A SMALL MOLECULE COMPOUND AND ITS USE AND PREPARATION METHOD

Abstract

The present invention relates to a small molecular compound and its application as an active ingredient in cosmetics, as well as its preparation method. The small molecular compound is represented by the following chemical formula:

##STR00001##

Wherein, R1 is selected from compounds represented by the following formula:

##STR00002##

C1 represents a five-or six-membered carbon ring or heterocycle, while R11 stands for alkyl or ester groups with multiple substitutions on the ring.

##STR00003##

C2 represents an aromatic ring, while R12 stands for alkyl, ester, carbonyl, ether groups, or cyclic substituents formed by bridging two adjacent substituents on the ring with multiple substitutions.

##STR00004##

R13, R13, R13, identical or different, are selected from hydrogen, ester groups, alkyl, alkenyl, and cycloalkenyl. R2 is chosen from

##STR00005##

This compound exhibits antioxidant properties when used as a reagent.

Claims

1. A type of small-molecule compound wherein the following structural formula: ##STR00028## Wherein, R1 is selected from compounds represented by the following formula: ##STR00029## C1 represents a five-membered or six-membered carbon ring or heterocycle, while R11 represents alkyl or ester groups with multiple substitutions on the ring; ##STR00030## C2 is an aromatic ring, while R12 represents alkyl, ester, carbonyl, ether groups, or cyclic substituents formed by two adjacent substituents on the ring with multiple substitutions. ##STR00031## R13, R13, R13, whether identical or different, are chosen from hydrogen, ester groups, alkyl, alkenyl, and cycloalkenyl. R2 is chosen from ##STR00032##

2. A small molecular compound as described in claim 1, wherein: R1 is chosen from ##STR00033##

3. A small molecular compound as described in claim 1, wherein: The small molecular compound is selected from compounds represented by the following structures: ##STR00034##

4. A small molecular compound as described in claim 1, wherein: The mentioned small molecular compound serves as an active ingredient in cosmetics.

5. A small molecular compound as described in claim 1, wherein: Containing at least one of the following uses: A. Serving as an antioxidant. B. Serving as an antioxidant for use in cosmetics.

6. A small molecular compound as described in claim 1, wherein: A. Serving as a DPPH radical scavenger. B. Serving as a DPPH radical scavenger for use in cosmetics.

7. A small molecular compound as described in claim 1, wherein: A. Serving as an inhibitor of intracellular reactive oxygen species (ROS). B. Serving as an inhibitor of intracellular reactive oxygen species (ROS) for use in cosmetics.

8. A small molecular compound as described in claim 1, wherein: Using a selenium-containing catalyst, under its catalytic action, carboxylic acids containing active unit A and alcohols containing active unit B react to form small molecular compounds containing both active units A and B simultaneously. Wherein, the carboxylic acid containing active unit A mentioned above has the general formula R2COOH; The alcohol containing active unit B mentioned above has the general formula R1OH.

9. A small molecular compound as described in claim 8, wherein: The structural formula of the selenium-containing catalyst is as follows: ##STR00035## Wherein, the ##STR00036## are benzene ring that may or may not contain substituents; these substituents are chosen from alkyl, alkoxy, fluoroalkyl, halogens, cyano, and amino groups.

Description

FIGURE DESCRIPTION

[0049] FIG. 1-1: Schematic of the Reaction Process;

[0050] FIG. 1-2: Mechanism Diagram;

[0051] FIG. 2-1: Proton NMR of Compound Corresponding to Example 2-1;

[0052] FIG. 2-2: Carbon NMR of Compound Corresponding to Example 2-1;

[0053] FIG. 2-3: Elemental Analysis of Compound Corresponding to Example 2-1;

[0054] FIG. 2-4: Mass Spectrum of Compound Corresponding to Example 2-1;

[0055] FIG. 3: Reference Curve for Ascorbic Acid (Vitamin C) DPPH Free Radical Scavenging Activity;

[0056] FIG. 4: Antioxidant Activity of Retinyl-trans-4-tert-butylcyclohexanol Ester against DPPH Free Radicals ***: p<0.001 compared to the control group;

