Whitening and anti-sensitivity compositions prepared from deep eutectic solvents and methods of making and using the same

Abstract

The present application relates to the field of cosmetic technology (IPC classification A61K36), and more particularly to a whitening and anti-allergic composition prepared by a eutectic solvent, a preparation method and application thereof, the composition comprising white peony flower, white water lily flower, white chrysanthemum and white plum blossom. The present application selects white flowers, white peony flower, white water lily flower, white chrysanthemum and white plum blossom, which are representative in spring, summer, autumn and winter respectively, as the plant components for whitening and anti-allergy, has low anthocyanin and cyanidin content, high extraction efficiency of active ingredients by microjet extraction process, and good flowability and stability of the extract.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology (IPC classification number A61K36), and more particularly to whitening and anti-allergic compositions prepared with eutectic solvents, their preparation methods, and applications. Background Technology

[0002] Fair skin has always been a popular aesthetic standard, leading to the addition of various whitening ingredients to skincare products. However, many whitening ingredients, such as niacinamide, vitamin C, and arbutin, are irritating and have low skin-friendly properties with long-term use. As society evolves, consumers' skincare needs are no longer limited to whitening alone; anti-allergy and soothing effects have become a trend in modern consumer demand.

[0003] Flower-based skincare is a time-honored skincare philosophy across cultures. While whitening and soothing plant compositions often contain floral ingredients, they are typically a combination of flower and leaf or fruit extracts; pure flower compositions are rare, and the proportion of floral components is generally low. Floral ingredients are rich in anthocyanins, flavonoids, polyphenols, vitamins, and other active ingredients with whitening and anti-allergic effects. Traditional water or dilute alcohol extraction methods produce flower extracts with low toxicity, but the solvent polarity range is limited, resulting in low active ingredient content and poor stability. Although low-temperature microfluidic technology can increase the content of effective plant components and offers better stability, its potential for improvement is limited. Therefore, there is an urgent need for a highly efficient, green, and safe low-temperature microfluidic extraction technology that combines extraction solvents. Eutectic solvents, as a novel low-melting-point solvent, utilize a new hydrogen bond force composed of hydrogen bond donors and acceptors. They offer advantages such as convenient preparation, low melting point, low toxicity, and biodegradability, attracting widespread attention from the chemical and biopharmaceutical industries. However, some hydrogen bond acceptors cannot be used in industries such as food and cosmetics, leading to the development of natural eutectic solvents, which have shown great potential.

[0004] Chinese patent CN112933131A discloses a method for preparing Eucommia ulmoides extract using natural eutectic solvents. The hydrogen bond donors used include L-lactic acid, sorbitol, urea, DL-malic acid, D-fructose, glucose, and citric acid, while the hydrogen bond acceptors include choline chloride, betaine, and citric acid. However, solvent systems such as lactic acid, citric acid, and choline chloride are highly irritating to the skin. Chinese patent CN113262179A discloses a plant extract composition with anti-allergic effects, its preparation method, and its uses. The eutectic solvent system using betaine, 1,3-butanediol, and 1,2-hexanediol in the technical solution has been verified to be unsuitable for pure flower plant systems. It is evident that few existing technologies possess a eutectic solvent system that can efficiently extract pure flower components while simultaneously achieving balanced whitening and soothing effects. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the first aspect of the present invention provides a whitening and anti-allergic composition prepared by a eutectic solvent, the composition comprising white peony flower, white water lily, Hangzhou white chrysanthemum, and white plum blossom.

[0006] In some embodiments, the composition comprises, by weight, 30-50 parts of white peony flowers, 10-20 parts of white water lily, 20-40 parts of Hangzhou white chrysanthemum, and 10-20 parts of white plum blossom.

[0007] A second aspect of the present invention provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent, the method comprising adding the composition to a betaine eutectic solvent, and extracting the resulting mixture using a microfluidic extractor to obtain the whitening and anti-allergic composition.

