Pyranol retinoate, its preparation method and use in cosmetics, skin topical

Abstract

The application discloses a pyranol retinol, and a structural formula of the pyranol retinol is shown as formula (I). The pyranol retinol has good stability, small skin irritation, good whitening, oxidation resistance, free radical elimination, anti-aging, wrinkle removal and acne removal effects, and can be applied to cosmetics and skin external medicines.

Description

Technical Field

[0001] This invention belongs to the field of cosmetics and pharmaceutical technology, and in particular relates to pyranol retinate, its preparation method and its application in cosmetics and topical skin medications. Background Technology

[0002] Retinoic acid, also known as retinoic acid, has the function of exfoliating the stratum corneum of the skin, promoting the division and proliferation of epidermal cells, and skin regeneration. Therefore, retinoic acid can promote the excretion of melanin from the periphery of the basal layer of the epidermis. In addition to effectively treating acne and blemishes (age pigmentation, seborrheic keratosis, etc.), retinoic acid also has anti-wrinkle effects. However, retinoic acid is irritating to the skin. Oral retinoic acid has strong teratogenic effects in experimental animals and humans. Topical application of retinoic acid has clear embryotoxicity and teratogenicity in mice, rats, hamsters, and rabbits in the embryonic sensitive period, and can cause maternal systemic toxicity, resulting in fetal malformations. Therefore, retinoic acid and its salts are prohibited raw materials in the 2021 version of the Cosmetic Safety Technical Specifications, and the detection limit concentration is set at 0.4 mg / kg.

[0003] In recent years, milder retinoic acid derivatives have become popular alternatives to retinoic acid. These derivatives are metabolized into their active forms by skin cells. Hydroxypinazone retinate (HPR) is synthesized from retinoic acid and a small molecule pinacol. Its key feature is that it retains the active group of retinoic acid, eliminating the need for conversion to retinoic acid and allowing it to directly bind to receptors to exert its effects. When used in products such as serums, lotions, and creams, isosorbide dimethyl ether, polyols, or polar oils are commonly used as solvents to dissolve hydroxypinazone retinate. However, in these products, hydroxypinazone retinate exhibits poor stability, with the retinoic acid content increasing rapidly over extended storage time.

[0004] Therefore, it is of great significance to develop retinoic acid derivatives with better stability, slow down their degradation rate in cosmetics, reduce the rate and content of retinoic acid production, and improve efficacy to expand their application range. Summary of the Invention

[0005] Based on this, the present invention provides a retinic acid derivative with good stability, low skin irritation, and good whitening, antioxidant, free radical scavenging, anti-aging, wrinkle removal and acne removal effects.

[0006] The present invention includes the following technical solutions.

[0007] On the one hand, the present invention provides a pyranol retinate, the structural formula of which is shown in formula (I):

[0008]

[0009] Formula (I).

[0010] On the other hand, the present invention provides three methods for preparing the pyranol retinate.

[0011] Method 1, the preparation method of the pyranol retinate includes the following steps:

[0012] Retinic acid reacts with dimethylmethoxybenzodihydropyranol to yield pyranol retinate.

[0013] Method 2, the preparation method of the pyranol retinate includes the following steps:

[0014] S1. Retinic acid reacts with an acyl halide reagent to yield retinyl chloride;

[0015] S2, retinyl chloride and dimethylmethoxybenzodihydropyranol react to give pyranol retinate.

[0016] Method 3, the preparation method of the pyranol retinate includes the following steps:

[0017] Methyl retinate and dimethylmethoxybenzodihydropyranol acetate undergo transesterification to yield pyranol retinate.

[0018] Thirdly, the present invention provides the following applications of the pyranol retinate:

[0019] The application of the pyranol retinate described in this invention in the preparation of cosmetics with whitening effects.

[0020] The application of the pyranol retinate described in this invention in the preparation of cosmetics with antioxidant effects.

[0021] The application of the pyranol retinate described in this invention in the preparation of cosmetics with acne-removing effects.

[0022] The application of the pyranol retinate described in this invention in the preparation of cosmetics with anti-aging and wrinkle-reducing effects.

[0023] The application of the pyranol retinate ester described in this invention in the preparation of a topical skin medicine with whitening effects.

[0024] The application of the pyranol retinate ester described in this invention in the preparation of topical skin medications with antioxidant effects.

[0025] The application of the pyranol retinate ester described in the invention in the preparation of topical skin medications with acne-reducing effects.

[0026] The application of the pyranol retinate ester described in this invention in the preparation of topical skin medications with anti-aging and wrinkle-reducing effects.

[0027] Fourthly, the present invention provides a cosmetic product in which the active ingredient comprises the pyranol retinate described in the present invention.

[0028] Fifthly, the present invention provides a topical skin medicine, wherein the active ingredient in the topical skin medicine comprises the pyranol retinate described in the present invention.