[0057] FIG. 5: Elimination of Intracellular Reactive Oxygen Species (ROS) by Retinyl-trans-4-tert-butylcyclohexanol Ester ###: p<0.001 compared to the Control group; ***: p<0.001 compared to the UVB control group; NS: indicates no statistical difference, p0.05

[0058] FIG. 6: Skin Responses of 30 Subjects under Different Observational Conditions;

[0059] FIG. 7: Stability Comparison of Retinyl-trans-4-tert-butylcyclohexanol Ester and Retinol under Light Conditions;

[0060] FIG. 8: Stability Comparison of Retinyl-trans-4-tert-butylcyclohexanol Ester and Retinol at 50 C.;

[0061] FIG. 9: Stability Comparison of Retinyl-trans-4-tert-butylcyclohexanol Ester and Retinol at 45 C.;

[0062] FIG. 10: Stability Comparison of Retinyl-trans-4-tert-butylcyclohexanol Ester and Retinol at Room Temperature;

[0063] FIG. 11: Stability Comparison of Retinyl-trans-4-tert-butylcyclohexanol Ester and Retinol at 4 C.

SPECIFIC IMPLEMENTATION METHODS

[0064] For the preparation method of the aforementioned small molecule compound, traditional esterification methods can be employed. However, their yield and purity might not be optimal, particularly when the compound possesses chirality. Therefore, in this specific case, a novel preparation method is proposed. This method involves the use of a selenium-containing catalyst, where, under its catalytic action, a carboxylic acid containing active unit A reacts with an alcohol containing active unit B, resulting in the formation of a small molecule compound containing both active units A and B simultaneously.

[0065] The carboxylic acid containing the aforementioned active unit A has the general formula R2COOH.

[0066] The alcohol containing the aforementioned active unit B has the general formula R1OH.

[0067] The reaction process is illustrated in FIG. 1-1.

[0068] During the selenium-catalyzed reaction process, the carboxylic acid containing active unit A and the alcohol containing active unit B, under the catalytic influence of a selenium-containing catalyst, undergo reflux reaction at 60-90 C. for 0.5-10 hours to obtain the desired product.

[0069] The molar ratio between the carboxylic acid containing active unit A and the alcohol containing active unit B is typically 1:1.

[0070] The catalyst is generally used in an amount of 1-10% based on the molar quantity of the carboxylic acid containing active unit A. The reaction is usually carried out in a solvent with a boiling point ranging from 60-90 C.

[0071] The structural formula of the aforementioned selenium-containing catalyst is as follows:

##STR00015##

[0072] Among which, the above

##STR00016##

are a benzene ring with or without substituents.

[0073] The above substituents are selected from alkyl, alkoxy, fluoroalkyl, halogens, cyano, and amino.

[0074] The above selenium-containing catalyst is preferably selected from at least one of the catalysts represented by catalyst 1 or catalyst 2

##STR00017##

[0075] The specific reaction mechanism is illustrated in FIG. 1-2.

[0076] The aforementioned selenium-containing catalyst is obtained from the reaction between ortho-hydroxybenzyl halide and diphenyldiselenide. The structure of the aforementioned ortho-hydroxybenzyl halide is as follows:

##STR00018##

[0077] The structure of the aforementioned diphenyldiselenide is as follows

##STR00019##

[0078] X is preferably Br. The specific reaction equation is as follows:

##STR00020##

[0079] The preparation method of the aforementioned selenium-containing catalyst is as follows: [0080] S1. At room temperature, add a reducing agent to diphenyldiselenide and stir until it clarifies. [0081] S2. At room temperature, add ortho-hydroxybenzyl halide to the reaction solution from S1. After reacting for 10-36 hours, the resulting solution undergoes acidification, extraction, drying, filtration, and concentration to obtain the catalyst intermediate. [0082] S3. Dissolve the catalyst intermediate obtained in S2, cool it to below 0 degrees Celsius, add NBS, and react for 1-10 hours. Then, quench the reaction with an alkali solution. Extract, dry, filter, concentrate, and recrystallize to obtain the target product.

[0083] Further, the method for preparing a small molecule compound provided by the present invention also comprises the following features:

[0084] The molar ratio of the aforementioned ortho-hydroxybenzyl halide to the diaryl diselenide ranges from 1:1 to 1:2.