[0008] Microfluidic technology is a novel nanoparticle preparation technique that offers advantages such as reducing the particle size of active ingredients without damaging them and achieving a uniform and concentrated particle size distribution. The resulting suspended particles can reach the nanometer range. During the research process, the applicant discovered that using a microfluidic extractor can significantly improve the extraction rate of active ingredients in the composition. However, the darker the color of the extracted substance, the worse the stability of the resulting extract. It is speculated that this may be because darker-colored flowers have a higher content of anthocyanins, resulting in poorer structural stability.

[0009] In some embodiments, the raw materials for preparing the betaine eutectic solvent include betaine, hydrogen bond donor, and water.

[0010] In some embodiments, the molar ratio of betaine to hydrogen bond donor is 1:(1-5), and examples include 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, and 1:5, but it is not limited to these examples.

[0011] Based on the design requirement that the composition consists of white flowers, the applicant found in the experiment that the extract obtained has the best stability when the molar ratio of betaine to hydrogen bond donor is 1:(1-5). When the content of hydrogen bond donor is too small, the solubility of betaine is poor and the two cannot form a clear and homogeneous solution. When the content of hydrogen bond donor is too large, although a clear and homogeneous solution can be formed, the extraction rate of active ingredients of plant materials is hardly improved.

[0012] In some embodiments, the hydrogen bond donor includes at least one of urea, polyol, and organic acid.

[0013] Furthermore, the hydrogen bond donor includes at least one of urea, glycerol, butylene glycol, hexanediol, pentanediol, sorbitol, xylitol, lactic acid, citric acid, salicylic acid, and malic acid.

[0014] The maximum allowable amount of organic acids in the "Cosmetic Safety Technical Specifications" (2015 edition) is 0.4 μg / g. If formulated into a eutectic solvent, the content of organic acids will far exceed the allowable amount. Therefore, the betaine-organic acid system is not considered.

[0015] To balance the extraction rate and the soothing effect, the hydrogen bond donor further includes at least one of urea, glycerol, and butylene glycol, and more specifically, the hydrogen bond donor is urea, glycerol, or butylene glycol.

[0016] Furthermore, the molar ratio of betaine to butylene glycol is 1:(1-3).

[0017] Furthermore, the molar ratio of betaine to glycerol is 1:(1-4).

[0018] Furthermore, the molar ratio of betaine to urea is 1:(1-5).

[0019] In some embodiments, the volume fraction of water in the betaine eutectic solvent is 10-60%.

[0020] In some embodiments, the micropore diameter of the microjets extractor is 500-800 μm.

[0021] To further enhance the extraction effect of active ingredients, in some embodiments, the mass ratio of the betaine eutectic solvent to the composition is (8-12):1.

[0022] Furthermore, such as Figure 1 As shown, the preparation method includes mixing the composition evenly, pulverizing, sieving, adding betaine eutectic solvent, placing the resulting mixture in a microfluidic extractor, stirring for 10-60 min at 20-25℃, 500-1000 rpm, and a feeding rate of 10-20 L / min, removing the filter residue using a horizontal screw centrifuge at 2000-5000 rpm, and removing other impurities using a high-speed separator at 5000-10000 rpm to obtain a filtrate, which is further filtered using a reverse osmosis membrane to obtain a whitening and anti-allergic composition.

[0023] A third aspect of the invention provides the application of the whitening and anti-allergic composition prepared by the eutectic solvent or the preparation method described herein in the preparation of cosmetics.

[0024] In some embodiments, the whitening and anti-allergic composition is added to the cosmetic at a ratio of 0.1-30 wt%, and the dosage form of the cosmetic includes, but is not limited to, creams, serums, gels, and lotions.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. This invention selects representative white flowers from each of the four seasons—white peony, white water lily, Hangzhou white chrysanthemum, and white plum blossom—as whitening and anti-allergic plant ingredients. The anthocyanin and anthocyanin content is low, the microfluidic extraction process has high extraction efficiency of active ingredients, and the extract has good fluidity and stability.

