026] The polarity of the organic solvent - water binary mixed solution used in S4 needs to be not less than that used in S2, that is, when the same organic solvent is selected, the proportion of the organic solvent is not less than that in S2, and when different organic solvents are selected but with the same proportion, they are selected according to the polarity order of methanol > ethanol > acetone.

[0027] Preferably, in the secondary elution, monitor the ratio of 3’,5 - dimethyl hesperidin and 3’ - methyl hesperidin as the elution endpoint.

[0028] A preparation method of a tyrosinase inhibitor, comprising the following steps:

[0029] 1. Select hesperidin raw material as the initial raw material, add a methylation reagent and heat to react to obtain a methylation product;

[0030] 2. Add an organic solvent aqueous solution of 10%-20% (the organic solvent can be one of ethanol, methanol, and acetone) to the methylation product in step 1, ultrasonically treat for 5-10 minutes, and prepare a methylation sample solution with a concentration of 10-15 mg per milliliter;

[0031] 3. Weigh 5-6 times the amount of resin material (the resin material is one of the macroporous resins with the models of HPD100, AB-8, and D101), add 95% ethanol until the resin material is completely immersed, soak for more than 10 hours, remove the ethanol by suction filtration, slowly add purified water 3-4 times the amount of ethanol to wash the resin material, and then place it in an electrothermal constant temperature blast drying oven to dry for 3-4 hours, and the drying temperature is 110-120 °C;

[0032] 4. After the resin material is activated, add the methylation sample solution in step 2, and place it in a water bath constant temperature oscillator for sample loading treatment for 1.5-2 hours. The sample loading treatment conditions are 30-40 °C and 180-220 revolutions per minute;

[0033] 5. Pack the resin material containing the methylation sample solution after the sample loading treatment. After washing off the residual solution in the column, elute with 2-3 times the column volume of purified water to remove water-soluble impurities, and then elute with an organic solvent aqueous solution of 5%-25%. Use the ultraviolet rapid detection method for dynamic monitoring, and monitor until the ratio of 3',5-dimethylhesperidin to 3'-methylhesperidin is about 1 as the elution end point;

[0034] 6. The operation of the ultraviolet rapid detection method is as follows:

[0035] A. Establishment of the linear relationship formula: Weigh anhydrous aluminum chloride and prepare a reaction reagent with methanol at 0.5 mg / ml; weigh the reference substances of 3',5-dimethylhesperidin and 3'-methylhesperidin respectively, and quantitatively prepare test solutions with different ratios (reference solution A / reference solution B: 0.5, 0.75, 1, 1.25, 1.5); immediately place it in a heating and stirring device with constant temperature after adding the reaction reagent for reaction, the water bath temperature is 50 °C, and the reaction time is 3 minutes; after detection by an ultraviolet-visible spectrophotometer (parameter settings: scanning range is 600-190 nm; photometric mode is Abs; scanning speed is fast; scanning interval is 0.2 nm), make a curve based on the ratio of the absorbance at the maximum absorption wavelength of 3',5-dimethylhesperidin and 3'-methylhesperidin and the actually prepared ratio, and perform linear regression analysis to obtain the linear relationship formula between the ultraviolet absorbance ratio and the actual ratio.

[0036] B. Rapid detection of eluted sample: Immediately add aluminum chloride methanol solution to the sample solution and then place it in a thermostatic heating and stirring device with heat collection for reaction. The water bath temperature is 50°C and the reaction time is 3 minutes. Immediately perform detection after the reaction is completed, calculate the ratio, and use the formula to obtain the actual proportion of the main components in the API.

[0037] 7. Collect the eluate and dry it using one or more of vacuum drying, spray drying, and freeze drying to obtain the composition product (the ratio of 3’,5-dimethylhesperidin to 3’-methylhesperidin is 0.8:1 - 1.25:1).

[0038] For the composition sample prepared by the above preparation method, the ratio of the main components 3’,5-dimethylhesperidin to 3’-methylhesperidin is between 0.8:1 and 1.25:1. Through the tyrosinase activity influence experiment, it is determined that under this ratio condition, 3’-methylhesperidin and 3’,5-dimethylhesperidin can have a synergistic additive effect, and the combination of the two at this ratio shows a better tyrosinase inhibitory effect than when used alone.

[0039] An application of 3’,5-dimethylhesperidin for preparing a drug for preventing or treating hyperpigmentation diseases of the skin.