[0085] The molar ratio of the aforementioned ortho-hydroxybenzyl halide to the reducing agent is between 1:2 to 1:3.

[0086] The molar ratio of the aforementioned ortho-hydroxybenzyl halide to N-bromosuccinimide (NBS) is between 1:2 to 1:5.

[0087] Based on the above scheme, specific experiments are conducted as shown below: )

Example 1. General Synthetic Steps of the Organic Selenium Catalyst

[0088] ##STR00021##

[0089] At room temperature, to a solution of 120 mmol of diaryl diselenide in 200 mL of tetrahydrofuran (THF), sodium borohydride (200 mmol) was added dropwise. The reaction continued until the solution became clear. Then, 100 mmol of 2-bromobenzyl alcohol was added, and the reaction was allowed to proceed for an additional 36 hours. Afterward, the reaction mixture was acidified with hydrochloric acid, extracted, dried, filtered, concentrated to yield the catalyst intermediate.

[0090] Subsequently, the obtained catalyst intermediate was dissolved in a mixture of methanol and dichloromethane. The solution was cooled to below 0 degrees Celsius, and N-bromosuccinimide (NBS) (200 mmol) was added. After a reaction time of 1 hour, the reaction was quenched using a 10% aqueous solution of sodium hydroxide. The resulting mixture was then extracted with dichloromethane, dried, filtered, and concentrated to obtain the crude product. Finally, the crude product was recrystallized from a mixture of n-hexane and dichloromethane to obtain the target product.)

Example 1-1. Preparation of Catalyst 1

[0091] ##STR00022##

[0092] According to the general synthesis procedure for the organic selenium catalyst, diphenyldiselenide and benzyl bromide without any other substituents were selected. This yielded Catalyst 1 with a purity of 98.5% and a yield of 83%.

[0093] Characterization of Compounds: .sup.1H NMR (400 MHz, CDCl.sub.3) =9.71 (s, 1H), 7.32-7.36 (m, 5H), 6.97-7.03 (m, 2H), 6.73-6.83 (m, 2H), 2.65 (s, 2H). .sup.13C NMR (400 MHz, CDCl.sub.3) =157.3, 135.1, 132.3, 131.1, 130.9, 127.5, 125.5, 124.7, 121.0, 116.2, 62.7. m/z =280.1. Elemental analysis: C, 56.01; H, 4.39.

Example 1-2. Preparation of Catalyst 2

[0094] ##STR00023##

[0095] Following the general synthesis steps for the organic selenium catalyst, diphenyldiselenide was chosen as the diselenide compound, and 2-bromo-4-methylphenol was used as the 4-hydroxybenzyl bromide. This procedure resulted in the formation of catalyst 2, with a yield of 81% and a purity of 99%.

[0096] Characterization of Compounds: .sup.1H NMR (400 MHz, CDCl.sub.3) =9.64 (s, 1H), 7.44 (d, J=7.5 Hz, 2H), 7.25 (d, J=7.5 Hz, 2H), 6.89-6.91 (m, 2H), 6.75 (d, J=7.5 Hz, 1H), 2.63 (s, 2H), 2.34 (s, 3H). .sup.13C NMR (400 MHz, CDCl.sub.3) =154.2, 134.6, 133.1, 132.0, 130.7, 128.9, 127.5, 124.5, 116.1, 63.5, 21.8. m/z =328.9. Elemental analysis: C, 51.43; H, 4.21.

Example 2

Example 2-1. Synthesis of trans-4-tert-butylcyclohexanol and Retinoic Acid

[0097] In a reaction flask, sequentially add 200 mL of toluene, retinoic acid (100 mol), trans-4-tert-butylcyclohexanol (100 mol), and organic selenium catalyst (5 mol). Reflux the mixture at elevated temperature for 18 hours, then extraction three times using water and ethyl acetate. Remove the aqueous layer and dry the organic layer with anhydrous sodium sulfate. Evaporate the solvent using a rotary evaporator, followed by purification through silica gel column chromatography to obtain the pure spliced product:

##STR00024##

[0098] According to the actual differences in reactants, the molar ratio between trans-4-tert-butylcyclohexanol and retinoic acid can be adjusted in the range of 1-2:1. The molar amount of the organic selenium catalyst is 2-20% based on the hydroxyl group-containing active compound. As a preferred embodiment: In this particular embodiment, the compound with the following structure was chosen as the catalyst:

##STR00025##

[0099] The above catalyst was synthesized according to the synthetic steps of the organic selenium catalyst in Example 1-2, and after synthesis, it exhibited a nuclear magnetic resonance (NMR) purity of 99%. Then, the aforementioned catalyst was utilized in a specific catalytic reaction experiment. Following the general steps for splicing two active compounds, the organic selenium catalyst chosen was the aforementioned catalyst 1, resulting in the production of retinoic acid trans-4-tert-butylcyclohexyl ester with a yield of 85% and a purity of 99.2%.

[0100] Characterization of Compounds (FIG. 2-1, 2-4): .sup.1H NMR (400 MHz, CDCl.sub.3) =7.01-6.94 (m, 1H), 6.29-6.11 (m, 4H), 5.75 (s, 1H), 4.68-4.66 (m, 1H), 2.34 (s, 3H), 2.06-1.83 (m, 4H), 1.99 (s, 3H), 1.71 (s, 3H), 1.80-1.58 (m, 6H), 1.48-0.81 (m, 5H), 1.00 (s, 6H), 0.86 (s, 9H).

[0101] .sup.13C NMR (400 MHz, CDCl.sub.3) =166.8, 152.4, 139.4, 137.8, 137.4, 135.4, 130.8, 130.0, 129.7, 128.6, 119.3, 77.5, 77.2, 76.8, 73.0, 47.3, 39.7, 34.4, 33.2, 32.4, 29.1, 27.7, 25.6, 21.9, 19.3, 13.9, 13.0. [0102] Elemental analysis: C, 82.00; H, 10.67. [0103] m/z: 439.4

Example 2-2. Synthesis of the Compound Resulting from the Splicing of Retinoic Acid and Hydroquinone

[0104] ##STR00026##

[0105] According to the general steps for splicing two active compounds, retinoic acid was chosen as the carboxylic acid active compound, hydroquinone was selected as the hydroxyl group active compound, and catalyst 1 was employed as the organic selenium catalyst. The target compound was obtained with a yield of 87% and a purity of 99.2%.)

[0106] Characterization of Compounds: .sup.1H NMR (400 MHz, CDCl.sub.3) =7.73 (d, J=8 Hz, 1H), 7.52 (s, 1H), 7.03-6.97 (m, 1H), 6.91 (d, J=8 Hz, 1H), 6.33-6.15 (m, 4H), 5.89 (s, 1H), 3.87 (s, 3H), 2.67 (s, 3H), 2.34 (s, 3H), 2.08-1.80 (m, 6H), 1.98 (s, 3H), 1.72 (s, 3H), 1.00 (s, 6H).

[0107] .sup.13C NMR (400 MHz, CDCl.sub.3) =197.1, 167.5, 166.8, 152.4, 150.4, 139.4, 137.8, 137.4, 135.4, 133.6, 130.8, 130.0, 129.7, 128.6, 119.3, 115.9, 112.3, 105.8, 73.0, 39.7, 34.4, 33.2, 32.4, 29.1, 21.9, 19.3, 13.9, 13.0. [0108] Elemental analysis: C, 77.41; H, 8.01. [0109] m/z: 449.2

Example 2-3. Synthesis of Compound from Retinoic Acid and Bisabolol

[0110] ##STR00027##

[0111] According to the general splicing steps for two active compounds, retinoic acid was used as the carboxylic acid active compound, and retinol was chosen as the hydroxyl active compound. Catalyst 1 was employed as the organic selenium catalyst. The target compound was obtained with a yield of 81% and a purity of 99.5%.

[0112] Characterization of Compounds: .sup.1H NMR (400 MHz, CDCl.sub.3) 7.03-6.97 (m, 1H), 6.91 (d, J=8 Hz, 1H), 6.33-6.15 (m, 4H), 5.89 (s, 1H), 5.33 (s, 1H), 5.02 (s, 1H), 2.34 (s, 3H), 2.12-1.83 (m, 13H), 1.98 (s, 3H), 1.82 (s, 3H), 1.72 (s, 3H), 1.68 (s, 3H), 1.64 (s, 3H), 1.61-1.33 (m, 4H), 1.43 (s, 3H), 1.00 (s, 6H).