[0029] The pyranol retinate of the present invention has good stability and low skin irritation. It has good whitening, antioxidant, free radical scavenging, anti-aging and wrinkle removal and acne removal effects, and can be used in cosmetics and topical skin medicines. Attached Figure Description

[0030] Figure 1 These are comparison images showing the melanin content on the body surface of zebrafish in different groups.

[0031] Figure 2 This is a bar chart showing the relative melanin content of each group of zebrafish.

[0032] Figure 3 This is a bar chart showing the relative inhibition rate of melanin in each group of zebrafish.

[0033] Figure 4 This is a bar chart showing the scavenging rate of DPPH free radicals for each sample. Detailed Implementation

[0034] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0035] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0037] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0038] In order to develop a retinoic acid derivative with good stability and while maintaining good whitening and anti-wrinkle effects, in one embodiment of the present invention, a pyranol retinoic acid ester (dimethylmethoxybenzodihydropyranol retinoic acid ester) is provided, the structural formula of which is shown in formula (I):

[0039]

[0040] Formula (I).

[0041] This pyranol retinyl ester has good stability and low irritation. It can effectively inhibit the formation of melanin and has a good whitening effect. At the same time, it has good antioxidant, free radical scavenging, anti-aging and wrinkle-reducing effects as well as acne-removing effects.

[0042] The pyranol retinate of the present invention can be prepared by conventional methods in the art, preferably by the following three methods, and more preferably by method three, which has a higher yield.

[0043] Method 1: Using retinoic acid and dimethylmethoxybenzodihydropyranol as reactants, the carboxyl group in retinoic acid and the hydroxyl group in dimethylmethoxybenzodihydropyranol undergo an esterification reaction to obtain pyranol retinate.

[0044] In some embodiments, the molar ratio of retinoic acid to dimethylmethoxybenzodihydropyranol is 1:1.01-1.4, more preferably 1:1.1-1.3, to ensure complete reaction of retinoic acid.

[0045] In some embodiments, the esterification reaction is carried out in the presence of a carboxyl activator (condensing agent), which is selected from any one or a combination of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), HCTU (6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate), BOP (benzotriazole-1-oxytris(dimethylamino)phosphide hexafluorophosphate), PyBOP (benzotriazole-1-yl-oxytripyrrolidinephosphide hexafluorophosphate), DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride), DIC (diisopropylcarbodiimide), DCC (dicyclohexylcarbodiimide), and EDCI (carbodiimide).

[0046] The molar ratio of retinoic acid to carboxyl activator is preferably 1:1-3.0, more preferably 1:1.5-2.0.

[0047] In some embodiments, the esterification reaction is carried out in the presence of a condensation aid selected from one or more combinations of HOBT (1-hydroxybenzotriazole), Cl-HOBt (6-chloro-1-hydroxybenzotriazole), HOAT (N-hydroxy-7-azabenzotriazole), Oyxma-Pure (ethyl 2-oxime cyanoacetate), Oxyma-B (1,3-dimethyl-2,4,5,6(1H,3H)-pyrimidinide-5-oxime), and DMAP (4-dimethylaminopyridine).

[0048] The molar ratio of retinoic acid to condensation aid is preferably 1:0.05-1.0, more preferably 1:0.08-0.2.

[0049] In some embodiments, the esterification reaction is carried out in a solvent, which can be a commonly used organic solvent in esterification reactions, such as any one or more combinations selected from aromatic hydrocarbons, halogenated hydrocarbons, ethers, esters, and amides; more preferably, the solvent is selected from any one or more combinations selected from toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, dichloroethane, methyl tert-butyl ether, methyl cyclopentyl ether, isopropyl ether, dibutyl ether, isopropyl acetate, ethyl butyl acetate, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, N-ethylpyrrolidone, and N-butylpyrrolidone.

[0050] In some embodiments, the esterification reaction is carried out at a temperature of 10°C-30°C for 1-3 hours.

[0051] After the esterification reaction is completed, the byproducts can be removed by extraction, washing with water and filtration. After concentration and drying, recrystallization yields a yellow solid product, namely pyranol retinate.

[0052] Preferably, the solvent for recrystallization is selected from one or more combinations of ethanol, isopropanol, butanol, and heptane.

[0053] In some embodiments, the preparation method of pyranol retinate according to Method 1 includes the following steps:

[0054] Retinic acid, dimethylmethoxybenzodihydropyranol, and condensation aids are added to a solvent, stirred and dissolved, then cooled to 5℃-10℃, a carboxyl activator is added, stirred evenly, and then heated to 10℃-30℃ for 1-3 hours.

[0055] Method 2: Retinyl acid first reacts with an acyl halide reagent to obtain retinyl chloride, which is then reacted with dimethylmethoxybenzodihydropyranol to obtain pyranol retinate. This involves the following two steps:

[0056] S1. Retinic acid reacts with an acyl halide reagent to yield retinyl chloride;

[0057] S2, retinyl chloride and dimethylmethoxybenzodihydropyranol react to give pyranol retinate.