[0040] Preferably, it is used for preparing a drug for inhibiting tyrosinase.

[0041] An application of a combination of 3’,5-dimethylhesperidin and 3’-methylhesperidin for preparing a drug for preventing or treating hyperpigmentation diseases of the skin.

[0042] Preferably, it is used for preparing a drug for inhibiting tyrosinase.

[0043] Compared with the prior art, implementing the present invention has the following beneficial effects:

[0044] 1. Aiming at the problems existing in the prior art, a tyrosinase inhibitor product with a fixed main component ratio range can be simply prepared, and under this component ratio range condition, the main components 3’-methylhesperidin and 3’,5-dimethylhesperidin of the product can have a synergistic effect and exert a better tyrosinase inhibitory effect than when used alone;

[0045] 2. This method has low cost and high safety and is applicable to industrial production. Detailed implementation mode

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, the following provides embodiments for further detailed description of the present invention, so that those skilled in the art can better understand the present invention and be able to implement it. However, the examples given are not intended to limit the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps, or conditions of the present invention all fall within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art.

[0047] Example 1

[0048] Through a tyrosinase inhibition test, compare the different effects of the single use of 3’,5-dimethylhesperidin monomer and 3’-methylhesperidin monomer and the combined use of compositions in different ratios on enzyme activity inhibition. Specifically, it includes the following steps:

[0049] (1) Weigh 14.33 g of Na2HPO4·12H2O and dissolve it in 200 mL of purified water; weigh 2.1 g of C6H8O7·H2O and dissolve it in 100 mL of water; measure 154.5 mL of the Na2HPO4·12H2O solution and add 45.5 mL of the C6H8O7·H2O solution to prepare a sodium bicarbonate-citric acid buffer solution.

[0050] (2) Weigh 1 mg of tyrosinase and dissolve it in 10 mL of buffer solution; weigh 20 mg of L-dopa and dissolve it in 20 mL of buffer solution.

[0051] (3) Weigh the reference substance of 3’-methylhesperidin and the reference substance of 3’,5-dimethylhesperidin, and prepare a single-substance test solution and a mixed test solution in different ratios (3’,5-dimethylhesperidin: 3’-methylhesperidin is 0.2, 0.4, 0.6, 0.8, 1, 1.25, 1.67, 2.5, 5) respectively.

[0052] (4) Add 100 μL of the test solution to a 96-well plate respectively, then add 50 μL of the enzyme solution and the buffer solution, shake evenly, and incubate at 40 °C for 10 minutes.

[0053] (5) Add 100 μL of the L-dopa solution respectively, vortex for 1 minute, and measure the absorbance at 475 nm in an enzyme-linked immunosorbent assay (ELISA) reader. The test results are shown in Table 1.

[0054] Table 1

[0055]

[0056] The detection results were statistically processed using SPSS software. One-way analysis of variance was employed, and P < 0.05 was considered statistically significant. In multiple comparisons (LSD), when the ratio of 3’,5-dimethylhesperidin to 3’-methylhesperidin was 0.8, 1, and 1.25, there were significant differences in the inhibition rates compared to when the monomers were used alone. Combining the detection results with the statistical results, when 3’,5-dimethylhesperidin and 3’-methylhesperidin were used in combination, they exhibited superior tyrosinase inhibitory effects compared to using the monomers alone. Additionally, the ratio of 3’,5-dimethylhesperidin to 3’-methylhesperidin affected the tyrosinase inhibitory effect when the two were used in combination. When the ratio of 3’,5-dimethylhesperidin to 3’-methylhesperidin was 0.8 - 1.25, the inhibitory effect of the mixture was optimal.

[0057] Example 2

[0058] A preparation method of a tyrosinase inhibitor. Specifically, it includes the following steps:

[0059] (1) Select a commercially available hesperidin raw material as the initial raw material, add a methylation reagent, and heat to react to obtain a methylation product;

[0060] (2) Add 100.0 mg of the methylation product to an aqueous ethanol solution of 10%, and ultrasonically treat for 10 minutes to prepare a methylation sample solution with a concentration of 10 mg per milliliter;

[0061] (3) Weigh 500 mg of styrene-type non-polar HPD100 macroporous resin material, add 95% ethanol until the resin material is completely immersed, soak for 14 hours, remove the ethanol by suction filtration, slowly add purified water three times the amount of ethanol to wash the resin material, and then place it in an electrothermal constant temperature forced air drying oven to dry for 3 hours, with a drying temperature of 115 °C;