[0113] .sup.13C NMR (400 MHz, CDCl.sub.3) =167.5, 166.8, 152.4, 139.4, 137.8, 137.4, 135.4, 134.2, 133.6, 131.3, 130.8, 130.0, 129.7, 128.6, 124.5, 121.4, 119.3, 74.7, 73.0, 42.6, 39.7, 34.4, 33.2, 32.4, 31.1, 29.1, 26.9, 24.3, 23.9, 22.4, 22.1, 21.9, 19.3, 13.9, 13.0. [0114] Elemental analysis: C, 83.31; H, 10.45. [0115] m/z: 505.8

Example 3. DPPH Antioxidant Assay of Retinyl-trans-4-tert-butylcyclohexanol Ester

3.1 Experimental Principle:

[0116] The DPPH radical scavenging assay is an in vitro method used to evaluate antioxidant activity. DPPH is a stable free radical in organic solvents and appears purple in methanol or ethanol, with maximum absorption at a wavelength of 517 nm. The DPPH assay is based on the ability of an antioxidant to donate an electron to the DPPH radical, converting its purple color to yellow. The degree of absorbance change at 517 nm corresponds to the extent of the free radical scavenging activity, meaning the stronger the scavenging ability of the antioxidant, the lower the absorbance.

3.2 Experimental Materials Reagents:

[0117] DPPH (Sigma), PBS (Gibco), anhydrous ethanol (National Pharmaceutical Reagent), petroleum ether (National Pharmaceutical Reagent), Vitamin C (CNW), anhydrous ethanol (National Pharmaceutical Reagent). Main Equipment: Microplate Reader (Tecan, Spark), Microplate Shaker (Kylin-Bell, TS-92).

3.3 In Vitro DPPH Free Radical Scavenging Assay

3.3.1 Preparation of the DPPH Free Radical Scavenging Reference Curve

[0118] Using ascorbic acid (VC) as a system reference, dilute it with PBS to obtain five concentrations: 12.5, 25, 50, 100, and 200 g/mL. Perform the test and calculations according to the procedure outlined in 3.3.2. Plot the reference curve with the concentration of the reference substance on the x-axis and the DPPH free radical scavenging rate on the y-axis.

3.3.2 In Vitro DPPH Free Radical Scavenging Test

[0119] Prepare the samples into the respective concentrations of test solutions. Prepare the reaction system by adding the specified amount of each reagent mentioned in Table 1, mix thoroughly. For each concentration, set up three replicate wells and one blank control well.

TABLE-US-00001 TABLE 1 Reaction System for DPPH Free Radical Scavenging Test (units in L) (L) C1 C2 T1 T2 DPPH Ethanol 180 0 180 0 Solution Test Sample 0 0 20 20 Anhydrous 0 180 0 180 Ethanol PBS 20 20 0 0

[0120] The reaction system is placed at room temperature and allowed to react in the dark for 30 minutes. After the reaction is complete, the absorbance (OD value) is measured at 515 nm. The formula to calculate the scavenging rate of the sample against DPPH free radicals is as follows:

[00001]$\mathrm{Sample}s\mathrm{DPPH}\mathrm{Free}\mathrm{Radical}\mathrm{Scavenging}\mathrm{Rate}=$$\left[\left(C1-C2\right)-\left(T1-T2\right)\right]/\left(C1-C2\right)100\%$ [0121] Where: [0122] C1=Absorbance value of the blank with DPPH [0123] C2=Absorbance value of the blank without DPPH [0124] T1=Absorbance value of the sample with DPPH [0125] T2=Absorbance value of the sample without DPPH

3.4 In Vitro DPPH Radical Scavenging Assay Result

3.4.1 In Vitro DPPH Free Radical Scavenging Test Results

[0126] The system reference curve for DPPH free radical scavenging is presented in Table 1 and FIG. 3.