[0058] The acyl halide reagent is preferably selected from at least one of thionyl chloride, oxalyl chloride, diphosgene, and triphosgene.

[0059] In some of these embodiments, the molar ratio of retinoic acid to the acyl halide reagent is 1:1-1.3.

[0060] In some embodiments, the reaction in step S1 is carried out in a solvent, preferably one or more combinations of toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, dichloroethane, methyl tert-butyl ether, methyl cyclopentyl ether, isopropyl ether, dibutyl ether, isopropyl acetate, ethyl butyl acetate, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, N-ethylpyrrolidone, and N-butylpyrrolidone.

[0061] In some embodiments, the reaction temperature in step S1 is 10°C-50°C, preferably 35°C-45°C, and the reaction time is 2h-6h.

[0062] In some of these embodiments, the molar ratio of retinyl chloride to dimethylmethoxybenzodihydropyranol is 1:1-1.3.

[0063] In some embodiments, the reaction in step S2 is carried out under the action of an acid-binding agent, which is preferably any one or a combination of triethylamine, diisopropylethylamine, pyridine, and methylmorpholine.

[0064] In some of these embodiments, the molar ratio of retinyl chloride to tannin-binding agent is 1:1-1.3.

[0065] In some embodiments, the reaction in step S2 is carried out in a solvent, preferably one or more combinations of toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, dichloroethane, methyl tert-butyl ether, methyl cyclopentyl ether, isopropyl ether, dibutyl ether, isopropyl acetate, ethyl butyl acetate, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, N-ethylpyrrolidone, and N-butylpyrrolidone.

[0066] In some embodiments, the preparation method of pyranol retinate according to Method 2 includes the following steps:

[0067] S10. Disperse and dissolve retinoic acid in a solvent, then add an acyl halide reagent, and after the reaction, retinoic acid chloride is obtained;

[0068] S20. Retinyl chloride dissolved in a solvent is added to a mixture of dimethylmethoxybenzodihydropyranol, solvent and acid-binding agent, and the reaction yields pyranol retinate.

[0069] In some of these embodiments, the temperature at which retinyl chloride is added is 0°C-10°C.

[0070] After the two-step reaction is completed, an appropriate amount of hydrochloric acid is added for washing, the mixture is concentrated to dryness, and then recrystallized to obtain a yellow solid product, namely pyranol retinate. The solvent used for recrystallization is preferably one or a combination of ethanol, isopropanol, butanol, and heptane.

[0071] Method 3: Using methyl retinoate and dimethyl methoxybenzodihydropyranol acetate as raw materials, a transesterification reaction is carried out to obtain pyranol retinate.

[0072] The preferred molar ratio of methyl retinate to dimethyl methoxybenzodihydropyranol acetate is 1:0.9-1:2.

[0073] In some embodiments, the transesterification reaction is carried out under the action of a base, preferably sodium methoxide, and the molar ratio of methyl retinoate to the base is preferably 1:0.03-0.1.

[0074] In some embodiments, the transesterification reaction is carried out at a temperature of 55°C-65°C.

[0075] In some embodiments, the transesterification reaction is carried out under reduced pressure, preferably 100 Pa to 1000 Pa, more preferably 500 Pa to 700 Pa.

[0076] In some embodiments, the transesterification reaction takes 2-4 hours.

[0077] In some embodiments, the preparation method of pyranol retinyl ester according to Method 3 includes the following steps: methyl retinoate, dimethyl methoxybenzodihydropyranol acetate and alkali are mixed and stirred evenly, then heated to 55℃-65℃, reduced to 100Pa-1000Pa, and reacted for 2h-4h. During the reaction, byproducts are removed by distillation to obtain the product.

[0078] After the transesterification reaction was completed, a solvent was added and recrystallized to obtain a yellow solid product, namely pyranol retinate.

[0079] Preferably, the solvent used for recrystallization is selected from one or more combinations of ethanol, isopropanol, butanol, and heptane.

[0080] In another embodiment of the present invention, the present invention also provides the application of pyranol retinyl ester in cosmetics and topical skin medications, so that the cosmetics and topical skin medications have whitening, antioxidant, free radical scavenging, anti-aging and wrinkle removal and acne removal effects.

[0081] In another embodiment of the present invention, the present invention also provides a cosmetic product in which the active ingredient comprises pyranol retinate as described in structural formula (I); the amount of which is preferably added is 0.01wt%-5wt%.

[0082] In another embodiment of the present invention, the present invention also provides a topical skin medicine in which the active ingredient comprises pyranol retinate as described in structural formula (I); the amount added is preferably 0.01wt%-5wt%.

[0083] The present invention will be further described in detail below with reference to specific embodiments.