[0027] 2. This invention selects biocompatible natural substances as hydrogen bond donors and acceptors. The prepared eutectic solvent can maintain good viscosity in aqueous solvents, further increasing the content of effective components in plant extracts. It is reusable, easily biodegradable, and the microfluidic extraction process does not require the addition of other chemical reagents or the use of complex processes. The extraction yield of effective components is significantly improved, while having no toxic side effects and low irritation, which is conducive to industrial production.

[0028] 3. Compared with hydrogen bond donors such as lactic acid and xylitol in the prior art, the present invention selects specific urea, butylene glycol and glycerol as hydrogen bond donors and a specific ratio of betaine to specifically extract the white flowers of the present invention. The extract has been tested in multiple tests and has good antioxidant and whitening effects, without allergic irritation problems. It achieves the multi-effect combination of high extraction rate, whitening and anti-allergy in the prior art.

[0029] 4. By combining microfluidic technology with specific flowers, the problem of easy product layering or turbidity is solved. The prepared whitening and anti-allergic composition is suitable for a variety of skin care products, and the addition amount can be as high as 30wt% while maintaining system stability.

[0030] 5. The preparation process of this invention is applicable to product development in modern cosmetic enterprises and has extremely high economic value. Attached Figure Description

[0031] Figure 1 This is a flowchart illustrating the preparation process of the whitening and anti-allergic composition of the present invention.

[0032] Figure 2 This is a comparison diagram of the solution states of Example 1 and Comparative Example 2.

[0033] Figure 3-11 This is the test report for the test case. Detailed Implementation

[0034] Example 1

[0035] The first aspect of this embodiment provides a whitening and anti-allergic composition prepared by a eutectic solvent, wherein the composition comprises, by weight, 40 parts of white peony flower, 15 parts of white water lily, 35 parts of Hangzhou white chrysanthemum, and 10 parts of white plum blossom.

[0036] The second aspect of this embodiment provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The preparation method includes mixing the composition evenly, pulverizing it, passing it through a 12mm sieve, adding betaine eutectic solvent, placing the resulting mixture in a microfluidic extractor (micropore size of 800μm), stirring it for 30min at 20℃, 600rpm, and a feeding rate of 15L / min, removing the filter residue using a horizontal screw centrifuge at 3000rpm, and removing other impurities using a high-speed separator at 8000rpm to obtain a filtrate, further filtering the filtrate using a reverse osmosis membrane (pore size of 0.5nm) to obtain the whitening and anti-allergic composition.

[0037] The raw materials for preparing the betaine eutectic solvent are an aqueous solution of 1,3-butanediol and betaine (molar ratio 2:1) with a water content of 30 wt%.

[0038] The mass ratio of the betaine eutectic solvent to the composition is 10:1.

[0039] The third aspect of this embodiment provides the application of the whitening and anti-allergic composition prepared by the eutectic solvent in the preparation of cosmetics.

[0040] Example 2

[0041] The first aspect of this embodiment provides a whitening and anti-allergic composition prepared by a eutectic solvent, wherein the composition comprises, by weight, 35 parts of white peony flower, 15 parts of white water lily, 35 parts of Hangzhou white chrysanthemum, and 15 parts of white plum blossom.

[0042] The second aspect of this embodiment provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The preparation method includes mixing the composition evenly, pulverizing it, passing it through a 12 mm sieve, adding betaine eutectic solvent, placing the resulting mixture in a microfluidic extractor (micropore size of 600 μm), stirring it for 50 min at 20°C, 500 rpm, and a feeding rate of 20 L / min, removing the filter residue using a horizontal screw centrifuge at 5000 rpm, and removing other impurities using a high-speed separator at 10000 rpm to obtain a filtrate, further filtering the filtrate using a reverse osmosis membrane (pore size of 0.5 nm) to obtain the whitening and anti-allergic composition.

[0043] The raw materials for preparing the betaine eutectic solvent are an aqueous solution of glycerol and betaine (molar ratio 1:1) with a water content of 40 wt%.

[0044] The mass ratio of the betaine eutectic solvent to the composition is 8:1.

[0045] The third aspect of this embodiment provides the application of the whitening and anti-allergic composition prepared by the eutectic solvent in the preparation of cosmetics.