TABLE-US-00002 TABLE 2 System Reference Results Analysis Concentration (g/mL) 200 100 50 25 12.5 (%)DPPH Free 101.28 101.32 64.58 30.26 20.55 Radical Scavenging 92.01 95.11 65.09 31.17 18.55 Rate (%) 108.75 97.07 62.73 32.98 20.32

3.4.2 Results of In Vitro DPPH Free Radical Scavenging Test

[0127] Refer to Table 3 and FIG. 4

TABLE-US-00003 TABLE 3 Analysis of DPPH Free Radical Scavenging Test Results DPPH Free Sample Radical Concentration Scavenging Sample (%v/v) Rate (%v/v) p Significance Retinyl 0 0.00 1.22 / / 4-trans-t-Butyl 4 83.96 1.22 <0.001 *** cyclohexanol 2 48.12 1.07 <0.001 *** 0.5 36.72 1.98 <0.001 *** 0.1 15.91 1.25 <0.001 *** 0.02 9.87 3.44 <0.001 *** Note: Data presented as mean SD. When conducting statistical analysis using the t-test, significance is denoted by *, where p < 0.001 is represented as *** when comparing different concentrations of Retinyl 4-trans-t-Butylcyclohexanol experimental groups with the blank control group.

[0128] Conclusion: Retinyl 4-trans-t-Butylcyclohexanol at concentrations ranging from 0.02% to 4% demonstrates a statistically significant increase in DPPH free radical scavenging compared to the control group (p<0.001), indicating its antioxidative capacity.

Example 4 Implementation of ROS Scavenging Test of Retinyl 4-trans-t-Butylcyclohexanol

4.1 Experimental Objective

[0129] Oxidation represents the greatest threat to skin aging, primarily caused by environmental stressors such as UV radiation, pollution, smoke, and daily stress. Among these factors, UV radiation stands as the most significant contributor. Skin exposed to UV radiation generates an excess of reactive oxygen species (ROS) within cells, triggering the expression of genes related to aging, instigating an inflammatory cascade, and reducing the expression of elastin and collagen proteins, leading to manifestations of photoaging like skin laxity and wrinkles. Photoaging predominantly occurs in the dermis. Therefore, this test utilizes fibroblast cells as a test system to evaluate changes in ROS levels and assess whether Retinyl 4-t-Butylcyclohexanol test samples possess antioxidant effects in reducing ROS levels.

4.2 Experimental Design

[0130] UVB irradiation is administered to immortalized human keratinocytes (HaCat cells) to induce intracellular reactive oxygen species (ROS) generation. The UVB dose applied to HaCat cells is 20 mJ/cm2. Following the UVB treatment, varying concentrations of Retinyl 4-t-Butylcyclohexanol test samples are added to the cells and incubated for 24 hours. ROS levels within the cells are assessed using a ROS detection kit.

4.3 Experimental Results

[0131] Graph Pad Prism was used for statistical plotting, and the results were presented as MeanSD. Statistical analysis was conducted using the t-test. A significance level of p<0.05 was considered statistically significant, where *p<0.05, ** p<0.0 1 (0.005<** p<0.01), and *** p<0.001, indicating increased significance as the p-value decreases. When employing the t-test for statistical analysis, the comparison between the UVB control group and the Control group was represented as # (p<0.001 indicated as ###). For the experimental groups with varying concentrations of Retinyl-trans-4-tert-Butylcyclohexanol compared to the UVB control group, significance was represented by * (ns indicated no statistical difference, p0.05; p<0.001 indicated as ***).

[0132] The results of the Retinyl-trans-4-tert-Butylcyclohexanol intracellular reactive oxygen species (ROS) scavenging experiment are illustrated in FIG. 5 below.

[0133] Conclusion: UVB significantly increased the ROS level in Hacat cells. Retinyl-trans-4-tert-Butylcyclohexanol effectively reduced the UVB-induced intracellular ROS levels compared to the control group, showing statistical significance (p<0.001), thereby demonstrating antioxidative capabilities.

Example 5. Retinyl-trans-4-tert-Butylcyclohexanol Patch Test

5.1 Materials and Methods

5.1.1 Test Substance:

[0134] 2% Retinyl-trans-4-tert-Butylcyclohexanol oil solution (the carrier oil here is Caprylic/Capric Triglyceride).

5.1.2 Negative Control:

[0135] Filter patch.