[0084] Example 1 Preparation of pyranol retinate

[0085]

[0086] In a 250 ml three-necked flask equipped with a stir bar and thermometer, dichloromethane (100 ml), retinoic acid (10.00 g, 33.33 mmol, 1 eq), dimethylmethoxybenzodihydropyranol (8.33 g, 40 mmol, 1.2 eq), and DMAP (0.41 g, 3.33 mmol, 0.1 eq) were added sequentially. The mixture was stirred until dissolved, cooled to 6 °C in an ice bath, and then EDCI (12.80 g, 66.67 mmol, 2 eq) was added. After stirring in an ice bath for 30 min, the mixture was heated to 25 °C and reacted for 2 h until complete. The mixture was washed with 50 ml of water, and the organic phase was concentrated to dryness. Recrystallization with ethanol yielded 15 g of a yellow solid product, namely pyranol retinate, with a yield of 92% and a purity of 99.5%.

[0087] 1 H NMR: (CDCl3, 500MHz) δ=1.04 (s, 6H), 1.3 (s, 6H), 1.46-1.79 (m, 11H), 2.03 (s, 3H), 2 .40(s, 3H), 2.69(t, 2H), 3.76(s, 3H), 6.03-6.42(m, 6H), 6.75(s, 1H), 7.05(q, 1H).

[0088] Example 2 Preparation of pyranol retinate

[0089]

[0090] In a 250 ml three-necked flask equipped with a stir bar, thermometer, and dropping funnel, 100 ml of dichloromethane and retinoic acid (10.00 g, 33.33 mmol, 1 eq) were added sequentially, followed by the addition of thionyl chloride (4.36 g, 36.67 mmol, 1.1 eq). After the addition was complete, the mixture was refluxed at 40 °C for 4 h. The solvent was then concentrated to obtain 10.62 g of retinyl chloride, which was used for the next reaction.

[0091]

[0092] In a 250 mL three-necked flask equipped with a stir bar, thermometer, and dropping funnel, 100 mL of dichloromethane, dimethylmethoxybenzodihydropyranol (7.60 g, 36.67 mmol, 1.1 eq), and triethylamine (4.04 g, 40 mmol, 1.2 eq) were added sequentially. The mixture was cooled to 6 °C in an ice bath, and a solution of retinyl chloride (10.62 g, 33.33 mmol, 1 eq) dissolved in 30 mL of dichloromethane was added dropwise. After the addition was complete, the temperature was raised to 25 °C and the reaction was carried out for 1 h until complete. The mixture was washed with 50 mL of 0.5 N hydrochloric acid, and the organic phase was concentrated to dryness. Ethanol was added for recrystallization to give 14.7 g of a yellow solid product, namely pyranol retinate, with a yield of 90.2% and a purity of 99.3%.

[0093] 1 H NMR: (CDCl3, 500MHz) δ=1.04 (s, 6H), 1.3 (s, 6H), 1.46-1.79 (m, 11H), 2.03 (s, 3H), 2 .40(s, 3H), 2.69(t, 2H), 3.76(s, 3H), 6.03-6.42(m, 6H), 6.75(s, 1H), 7.05(q, 1H).

[0094] Example 3 Preparation of pyranol retinate

[0095]

[0096] In a 250 ml three-necked flask equipped with a stir bar and thermometer, methyl retinoate (10.0 g, 31.85 mmol, 1 eq), dimethyl methoxybenzodihydropyranol acetate (7.97 g, 31.85 mmol, 1 eq), and sodium methoxide (0.09 g, 1.6 mmol, 0.05 eq) were added sequentially. The mixture was heated to 60 °C and then reduced to 600 Pa. Byproducts were removed by distillation. After 3 h, the reaction was complete. Ethanol was added and recrystallized to give 15.0 g of a yellow solid product, namely pyranol retinate, with a yield of 96% and a purity of 99.6%.

[0097] 1 H NMR: (CDCl3, 500MHz) δ=1.04 (s, 6H), 1.3 (s, 6H), 1.46-1.79 (m, 11H), 2.03 (s, 3H), 2 .40(s, 3H), 2.69(t, 2H), 3.76(s, 3H), 6.03-6.42(m, 6H), 6.75(s, 1H), 7.05(q, 1H).

[0098] Example 4 Stability Test

[0099] According to Table 1, the pyranol retinate ester prepared in Example 1, as well as the commercially available tocopherol retinate ester and hydroxypinazone retinate ester, were dispersed in a solvent composed of isosorbide dimethyl ether and phenoxyethanol, respectively, as Sample 1 and Controls 1-2. Then, the changes in the content of retinate ester (active ingredient) and retinoic acid in each sample were tested under light-protected conditions at 25°C, under constant temperature and humidity incubator (40±2°C, protected from light), and under ultraviolet light at 25°C. The test results are shown in Tables 2-4.