[0046] Example 3

[0047] The first aspect of this embodiment provides a whitening and anti-allergic composition prepared by a eutectic solvent, wherein the composition comprises 30 parts by weight of white peony flower, 20 parts by weight of white water lily, 30 parts by weight of Hangzhou white chrysanthemum, and 20 parts by weight of white plum blossom.

[0048] The second aspect of this embodiment provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The preparation method includes mixing the composition evenly, pulverizing it, passing it through a 12 mm sieve, adding betaine eutectic solvent, placing the resulting mixture in a microfluidic extractor (micropore size of 700 μm), stirring it for 60 min at 20°C, 1000 rpm, and a feeding rate of 20 L / min, removing the filter residue using a horizontal screw centrifuge at 5000 rpm, and removing other impurities using a high-speed separator at 8000 rpm to obtain a filtrate, and further filtering the filtrate using a reverse osmosis membrane (pore size of 0.5 nm) to obtain the whitening and anti-allergic composition.

[0049] The raw material for preparing the betaine eutectic solvent is an aqueous solution of urea and betaine (molar ratio 1:1) with a water content of 40 wt%.

[0050] The mass ratio of the betaine eutectic solvent to the composition is 8:1.

[0051] The third aspect of this embodiment provides the application of the whitening and anti-allergic composition prepared by the eutectic solvent in the preparation of cosmetics.

[0052] Example 4

[0053] The first aspect of this embodiment provides a whitening and anti-allergic composition prepared with a eutectic solvent, and the specific implementation method is the same as in Example 1.

[0054] The second aspect of this embodiment provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The specific implementation method is the same as in Example 1, except that the raw material for preparing the betaine eutectic solvent is an aqueous solution of 1,3-butanediol and betaine (molar ratio 3:1) with a water content of 30 wt%.

[0055] The third aspect of this embodiment provides the application of the whitening and anti-allergic composition prepared by the eutectic solvent in the preparation of cosmetics.

[0056] Example 5

[0057] The first aspect of this embodiment provides a whitening and anti-allergic composition prepared with a eutectic solvent, and the specific implementation method is the same as in Example 2.

[0058] The second aspect of this embodiment provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The specific implementation method is the same as in Example 2, except that the raw material for preparing the betaine eutectic solvent is an aqueous solution of glycerol and betaine (molar ratio 4:1) with a water content of 40 wt%.

[0059] The third aspect of this embodiment provides the application of the whitening and anti-allergic composition prepared by the eutectic solvent in the preparation of cosmetics.

[0060] Example 6

[0061] The first aspect of this embodiment provides a whitening and anti-allergic composition prepared with a eutectic solvent, and the specific implementation method is the same as in Example 3.

[0062] The second aspect of this embodiment provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The specific implementation method is the same as in Example 3, except that the raw material for preparing the betaine eutectic solvent is an aqueous solution of urea and betaine (molar ratio 5:1) with a water content of 40 wt%.

[0063] The third aspect of this embodiment provides the application of the whitening and anti-allergic composition prepared by the eutectic solvent in the preparation of cosmetics.

[0064] Comparative Example 1

[0065] The first aspect of this comparative example provides a whitening and anti-allergic composition prepared with a eutectic solvent, and the specific implementation method is the same as in Example 1.

[0066] The second aspect of this comparative example provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The specific implementation method is the same as in Example 1, except that the raw material for preparing the betaine eutectic solvent is pure water.

[0067] The third aspect of this comparative example provides the application of the whitening and anti-allergic composition prepared with the aforementioned eutectic solvent in the preparation of cosmetics.

[0068] Comparative Example 2

[0069] The first aspect of this comparative example provides a whitening and anti-allergic composition prepared with a eutectic solvent, and the specific implementation method is the same as in Example 3.

[0070] The second aspect of this comparative example provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The specific implementation method is the same as in Example 3, except that the raw material for preparing the betaine eutectic solvent is an aqueous solution of 1,3-butanediol and betaine (molar ratio 0.5:1) with a water content of 40 wt%.