[0062] (4) After activating the resin material, add the methylation sample solution described in (2), and place it in a water bath constant temperature oscillator for sample loading treatment for 1.5 hours. The sample loading treatment conditions are 30 °C and 200 revolutions per minute;

[0063] (5) Pack the resin material containing the methylation sample solution after sample loading treatment into a column. After washing to remove the residual solution in the column, elute with three times the column volume of purified water to remove water-soluble impurities, and then elute with a 20% organic solvent aqueous solution. Use the ultraviolet rapid detection method for dynamic monitoring, and monitor until the ratio of 3’,5-dimethylhesperidin to 3’-methylhesperidin is approximately 1 as the elution end point;

[0064] (6) Collect the eluate, perform vacuum drying to obtain 15.3 mg of product, and the ratio of 3’,5-dimethylhesperidin to 3’-methylhesperidin was detected by HPLC to be 1.0547.

[0065] Example 3

[0066] A preparation method of a tyrosinase inhibitor. Specifically, it includes the following steps:

[0067] (1) Select the commercially available hesperidin raw material as the initial raw material, add a methylation reagent and heat to react to obtain a methylation product;

[0068] (2) Add 200.0 mg of the methylation product to an aqueous ethanol solution of 10%, and ultrasonically treat for 10 minutes to prepare a methylation sample solution with a concentration of 10 mg per milliliter;

[0069] (3) Weigh 1100 mg of styrene-type non-polar D101 macroporous resin material, add 95% ethanol until the resin material is completely immersed, soak for 10 hours, remove the ethanol by suction filtration, slowly add purified water three times the amount of ethanol to wash the resin material, and then place it in an electrothermal constant temperature forced air drying oven to dry for 3 hours, and the drying temperature is 115 °C;

[0070] (4) After the resin material is activated, add the methylation sample solution described in (2), and place it in a water bath constant temperature oscillator for sample loading treatment for 2 hours. The sample loading treatment conditions are 35 °C and 210 revolutions per minute;

[0071] (5) Pack the resin material containing the methylation sample solution after the sample loading treatment into a column. After washing off the residual solution in the column, elute with three times the column volume of purified water to remove water-soluble impurities, and then elute with an aqueous solution of 25% organic solvent. Use the ultraviolet rapid detection method for dynamic monitoring, and monitor until the ratio of 3',5-dimethylhesperidin to 3'-methylhesperidin is about 1 as the elution end point;

[0072] (6) Collect the eluate, perform vacuum drying to obtain 75.9 mg of the product, and the ratio of 3',5-dimethylhesperidin to 3'-methylhesperidin is 0.8775 by HPLC detection.

[0073] Example 4

[0074] A preparation method of a tyrosinase inhibitor. Specifically, it includes the following steps:

[0075] (1) Select the commercially available hesperidin raw material as the initial raw material, add a methylation reagent and heat to react to obtain a methylation product;

[0076] (2) Add 1.00 g of the methylation product to an aqueous ethanol solution of 20%, and ultrasonically treat for 10 minutes to prepare a methylation sample solution with a concentration of 10 mg per milliliter;

[0077] (3) Weigh 7 grams of styrene-based non-polar HPD100 macroporous resin material, add 95% ethanol until the resin material is completely immersed, soak for 15 hours, remove the ethanol by suction filtration, slowly add purified water three times the amount of ethanol to wash the resin material, and then place it in an electrothermal constant temperature blast drying oven to dry for 3 hours, with the drying temperature being 115 degrees Celsius;

[0078] (4) After the resin material is activated, add the methylated sample solution described in (2), and place it in a water bath constant temperature oscillator for sample loading treatment for 2 hours. The sample loading treatment conditions are 30 degrees Celsius and 180 revolutions per minute;

[0079] (5) Pack the resin material containing the methylated sample solution after the sample loading treatment into a column. After washing off the residual solution in the column, elute with purified water three times the column volume to remove water-soluble impurities, and then elute with a 20% organic solvent aqueous solution. Use the ultraviolet rapid detection method for dynamic monitoring, and monitor until the ratio of 3’,5-dimethylhesperidin to 3’-methylhesperidin is about 1 as the elution end point;

[0080] (6) Collect the eluate, perform vacuum drying to obtain 521.1 milligrams of product. The ratio of 3’,5-dimethylhesperidin to 3’-methylhesperidin detected by HPLC is 1.0998.