[0100] Table 1. Formulations of each sample

[0101]

[0102] Table 2. Stability test results of each sample under light-protected conditions at 25℃

[0103]

[0104] Table 3. Stability test results of each sample under light-protected conditions at 40±2℃

[0105]

[0106] Table 4. Stability test results of various samples under 25℃ ultraviolet light irradiation.

[0107]

[0108] As shown in Tables 2-4, under 25°C and light-protected conditions for 60 days, the active ingredient content of Sample 1 (pyranol retinate) decreased by no more than 6.5%, significantly better than Controls 1-2. Under 40±2°C and light-protected conditions, the active ingredient content of Sample 1 decreased by less than 5% within 30 days. After 60 days, the decrease rate of retinate content in Sample 1 did not exceed 7.5%, while the decrease rate of retinate content in Controls 1-2 reached about 20%. Under 25°C and ultraviolet light, the active ingredient content of the sample showed a significant decrease, but after 60 days, the decrease in retinate content was significantly lower than that in Controls 1-2. Furthermore, under 25°C and light-protected conditions, Sample 1 did not produce retinoic acid within 60 days, and the amount of retinoic acid produced under 40±2°C and light-protected conditions and under 25°C and ultraviolet light conditions was also much lower than that in Controls 1-2. This indicates that the stability of the pyranol retinate of the present invention is significantly better than that of tocopherol retinate and hydroxypinazone retinate.

[0109] Example 5: Detection of melanin inhibition ability in zebrafish

[0110] Using wild-type zebrafish of the AB line as a model, the melanin inhibition ability of pyranol retinate, tocopherol retinate, hydroxypinazone retinate, and dimethylmethoxybenzodihydropyran palmitate (hereinafter referred to as pyran whitening agent) in Example 1 was tested according to T / SHRH 036-2021 "Melanin Inhibition Test for Cosmetics - Zebrafish Embryo Test Method".

[0111] Test samples: Pyranol retinate (Example 1), tocopherol retinate, hydroxypinazone retinate, and pyran whitening agent were diluted with 1% DMSO and used for later use. The sample concentrations are shown in Table 5.

[0112] Table 5. Sample Dosage Table

[0113]

[0114] Toxicity test: Normally developing AB strain wild-type zebrafish 22 hpf embryos were randomly selected and placed in 96-well plates, with 5 replicates per group and 2 embryos per well. Drugs were administered according to the groups listed in Table 5, with 100 μL of different concentrations of sample solution added. The blank control group was added with 100 μL of 1% DMSO solution. The plates were incubated in a biochemical incubator at 28±1℃. After 24 h, embryonic development was observed under an optical microscope, and the number of malformations was recorded. The safe concentration was determined to be 60 μmol / L.

[0115] Zebrafish melanin inhibition assay: Normally developing AB strain wild-type zebrafish embryos (22 hpf) were randomly selected and placed in 96-well plates, with 10 replicates per group and 2 embryos per well. 100 μl of sample solution (1% DMSO solution as solvent, sample concentration 60 μmol / L) was added, along with 100 μl of 1% DMSO solution as a blank control. The plates were incubated in a biochemical incubator at 28±1℃ in the dark. After 24 hours, the development of melanin on the embryonic surface was observed under a stereomicroscope. At least 5 zebrafish embryos from each group were randomly selected and placed on slides, fixed with 4% methylcellulose fixative, and top-view images were taken (requiring symmetrical eyes and yolk sacs, and a straight trunk). One image from each group was selected for comparison. Figure 1 As shown.

[0116] from Figure 1 It can be found that the melanin-inhibiting ability of the pyranol retinate of the present invention is comparable to that of hydroxypinazone retinate, and is significantly superior to that of tocopherol retinate and pyran whitening agents.

[0117] Quantitative analysis of zebrafish melanin: ImageJ software was used to identify melanin regions on the zebrafish body surface. The threshold was adjusted to 118, and the area of ​​the melanin region was calculated. The relative content of zebrafish melanin was calculated according to formula (1), and the relative inhibition rate of zebrafish melanin was calculated according to formula (2). The calculation results are expressed as mean ± standard deviation (mean ± SD). The results are shown in Table 6 and below. Figure 2-3 One-way ANOVA was performed using GraphPad Prism 8.0 to analyze the significance of differences between different groups, and a p-value < 0.05 was considered statistically significant.

[0118] Relative melanin content (%) in zebrafish = (1)

[0119] Zebrafish melanin relative inhibition rate (%) = (2)

[0120] In equations (1) and (2):

[0121] V e —Average melanin content of the sample group;

[0122] Vc —The average melanin content in the blank control group.

[0123] Table 6. Relative melanin content and inhibition rate of zebrafish in each group at a sample concentration of 60 μmol / L

[0124]

[0125] From Table 6 and Figure 2-3 The results showed that pyranol retinate inhibited zebrafish melanin production by 43.80% at a concentration of 60 μmol / L, which was statistically different from the blank control group, indicating that pyranol retinate has a whitening effect. Furthermore, at a concentration of 60 μmol / L, pyranol retinate's ability to inhibit zebrafish melanin was superior to that of tocopherol retinate and pyran whitening agents, and showed a significant difference compared to pyran whitening agents.