[0071] The third aspect of this comparative example provides the application of the whitening and anti-allergic composition prepared with the aforementioned eutectic solvent in the preparation of cosmetics.

[0072] Comparative Example 3

[0073] The first aspect of this comparative example provides a whitening and anti-allergic composition prepared with a eutectic solvent, and the specific implementation method is the same as in Example 1.

[0074] The second aspect of this comparative example provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The specific implementation method is the same as in Example 1, except that the raw material for preparing the betaine eutectic solvent is an aqueous solution of 1,3-butanediol and betaine (molar ratio 7:1) with a water content of 30 wt%.

[0075] The third aspect of this comparative example provides the application of the whitening and anti-allergic composition prepared with the aforementioned eutectic solvent in the preparation of cosmetics.

[0076] Comparative Example 4

[0077] The first aspect of this comparative example provides a whitening and anti-allergic composition prepared with a eutectic solvent, and the specific implementation method is the same as in Example 1.

[0078] The second aspect of this comparative example provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The specific implementation method is the same as in Example 1, except that the raw material for preparing the betaine eutectic solvent is an aqueous solution of 1,2-hexanediol and betaine (molar ratio 2:1) with a water content of 30 wt%.

[0079] The third aspect of this comparative example provides the application of the whitening and anti-allergic composition prepared with the aforementioned eutectic solvent in the preparation of cosmetics.

[0080] Comparative Example 5

[0081] The first aspect of this comparative example provides a whitening and anti-allergic composition prepared with a eutectic solvent, and the specific implementation method is the same as in Example 1.

[0082] The second aspect of this comparative example provides a method for preparing a whitening and anti-allergic composition using a eutectic solvent. The specific implementation method is the same as in Example 1, except that the raw material for preparing the betaine eutectic solvent is an aqueous solution of xylitol and betaine (molar ratio 1:1) with a water content of 30 wt%.

[0083] The third aspect of this comparative example provides the application of the whitening and anti-allergic composition prepared with the aforementioned eutectic solvent in the preparation of cosmetics.

[0084] Test case

[0085] This test example provides an essence cream with the following formula as shown in the table below. The preparation method is as follows: in phase A, glycerin and the ingredients except water are pre-dissolved, water is added after dissolution, and the mixture is heated to 85°C; phase B is mixed and then heated to 85°C to dissolve, and then phases A and B are mixed and homogenized, stirred and cooled; phase C is weighed and dissolved separately until clear and transparent, and added after the mixture of phases A and B has cooled to below 50°C; phase D is added after the mixture of phases A, B, and C has cooled to room temperature.

[0086]

[0087] Performance testing

[0088] The whitening and anti-allergic compositions obtained from the above examples and comparative examples were subjected to the following tests.

[0089] 1. In vitro antioxidant assay - ABTS method

[0090] (1) Reagent preparation:

[0091] PBS solution: Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 (3.63g Na2HPO4·12H2O) and 0.24g KH2PO4, adjust pH to 7.2 and dilute to 1000mL.

[0092] ABTS + Prepare a 2.45 mmol / L potassium persulfate solution, dissolve ABTS in potassium persulfate to prepare a 7 mmol / L ABTS stock solution, and let it stand for 12 hours at room temperature in the dark.

[0093] 2.45 mmol / L potassium persulfate: To prepare 100 mL, take 0.0662 g of potassium persulfate and dilute to 100 mL with water.

[0094] 7 mmol / L ABTS stock solution: Dissolve 0.0384 g of ABTS in 2.45 mmol / L potassium persulfate and bring the volume up to 10 mL. The solution is dark green.

[0095] ABTS + Assay solution: ABTS + The stock solution was diluted with phosphate buffer (pH=7.2) to achieve an absorbance of 0.700±0.020 at a wavelength of 734 nm.

[0096] The whitening and anti-allergic compositions of each example and comparative example were prepared as aqueous solutions with mass concentrations of 0.1% and 0.5%, respectively, for testing.