5.1.3 Subjects:

[0136] A total of 30 individuals, 15 males and 15 females, aged between 22 to 55 years old, with an average age of 40.731.76 years old, meeting the selection criteria for volunteers. (To prevent the possibility of subjects dropping out during the study, two alternative candidates, one male and one female, were selected for this experiment.

5.1.4 Patch Test Method:

[0137] Qualified patch test equipment was used, employing a closed patch test method. Approximately 0.020 mL to 0.025 mL (liquid) of the test substance was placed onto the patch test apparatus and applied on the subjects' backs using hypoallergenic adhesive tape. After 24 hours, the test substance was removed, and skin reactions were observed at 0.5, 24, and 48 hours post-removal. Skin reactions were recorded according to the current effective standards for grading skin reactions in accordance with established technical specifications.

5.2 Test Results

[0138] The patch test results on human skin showed negative reactions for all 30 subjects. The summarized results are presented in Table 4.

TABLE-US-00004 TABLE 4 Summary of Human Skin Patch Test Results for Cosmetics Number Number of Subjects with Different of Observation Skin Reactions in Patch Test Groups Subjects Time 0 1 2 3 4 Test 30 0.5 30 0 0 0 0 Substance 24 30 0 0 0 0 48 30 0 0 0 0 Control 30 0.5 30 0 0 0 0 24 30 0 0 0 0 48 30 0 0 0 0 Note: Skin reaction observations for the first 30 subjects at different observation times are depicted in FIG. 6.

6. Stability Comparison Between Retinyl-trans-4-tert-Butylcyclohexanol and Retinol

6.1 Experimental Purpose

[0139] To compare the stability of Retinyl-trans-4-tert-Butylcyclohexanol and retinol (selected as a representative of retinol) under different conditions.

6.2 Experimental Design

[0140] A certain amount of retinol and Retinyl-trans-4-tert-Butylcyclohexanol is taken and placed under conditions of light exposure, 4 C., 25 C., 45 C., and 50 C. for 28 days. Samples are periodically collected to determine their content and examine the influence of light and temperature on their stability.

6.3 Instruments and Equipment

[0141] High-Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD), Electronic Balance (precision of 0.01 mg), Chromatographic Column (Welch Ultimate series XB-C18 column, 250 mm*4.6 mm, 5 m), Several volumetric flasks.

6.4 Reagents, Solutions, and Control Samples

[0142] Reagents: Acetonitrile (chromatographic grade), Formic acid (chromatographic grade), Ultrapure water. [0143] Control Sample 1: Retinol, purity above 99.5%, Manufacturer: Self-made by Coachchem Laboratory (20 C., light-resistant and vacuum-sealed). [0144] Control Sample 2: Retinyl-trans-4-tert-Butylcyclohexanol, purity above 99.0%, Manufacturer: Self-made by Kechin Laboratory (20 C., light-resistant and vacuum-sealed).

[0145] Blank Solvent: Acetonitrile-0.1% Formic acid water solution (95/5).

6.5 Sample Preparation

6.5.1 Control Sample 1 Solution:

[0146] Accurately weigh approximately 10.0 mg of retinol crystal pure substance control sample into a 20 mL brown volumetric flask. Add antioxidant BHT (butylated hydroxytoluene) accordingly, then add an appropriate amount of acetonitrile, ultrasonicate while avoiding light, dilute to the mark, shake well, and prepare two parallel samples.

6.5.2 Control Sample 2 Solution:

[0147] Accurately weigh approximately 10.0 mg of Retinyl-trans-4-tert-Butylcyclohexanol crystal pure substance control sample into a 20 mL brown volumetric flask. Add antioxidant BHT accordingly, then add an appropriate amount of acetonitrile, ultrasonicate while avoiding light, dilute to the mark, shake well, and prepare two parallel samples.

6.5.3 Retinyl-trans-4-tert-Butylcyclohexanol Sample Solution:

[0148] Accurately weigh approximately 0.1g of Retinyl-trans-4-tert-Butylcyclohexanol samples obtained under conditions of light exposure, 4 C., 25 C., 45 C., and 50 C. into a 100 mL brown volumetric flask. Add an appropriate amount of acetonitrile while avoiding light, ultrasonicate, dilute to the mark, shake well, and prepare two parallel samples for each condition.