[0126] Example 6: DPPH free radical scavenging ability test

[0127] The DPPH free radical scavenging ability of pyranol retinyl ester (Example 1), hydroxypinazone retinyl ester, and pyran whitening agent was tested according to GB / T 39100-2020 "Determination of Antioxidant Properties of Peptides - DPPH and ABTS Methods" and T / SHRH 006-2018 "Cosmetics - Experimental Method for Free Radical (DPPH) Scavenging".

[0128] Test samples: Pyranol retinate (Example 1), hydroxypinazone retinate, and pyran whitening agent were diluted with anhydrous ethanol to prepare test sample solutions with concentrations of 30.6 μmol / g and 61.2 μmol / g, respectively.

[0129] In vitro DPPH free radical scavenging assay: Weigh 3.9 mg of DPPH, dissolve it in an appropriate amount of anhydrous ethanol, and sonicate it in the dark until fully dissolved. Make up to 100 ml with anhydrous ethanol to prepare a 0.1 mmol / L DPPH working solution. Prepare the corresponding concentration of the sample as the test solution. In a 96-well plate, prepare the reaction system according to the reagent addition amounts in Table 7, mix well, and set up 3 replicates and 1 background control well for each concentration.

[0130] Table 7. DPPH free radical scavenging test reaction system

[0131]

[0132] After incubation at room temperature for 5 min, the absorbance (OD) value of each well at 517 nm was measured using an ELISA reader. The scavenging rate of DPPH free radicals for each sample was calculated according to formula (3):

[0133] DPPH free radical scavenging rate (%) = (3)

[0134] In the formula: C1—OD value of the blank group containing DPPH system

[0135] C2—OD value of the blank group without DPPH system

[0136] T1—OD value of the DPPH system in the sample group

[0137] T2—OD value of the DPPH-free system in the sample group

[0138] The results are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was performed on the differences between groups using GraphPad Prism 8.0, and a p < 0.05 was considered statistically significant.

[0139] Table 8. Scavenging rate of DPPH free radicals for each sample

[0140]

[0141] From Table 8 and Figure 4 It can be seen that pyranol retinate can improve the scavenging rate of DPPH free radicals at a concentration of 30.6 μmol / g, indicating that pyranol retinate has antioxidant effects.

[0142] At a concentration of 61.2 μmol / g, pyranol retinate showed significantly better DPPH free radical scavenging ability than pyran whitening agents and hydroxypinazone retinate.

[0143] Example 7 Evaluation of Zebrafish's Reactive Oxygen Species (ROS) Inhibition Capacity

[0144] Using wild-type zebrafish of the AB line as a model, the inhibitory effects of pyranol retinyl ester (Example 1), tocopherol retinyl ester, hydroxypinazone retinyl ester, and pyran whitening agent on reactive oxygen species (ROS) in zebrafish were tested according to T / AHPCA 044-2023 "Detection Method for Anti-wrinkle and Firming Efficacy of Cosmetics (Zebrafish Embryo Oxidative Stress Model)".

[0145] Zebrafish ROS Inhibition Assay: One hour after feeding the day before the experiment, healthy adult AB strain wild-type zebrafish, half male and half female, were selected and placed in mating tanks, one pair per tank, separated by a partition. At 9:00 AM the following morning, the partition was removed, and embryos were collected one hour after free mating. Normally developed fertilized eggs were selected and placed in culture dishes, with an appropriate amount of embryo culture medium containing 0.03 g / L PTU added. Each dish contained no more than 100 embryos and was incubated in a biochemical incubator at 28±1℃. 72 hpf embryos of normally developing AB strain wild-type zebrafish were randomly selected and placed in 48-well plates, with 4 replicates per group and 5 embryos per well. The modeling agent was mixed with the sample solution and administered, with the final concentration according to the groupings in Table 9. 500 μl of the solution was added to each well and incubated in a biochemical incubator at 28±1℃ in the dark for 2 hours. Discard the original solution, wash with embryo culture medium, add 300 μl of DCFH-DA working solution to each well, and incubate in the dark for 1 h. After treatment, wash with embryo culture medium, place under an upright fluorescence microscope, and photograph. Randomly select at least 4 zebrafish embryos from each group, place them on a glass slide, add 4% methylcellulose fixative, fix, and photograph.

[0146] Table 9. Dosage Table

[0147]

[0148] The average fluorescence intensity of a specified area in the photographs was calculated using ImageJ software. The relative ROS content of zebrafish was calculated according to Equation (4), and the relative ROS inhibition rate of zebrafish was calculated according to Equation (5). The results are expressed as mean ± standard deviation (mean ± SD), see Table 10-11 (One-way ANOVA was performed between different groups using GraphPad Prism 8.0, and P < 0.05 was considered statistically significant):

[0149] Zebrafish ROS relative content (%) = (4)

[0150] In equation (4):

[0151] V e —The average fluorescence intensity of ROS in the sample group or model group;

[0152] V c —Mean ROS fluorescence intensity in the blank control group.