[0097] (2) Measurement method:

[0098] Take 4 mL of ABTS +Working solution → 1 mL sample → mix for 10 s → incubate at 25℃ in the dark for 6 min → measure absorbance at 734 nm. Use 4 mL PBS + 1 mL sample as a reference, and denote it as sample A;

[0099] Take 4 mL of ABTS + Working solution → 1 mL PBS → Mix for 10 seconds → Incubate at 25°C in the dark for 6 minutes → Measure absorbance at 734 nm. Use PBS as a reference and record it as A0.

[0100]

[0101] Table 1. Clearance rate of ABTS by the whitening and anti-allergic compositions of the examples and comparative examples.

[0102]

[0103]

[0104] 2. In vitro antioxidant assay - DPPH method

[0105] (1) Accurately weigh 0.0100 g of DPPH powder, place it in a 250 mL volumetric flask, dissolve it with 95 wt% ethanol aqueous solution and make up to 250 mL to obtain DPPH reagent with a concentration of 0.1 mmol / L.

[0106] (2) Measurement method:

[0107] Add 4.0 mL of prepared DPPH solution and 1.0 mL of 95 wt% ethanol aqueous solution to a 10 mL test tube, mix and shake well, react in the dark for 30 min, and after stabilization, measure the absorbance at 517 nm using 95 wt% ethanol aqueous solution as a reference, and record it as A0.

[0108] Add 4.0 mL of prepared DPPH solution and 1.0 mL of the test sample solution to a 10 mL test tube, mix and shake well, react in the dark for 30 min, and after stabilization, measure the absorbance at 517 nm using 95% ethanol as a reference, and record it as Ar.

[0109] Add 4.0 mL of 95% ethanol solution and 1.0 mL of the test sample solution to a 10 mL test tube, mix and shake well, react in the dark for 30 min, and after stabilization, measure the absorbance at 517 nm using 95 wt% ethanol aqueous solution as a reference, and record it as As;

[0110] The whitening and anti-allergic compositions of each embodiment and comparative example were prepared into aqueous solutions with mass concentrations of 1% and 3% respectively for testing, serving as test sample solutions.

[0111]

[0112] Table 2 shows the DPPH scavenging rate of the whitening and anti-allergic compositions in the examples and comparative examples.

[0113]

[0114] 3. In vitro whitening efficacy test - inhibition of tyrosinase activity

[0115] (1) Reagent preparation

[0116] Preparation of PBS buffer (pH=6.8): Accurately weigh 6.80 g of potassium dihydrogen phosphate and dissolve it in 250 mL of pure water to obtain a 0.2 mol / L potassium dihydrogen phosphate solution; accurately weigh 0.94 g of sodium hydroxide and dissolve it in 118 mL of pure water. Take 250 mL of the 0.2 mol / L potassium dihydrogen phosphate solution and 118 mL of the 0.2 mol / L sodium hydroxide solution, and dilute with water to 1 L to obtain the PBS buffer (pH=6.8).

[0117] Preparation of L-tyrosine solution: Prepare a 1.5 mmol / L L-tyrosine solution with PBS, pH=7;

[0118] Preparation of tyrosinase solution: Prepare a 200 U / mL tyrosinase solution using PBS.

[0119] (2) Experimental procedure

[0120] The total reaction volume was 4 mL. Accurately measure 0 mL of the sample solution, 2.0 mL of PBS, and 1.0 mL of tyrosinase solution, mix well, and record as A; accurately measure 0 mL of the sample solution, 3.0 mL of PBS, and 0 mL of tyrosinase solution, mix well, and record as B; accurately measure 1.0 mL of the sample solution, 1.0 mL of PBS, and 1.0 mL of tyrosinase solution, mix well, and record as C; accurately measure 1.0 mL of the sample solution, 2.0 mL of PBS, and 0 mL of tyrosinase solution, mix well, and record as D. After mixing, incubate at 37°C for 10 min. Then add 1.0 mL of tyrosine solution to each mixture, react for 15 min, and measure the absorbance at 490 nm. Use B as a reference to read the absorbance of A, and use D as a reference to read the absorbance of C.