[0081] Example 5

[0082] A preparation method of a tyrosinase inhibitor. Specifically, it includes the following steps:

[0083] (1) Select commercially available hesperidin raw material medicine as the initial raw material, add a methylation reagent and heat to react to obtain a methylation product;

[0084] (2) Add 1.015 kilograms of the methylation product to a 10% ethanol aqueous solution, ultrasonically treat for 20 minutes, and prepare a methylation sample solution with a concentration of 10 milligrams per milliliter;

[0085] (3) Weigh 5.455 kilograms of styrene-based non-polar HPD100 macroporous resin material, add 95% ethanol until the resin material is completely immersed, soak for 10 hours, remove the ethanol by suction filtration, slowly add purified water three times the amount of ethanol to wash the resin material, and then place it in an electrothermal constant temperature blast drying oven to dry for 5 hours, with the drying temperature being 115 degrees Celsius;

[0086] (4) After the resin material is activated, add the methylated sample solution described in (2), and place it in a water bath constant temperature oscillator for sample loading treatment for 2 hours. The sample loading treatment conditions are 40 degrees Celsius and 200 revolutions per minute;

[0087] (5) The resin material containing the methylated sample solution after sample loading treatment is packed into a column. After washing away the residual solution in the column, it is eluted with 3 column volumes of purified water to remove water-soluble impurities, and then eluted with a 20% aqueous solution of organic solvent. The dynamic monitoring is carried out by the ultraviolet rapid detection method, and the elution end point is monitored until the ratio of 3’,5-dimethyl hesperidin to 3’-methyl hesperidin is about 1.

[0088] (6) The eluate is collected and dried under reduced pressure to obtain 0.1961 kg of product. The ratio of 3’,5-dimethyl hesperidin to 3’-methyl hesperidin detected by HPLC is 1.1029.

[0089] Comparative Example 1

[0090] An experiment was conducted to compare the effects of the methylated product and the product obtained in Example 2 on tyrosinase activity. The methylated product was prepared by referring to the method in the literature (Chen Jianqiu, Yang Ke, Hu Zhijun. Study on the Derivatization Modification and Free Radical Scavenging of Hesperidin [J]. Journal of Anhui Agricultural Sciences, 2009, 37(30): 14544-14546), and the specific steps are as follows:

[0091] (1) Select the commercially available hesperidin raw material as the initial raw material, weigh 50.1 mg, dissolve it in 10 mL of sodium hydroxide solution, add 44.4 mg of dimethyl carbonate and 5.2 mg of zinc chloride, and place it in a thermostatic heating and stirring device with heat collection for reaction. The reaction temperature is 70 °C and the reaction time is 3 hours.

[0092] (2) After the reaction is completed, pour the reaction system into an ice-water solution of concentrated hydrochloric acid, let it stand for 2 hours, and then filter. The filter residue is dried to obtain 13.5 mg of the methylated product.

[0093] The methylated product obtained in (2) was detected by HPLC. It was found that the proportion of 3’,5-dimethyl hesperidin was 12.90%, and the proportion of 3’-methyl hesperidin was 47.54%. In addition to these two signal peaks, there were 5 other signal peaks, with proportions of 5.24%, 0.39%, 2.55%, 31.04% and 0.34% respectively.

[0094] The comparative experiment on the effect of tyrosinase activity specifically includes the following steps:

[0095] (1) Weigh 14.33 g of Na2HPO4·12H2O and dissolve it in 200 mL of purified water; weigh 2.1 g of C6H8O7·H2O and dissolve it in 100 mL of water; measure 154.5 mL of the Na2HPO4·12H2O solution and add 45.5 mL of the C6H8O7·H2O solution to prepare a sodium bicarbonate-citric acid buffer solution.

[0096] (2) Weigh 1 mg of tyrosinase and dissolve it in 10 mL of buffer solution; weigh 20 mg of L-dopa and dissolve it in 20 mL of buffer solution;

[0097] (3) Weigh 3 mg of the methylation product and the product obtained in Example 1 respectively, and prepare sample solutions with a concentration of 1 mg / mL;

[0098] (4) Set up sample tubes (T), sample backgrounds (T0), enzyme reaction tubes (C), and solvent backgrounds (C0). The specific liquid addition requirements for each tube are shown in Table 2.