[0153] Zebrafish ROS relative inhibition rate (%) = (5)

[0154] In equation (5):

[0155] V e —The average fluorescence intensity of ROS in the sample group;

[0156] V m —Mean ROS fluorescence intensity in the blank control group.

[0157] Table 10. Relative ROS content and inhibition rate of zebrafish in each group at a sample concentration of 40 μmol / L

[0158]

[0159] Table 11. Relative ROS content and inhibition rate of zebrafish in each group at a sample concentration of 60 μmol / L

[0160]

[0161] As shown in Tables 10-11, pyranol retinyl ester inhibited the ROS levels of zebrafish under oxidative stress by 31.78% and 31.83% at concentrations of 40 μmol / L and 60 μmol / L, respectively, which were statistically different from the model group, indicating that pyranol retinyl ester has anti-wrinkle and firming effects.

[0162] At a concentration of 40 μmol / L, pyranol retinate showed significantly better ROS inhibition ability than pyran whitening agents in zebrafish under oxidative stress.

[0163] Example 8 Stimulation Test

[0164] Thirty participants were included in the experiment, and all subjects met the standards described in the "Cosmetic Safety Technical Specifications". The product from Example 1 was prepared into a solution containing 0.3% test substance using caprylic / capric triglyceride. 20 μL of the test substance solution was added to the chamber of the waterproof spot applicator. The blank control well contained physiological saline, and the solvent control well contained caprylic / capric triglyceride. The spot applicator containing the test substance solution was applied to the flexor side of the subject's forearm, and gently pressed with the palm to ensure even application to the skin for 24 hours. Skin reactions were observed according to the standards in Table 12 at 30 min (after the indentation disappeared), 24 h, and 48 h after removing the spot applicator, and the results were recorded (see Table 13).

[0165] Table 12. Grading Criteria for Skin Reactions in Skin Occlusive Patch Tests

[0166]

[0167] Table 13. Results of human skin patch test

[0168]

[0169]

[0170] As shown in Table 13, neither 0.3% pyranol retinate nor tocopherol retinate is irritating to the skin.

[0171] Example 9: Evaluation of the efficacy of an in vitro skin model

[0172] The efficacy evaluation of the in vitro skin model was conducted in accordance with the "CALT / TM / SOP287-01 Standard Operating Procedure for Efficacy Evaluation of In Vitro Skin Models".

[0173] The test samples are as follows:

[0174] Negative group: PBS;

[0175] Blank group: Isonononyl isononanoate solution;

[0176] Positive group: Hydroxypinazone retinate was dissolved in isononyl isononanoate solution at a concentration of 2.53 mmol / L;

[0177] Sample group: The pyranol retinate from Example 1 was dissolved in isononyl isononanoate solution at a concentration of 2.53 mmol / L.

[0178] Experimental Methods: Pre-warmed culture medium was added to 6-well plates (2 mL / well). The whole-skin model was transferred to a 6-well plate containing culture medium and incubated overnight. 22.5 ± 2.5 μL of test sample was evenly applied to the surface of the whole-skin model. The models were divided into experimental groups and exposed at room temperature. After the specified exposure time, the samples were thoroughly rinsed with PBS until no residue remained, and then placed in a blue deep-well plate containing fresh culture medium for further incubation for 48 h. After the specified incubation time, the culture medium from each well was collected and stored at -80℃. The Collagen I content was determined using an ELISA kit. Data are expressed as mean ± standard deviation and analyzed using SPSS. The relative content of Collagen I in each group was calculated with the control group (NC) as 100% as the baseline. The results are shown in Table 14.

[0179] Table 14. Results of Collagen I content test

[0180]

[0181] As can be seen from Table 14, compared with the negative group, both the hydroxypinazone retinate solution (as the positive group) and the pyranol retinate solution (as the sample group) significantly increased Collagen I production in the whole-skin model; and the pyranol retinate of the present invention showed a significantly better Collagen I production-promoting effect than the hydroxypinazone retinate.

[0182] Example 10: Evaluation of Acne-Clearing Efficacy

[0183] After activating the frozen Propionibacterium acnes, a solution with a concentration of 1×10⁻⁶ was prepared using a turbidimetric method. 6CFU / mL bacterial suspension. 100 μl of bacterial suspension per well was added to each well of a 96-well plate, along with 100 μl of sample solution (pyranol retinate (Example 1) and hydroxypinazone retinate) per well. 100 μl of erythromycin solution was added to the positive control group. The plates were incubated at 37°C for 48 h in an anaerobic incubator. Wells with clear culture medium corresponded to the minimum inhibitory concentration (MIC) of the sample against *Propionibacterium acnes*. The results are shown in Table 15.