6.5.4 Retinol Sample Solution:

[0149] Accurately weigh approximately 0.1 g of retinol samples obtained under conditions of light exposure, 4 C., 25 C., 45 C., and 50 C. into a 100 mL brown volumetric flask. Add an appropriate amount of acetonitrile while avoiding light, ultrasonicate, dilute to the mark, shake well, and prepare two parallel samples for each condition.

6.6 Chromatographic Conditions

6.6.1 Retinol Chromatographic Conditions Mobile Phase:

[0150] Phase A of acetonitrile; phase B of 0.1% formic acid-water. [0151] Column Temperature: 25 C. [0152] Flow Rate: 1.0 mL/min [0153] Wavelength: 325 nm [0154] Injection Volume: 10 L Gradient elution program as per the table below:

TABLE-US-00005 Retinol Elution Program Table Time (min) Phase A (%) Phase B (%) 0 95 5 25 95 5 Retention time of retinol t = 9.2 min approximately 6.6.2 Chromatographic Conditions for Retinyl-trans-4-tert-Butylcyclohexanol Mobile Phase: A Phase Acetonitrile; B Phase 0.1% Formic Acid-Water Column Temperature: 35 C. Flow Rate: 1.5 mL/min Wavelength: 355 nm Injection Volume: 10 L Isocratic Elution Program as per the following table:)

TABLE-US-00006 Retinyl-trans-4-tert-Butylcyclohexanol Elution Program Table Time (min) Phase A (%) Phase B (%) 0 100 0 25 100 0 Retention time of Retinyl-trans-4-tert-Butylcyclohexanol t = 17.0 min approximately

6.7 Testing

[0155] Operate according to the procedures for high-performance liquid chromatography (HPLC) usage and maintenance: [0156] I) Run the blank solvent, inject 2 times, record the chromatogram for up to 30 minutes; [0157] II) Run at least 3 injections of the control solution 1, record the chromatogram for up to 30 minutes; [0158] III) Run the control solution 2, inject 2 times, record the chromatogram for up to 30 minutes; [0159] IV) Inject 2 samples of the test solution, record the chromatogram for up to 30 minutes; [0160] V) Adjust the chromatogram scale, integrate, and print.

6.8 System Suitability Test

[0161] The RSD (relative standard deviation) of the main peak correction factor of the control solution should not exceed 3.0%;

[0162] The theoretical plate number in the chromatogram of the control solution, calculated based on retinol, should not be less than 3000, and the tailing factor should not exceed 2.0.

6.9 Content Calculation

[0163] [00002]$\mathrm{Cr}=\frac{\mathrm{Mr}p}{\mathrm{Vr}};F=\frac{\mathrm{Cr}}{\mathrm{Ar}};\mathrm{Content}\left(\%\right)=\frac{\mathrm{As}F\mathrm{Vs}}{\mathrm{Ms}\left(1-\mathrm{Ws}\right)}100\%$

[0164] The formula is as follows: [0165] Cr represents the concentration of the reference solution (mg/mL); [0166] Mr represents the weighed amount of the reference material (mg); [0167] P represents the assigned content of the reference material; in this case, as provided by Coachchem Laboratory, P=99.0%; [0168] Vr represents the dilution factor of the reference solution; [0169] F represents the correction factor for retinol; [0170] Ar represents the peak area of the reference solution; [0171] As represents the peak area of the sample solution; [0172] Vs represents the dilution factor of the sample solution; [0173] Ms represents the weighed amount of the sample material (mg); [0174] Ws represents the moisture content of the sample material. If there is no moisture, then Ws=0.00%.

6.10 The Stability Test Results

[0175] The stability test results indicate that, except for the condition at 4 C., the stability of Retinol trans-4-t-Butylcyclohexanol ester is significantly higher than that of retinol under other conditions of light exposure, room temperature, 45 C., and 50 C. After being placed for 28 days under conditions of 4 C. and room temperature, its content remains above 98%, showing minimal degradation and demonstrating good stability. The results are shown in FIGS. 7 to 11.

[0176] The above descriptions represent specific embodiments of the present invention. However, the scope of protection of the present invention is not limited thereto. Any modifications or substitutions readily conceived by those skilled in the art within the technical scope disclosed in the present invention shall be encompassed within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the scope of protection as set forth in the claims.