[0121] The whitening and anti-allergic compositions of each embodiment and comparative example were prepared into aqueous solutions with mass concentrations of 5% and 10% for testing, respectively, as sample solutions.

[0122] Calculation formula:

[0123] Table 3 shows the inhibition rate of tyrosinase by the whitening and anti-allergic compositions of the examples and comparative examples.

[0124]

[0125]

[0126] 4. In vitro anti-allergic efficacy test - inhibition of hyaluronidase activity

[0127] (1) Reagent preparation

[0128] Preparation of acetate buffer solution (pH=5.6): Dilute 1.155 mL of glacial acetic acid with pure water to 100 mL, and take 4.8 mL as solution A; weigh 2.72 g of sodium acetate crystals, dissolve them in water and bring the volume to 100 mL, and take 45.2 mL as solution B; mix solutions A and B, and bring the volume to 100 mL with water. Adjust the pH to 5.6.

[0129] Preparation of hyaluronidase solution: Weigh 10 mg of hyaluronidase into a beaker and add 4 ml of acetate buffer solution with a concentration of 1250 units / mL;

[0130] Preparation of sodium hyaluronate solution: Weigh 5.0 mg of sodium hyaluronate and add 10 mL of acetate buffer solution to obtain sodium hyaluronate solution (0.5 mg / mL);

[0131] Preparation of Ehrlich's reagent: Weigh 0.8 g of p-dimethylaminobenzaldehyde and dissolve it in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol;

[0132] Preparation of acetylacetone solution: Dissolve 3.5 mL of acetylacetone in 50 mL of sodium carbonate aqueous solution (1.0 mol / L);

[0133] Preparation of sodium hydroxide solution: Weigh 16g of sodium hydroxide and dissolve it in pure water, then dilute to 1L with pure water.

[0134] (2) Experimental procedure

[0135] Take 0.1 mL of 0.25 mol / L CaCl2 solution and 0.5 mL of hyaluronidase solution and incubate at 37℃ for 20 min; add 0.5 mL of test solution and continue incubation at 37℃ for 20 min; add 0.5 mL of sodium hyaluronate solution and incubate at 37℃ for 30 min, then let stand at room temperature for 5 min; add 0.1 mL of NaOH solution (0.4 mol / L) and 0.5 mL of acetylacetone solution, heat in a boiling water bath for 15 min, and then immediately cool with ice water for 5 min; add 1.0 mL of Ehrlich reagent and dilute with 3.0 mL of anhydrous ethanol, let stand for 20 min for color development, and measure its absorbance value with a spectrophotometer.

[0136]

[0137] In the formula: A—ABS value of the control solution (using acetate buffer solution instead of the sample solution); B—ABS value of the control blank solution (using acetate buffer solution instead of the sample solution and enzyme solution); C—ABS value of the sample solution; D—ABS value of the sample blank solution (using acetate buffer solution instead of the enzyme solution).

[0138] During the experiment, the A group samples were first scanned at wavelengths of 400–900 nm to determine the maximum absorption wavelength as 420 nm. Then, using deionized water as a reference, the ABS value was measured at the maximum absorption wavelength.

[0139] The whitening and anti-allergic compositions of each embodiment and comparative example were prepared into aqueous solutions with mass concentrations of 5% and 10% for testing, respectively, as sample solutions.

[0140] Table 4 shows the inhibition rate of hyaluronidase by the whitening and anti-allergic compositions of the examples and comparative examples.

[0141]

[0142] 5. High Performance Liquid Chromatography - Paeoniflorin Content

[0143] The concentration of paeoniflorin standard was 0.22 mg / mL. The whitening and anti-allergic compositions of Examples 1-2 and Comparative Example 1 were diluted 10 times with pure water and analyzed by high performance liquid chromatography (HPLC). A Supersil ODS C18 column (4.6 mm × 250 mm, 5 μm) was used, and the method described in the literature was followed: mobile phase conditions: phase A was acetonitrile, phase B was 0.1% phosphoric acid water, and the volume ratio of phase A to phase B was 14:86; column temperature: 30℃; detection wavelength: 230 nm; detection time: 25 min; injection volume: 10 μL. The paeoniflorin content in the examples and comparative examples was estimated using the single-point method, and the results are shown in Table 5.