[0184] Table 15. Minimum inhibitory concentrations of each sample

[0185]

[0186] As shown in Table 15, the pyranol retinate of the present invention has a good inhibitory effect on Propionibacterium acnes, indicating that the pyranol retinate has acne-removing effects.

[0187] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0188] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A pyranol retinate, characterized in that, Its structural formula is shown in equation (I): Formula (I).

2. A method for preparing the pyranol retinate ester according to claim 1, characterized in that, Includes the following steps: Retinic acid reacts with dimethylmethoxybenzodihydropyranol to yield pyranol retinate; The molar ratio of retinoic acid to dimethylmethoxybenzodihydropyranol is 1:1.01-1.4; The reaction is carried out under the action of a carboxyl activator, which is selected from any one or more combinations of HBTU, HATU, HCTU, BOP, PyBOP, DMTMM, DIC, DCC, and EDCI. The reaction is carried out under the action of a condensation aid, which is selected from any one or a combination of HOBT, Cl-HOBt, HOAT, Oyxma-Pure, Oxyma-B, and DMAP. The reaction temperature is 10℃-30℃, and the reaction time is 1h-3h.

3. The method for preparing pyranol retinate according to claim 2, characterized in that, The reaction is carried out in a solvent selected from one or more combinations of toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, dichloroethane, methyl tert-butyl ether, methyl cyclopentyl ether, isopropyl ether, dibutyl ether, isopropyl acetate, ethyl butyl acetate, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, N-ethylpyrrolidone, and N-butylpyrrolidone.

4. The method for preparing pyranol retinate according to claim 2, characterized in that, The molar ratio of retinoic acid to carboxyl activator is 1:1-3.

0.

5. The method for preparing pyranol retinate according to claim 2, characterized in that, The molar ratio of retinoic acid to condensation aid is 1:0.05-1.

0.

6. A method for preparing the pyranol retinate ester according to claim 1, characterized in that, Includes the following steps: S1. Retinic acid reacts with an acyl halide reagent to yield retinyl chloride; S2. The retinyl chloride and dimethylmethoxybenzodihydropyranol react to give pyranol retinate; The molar ratio of retinoic acid to the acyl halide reagent is 1:1-1.3; The reaction in step S1 is carried out at a temperature of 10℃-50℃ for 2h-6h. The molar ratio of retinyl chloride to dimethylmethoxybenzodihydropyranol is 1:1-1.3; The reaction in step S2 is carried out under the action of an acid-binding agent, which is selected from any one or a combination of triethylamine, diisopropylethylamine, pyridine, and methylmorpholine.

7. The method for preparing pyranol retinate according to claim 6, characterized in that, The acyl halide reagent is selected from at least one of thionyl chloride, oxalyl chloride, diphosgene, and triphosgene; and / or, The reaction described in step S1 is carried out in a solvent selected from any one or more combinations of toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, dichloroethane, methyl tert-butyl ether, methyl cyclopentyl ether, isopropyl ether, dibutyl ether, isopropyl acetate, ethyl butyl acetate, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, N-ethylpyrrolidone, and N-butylpyrrolidone; and / or, The reaction described in step S2 is carried out in a solvent selected from any one or more combinations of toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, dichloroethane, methyl tert-butyl ether, methyl cyclopentyl ether, isopropyl ether, dibutyl ether, isopropyl acetate, ethyl butyl acetate, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, N-ethylpyrrolidone, and N-butylpyrrolidone.

8. The method for preparing pyranol retinate according to claim 6, characterized in that, The molar ratio of retinyl chloride to the acid-binding agent is 1:1-1.

3.

9. A method for preparing the pyranol retinate ester according to claim 1, characterized in that, Includes the following steps: Methyl retinate and dimethylmethoxybenzodihydropyranol acetate undergo transesterification to yield pyranol retinate. The molar ratio of methyl retinoate to dimethyl methoxybenzodihydropyranol acetate is 1:0.9-1:2; The transesterification reaction is carried out in the presence of a base, wherein the base is sodium methoxide. The transesterification reaction was carried out at a temperature of 55℃-65℃. The transesterification reaction was carried out under reduced pressure, with a pressure of 100 Pa to 1000 Pa. The transesterification reaction takes 2-4 hours.

10. The method for preparing pyranol retinate according to claim 9, characterized in that, The molar ratio of methyl retinoate to alkali is 1:0.03-0.

1.

11. The use of the pyranol retinate ester according to claim 1 in the preparation of cosmetics or topical skin medicines with whitening, antioxidant, anti-aging, wrinkle-reducing and / or acne-reducing effects.

12. A cosmetic product, characterized in that, The active ingredient in the cosmetic product includes the pyranol retinate as described in claim 1.

13. A topical skin medication, characterized in that, The active ingredient in the topical skin medication comprises the pyranol retinate as described in claim 1.

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