[0099] Table 2

[0100] T T0 C C0 Sample solution 1 1 / / Tyrosinase solution 0.5 / 0.5 / Buffer solution / 0.5 / 0.5 L-DOPA solution 2 2 2 2

[0101] (5) First, add the sample solution and the tyrosinase solution and mix them evenly, then incubate in a 40 °C water bath for 10 minutes;

[0102] (6) Add the L-dopa solution to each tube in sequence, control the reaction time to be 5 minutes, immediately transfer the solution in each tube into a cuvette, measure the absorbance at 480 nm, and calculate the inhibition rate according to the formula (Inhibition rate = [1 - (T - T0) / (C - C0)] * 100%);

[0103] (7) It is calculated that the tyrosinase inhibition rate of the methylation product is 36.62%, and the tyrosinase inhibition rate of the product of Example 2 is 49.78%, with an improvement of 35.93%. This shows that the product prepared by the preparation method of the present invention has a more excellent tyrosinase inhibitor.

[0104] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. Use of 3′,5-dimethylhesperidin, characterized in that, For preparing a preparation for preventing or treating skin hyperpigmentation.

2. The application of 3′,5-dimethyl hesperidin according to claim 1, characterized in that For preparing a preparation for inhibiting melanogenesis.

3. The application of 3’,5-dimethyl hesperidin according to claim 1, characterized in that, For preparing a preparation for inhibiting tyrosinase.

4. A preparation containing 3′,5-dimethylhesperidin as described in claim 1, characterized in that, Comprising the 3’,5-dimethylhesperidin and 3’-methylhesperidin.

5. The preparation according to claim 4, characterized in that, The molar ratio of the 3’,5-dimethylhesperidin and 3’-methylhesperidin is 0.8 - 1.25:

1.

6. A method for preparing the preparation according to claim 1, characterized in that, Comprising the following steps: S1. Using hesperidin raw material as the initial raw material, adding a methylation reagent and heating for reaction to obtain a methylation product; S2. Adding the methylation product into an organic solvent-water binary mixed solution, and formulating into a raw material sample solution after mixing treatment; S3. Adding the raw material sample solution to a resin material for sample loading treatment to obtain a sample-loaded solution; S4. Loading the sample-loaded solution onto a column, first performing a primary elution with purified water, then performing a secondary elution with an organic solvent-water binary mixed solution, monitoring the elution end point by ultraviolet rapid detection method, collecting the eluate of the secondary elution and drying to obtain the preparation.

7. The preparation method according to claim 6, characterized in that, In the organic solvent-water binary mixed solution, the volume proportion of the organic solvent is 5% - 25%; the organic solvent includes one or more of methanol, ethanol, and acetone; the concentration of the raw material sample solution is 10 - 15 mg / mL; the resin material includes one or more of macroporous resins such as HPD100, AB-8, and D101.

8. The preparation method according to claim 6, characterized in that, In the organic solvent-water binary mixed solution, the volume proportion of the organic solvent is 10% - 20%; the operation of step S1 includes: dissolving the initial raw material with a sodium hydroxide solution, adding dimethyl carbonate and zinc chloride, and reacting in a heat-collecting type constant temperature heating stirrer, the reaction temperature is 70 degrees Celsius, and the reaction time is 3 hours; in step S2, the mixing treatment includes ultrasonic treatment; in step S3, the resin material is pretreated before use; the sample loading treatment includes oscillation treatment.

9. The preparation method according to claim 8, characterized in that, In step S2, the operation of the ultrasonic treatment includes: ultrasonic treatment at 300 W for 5 - 10 minutes; in step S3, the operation of the pretreatment includes: weighing the resin material in an amount 5 - 6 times the volume of the raw material sample solution, adding 95% ethanol until the resin material is completely immersed, soaking for more than 10 hours, removing the ethanol by suction filtration, slowly adding purified water in an amount 3 - 4 times the volume of ethanol to wash the resin material, and then drying in an electrothermal constant temperature forced air drying oven for 3 - 4 hours, the drying temperature is 110 - 120 degrees Celsius; the operation of the oscillation treatment includes: water bath constant temperature oscillation, the water bath temperature is 30 - 40 degrees Celsius, the oscillation speed is 180 - 220 revolutions per minute, and the time is 1.5 - 2 hours; in step S4, the operation of the primary elution includes: eluting with 2 - 3 times the column volume of purified water; the polarity of the organic solvent-water binary mixed solution in step S4 is not less than that in step S2.

10. The preparation method according to claim 6, characterized in that, In the secondary elution, monitoring the ratio of the 3’,5-dimethylhesperidin and 3’-methylhesperidin as the elution end point.

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