[0144] Table 5. Paeoniflorin content of the whitening and anti-allergic compositions of the examples and comparative examples.

[0145]

[0146] 6. High Performance Liquid Chromatography - Ferulic Acid Content

[0147] The ferulic acid standard concentration was 0.18 mg / mL. Examples 1-2 and Comparative Example 1 were diluted 10-fold and detected by high-performance liquid chromatography (HPLC). A Supersil ODS C18 column (4.6 mm × 250 mm, 5 μm) was used. The mobile phase conditions were as follows, according to the literature: phase A was methanol, phase B was pure water, and the volume ratio of phase A to phase B was 30:70; the column temperature was 35℃; the detection wavelength was 313 nm; the detection time was 15 min; and the injection volume was 10 μL. The ferulic acid content in the examples and comparative examples was estimated using the single-point method, and the results are shown in Table 6.

[0148] Table 6. Ferulic acid content of the products obtained in Examples 1-2 and Comparative Example 1

[0149]

[0150]

[0151] 7. Human efficacy trials

[0152] The test cases were tested for whitening efficacy on human skin in accordance with the "Cosmetic Safety Technical Specifications" (2015 edition). The test report is available at [link to report]. Figure 3-11 .

[0153] 24 hours prior to the test, the small erythema dose (MED) of the subject's skin to ultraviolet radiation was predicted. The skin was then irradiated once daily for four consecutive days at the same irradiation point using a daylight lamp at 0.75 times the MED dose. Four days after the irradiation ended, the skin underwent a melanization period without any treatment. On the fifth day after irradiation, the skin color of each test area was visually assessed and measured using a skin color instrument. Test areas with poor consistency (areas whose ITA value differed from the mean of all test areas by more than 5) were removed. Starting that day, the corresponding test substance was applied to each melanized test area according to a random table, and the application continued for four weeks, with 2g of sample applied each time. Skin color was visually assessed and measured using an instrument at 1, 2, 3, and 4 weeks after application, and the results were recorded.

[0154] Statistical methods: Origin 2018 software was used for statistical analysis of the data. Quantitative data were expressed as mean and standard deviation, and were tested for normality. Paired t-tests were used for comparisons of data before and after the same event; otherwise, a two-sample rank-sum test was used. Ordinal data were compared before and after the same event using a two-sample rank-sum test. Independent samples t-tests or rank-sum tests were used for comparisons between the experimental product and the control group.

[0155] Table 7. Regression coefficients (slope K-values) of various parameters in the whitening efficacy test on human bodies over time.

[0156]

[0157]

[0158] Note: P<0.05 indicates a significant difference.

Claims

1. A method for preparing a whitening and anti-allergic composition using a eutectic solvent, characterized in that, The preparation method includes adding composition A to a betaine eutectic solvent, and extracting the resulting mixture using a microfluidic extractor to obtain a whitening and anti-allergic composition. By weight, composition A comprises 30-50 parts of white peony flower, 10-20 parts of white water lily, 20-40 parts of Hangzhou white chrysanthemum and 10-20 parts of white plum blossom; The raw materials for preparing the betaine eutectic solvent include betaine, a hydrogen bond donor, and water; the molar ratio of betaine to hydrogen bond donor is 1:(1-5). The hydrogen bond donor is 1,3-butanediol.

2. The method for preparing the whitening and anti-allergic composition using a eutectic solvent according to claim 1, characterized in that, The volume fraction of water in the betaine eutectic solvent is 10-60%.

3. The method for preparing the whitening and anti-allergic composition using a eutectic solvent according to claim 1, characterized in that, The microfluidic extractor has a pore size of 500-800 μm.

4. The method for preparing the whitening and anti-allergic composition using a eutectic solvent according to claim 1, characterized in that, The mass ratio of the betaine eutectic solvent to composition A is (8-12):

1.

5. The application of the preparation method according to any one of claims 1-4 in the preparation of cosmetics.

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