Patent ID: 12251458

DETAILED DESCRIPTION

Hereinafter, various exemplary embodiments of the present disclosure are described in detail, and this detailed description is not to be construed as limiting the present disclosure, but rather as describing certain aspects, features, and implementations of the present disclosure in more details.

It is to be understood that the term used herein is merely for describing particular embodiments rather than limiting the present disclosure. Additionally, for numerical range mentioned in the present disclosure, it is to be understood that each intermediate value in the upper and lower limits of the range is also specifically disclosed. Intermediate values within any stated values or stated ranges, as well as any smaller ranges in any stated values or any stated intermediate values within the range are also included in the present disclosure. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary skilled in the technical field to which the present disclosure belongs. Although only preferred methods and materials are described in the present disclosure, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present disclosure.

Without departing from the scope or spirit of the present disclosure, various improvements and variations may be made to the specific embodiments of the specification in the present disclosure, which are apparent to those skilled in the art, and other embodiments derived from the specification of the present disclosure will be also apparent to those skilled in the art. The specification and examples of the present disclosure are merely illustrative.

The terms “containing”, “including”, “having”, “comprising,” and the like used herein are in the open form, meaning including but not limited to.

The “%” mentioned in the present disclosure, unless otherwise specified, represents mass percentage.

In a first aspect of the present disclosure, an application of a 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and a salt thereof in preparing an anti-aging preparation for skin is provided.

In the present disclosure, a 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid salt may be sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate.

Since discovery, 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate (structural formula)

have not been disclosed or implied to have anti-aging and wrinkle-removing effects on skin. In the present disclosure, after repeated and in-depth research, it is the first time to discover that 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and a salt thereof (sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate) have the effect of promoting the synthesis of type I collagen, which, after being added to a drug, a cosmetic, a skincare product, or a cosmeceutical, if applied to skin, can increase the skin elasticity, reduce wrinkles, and realize the anti-aging effect.

In the present disclosure, a series of experiments prove that sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate has no damage to fibroblasts of mice and can promote the expression of COL1A2 in fibroblasts. Applying an aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate to the hair-removed skin on the backs of mice can downregulate the MMP-13 content in the treated skin, increase the COL1A2 content and the collagen fiber content in the treated skin, resulting in a thicker dermis of the treated skin on the backs of mice.

On the basis of the above studies, the present disclosure provides a new anti-aging preparation for skin containing a 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate. The anti-aging preparation for skin can be prepared by adding the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate to medically acceptable solvent carriers (such as purified water, glycerin, and mineral fat) for skin anti-aging.

Further, a person skilled in the art can understand that, in view of the anti-aging effect of 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate on skin, the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate can be easily added into skincare products, cosmeceuticals, or other cosmetics to prepare anti-aging skincare products, cosmeceuticals, or other cosmetics for skin containing 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate.

In addition, a person skilled in the art can understand that, except for being applied in the above-described skincare products, cosmetics and cosmeceuticals, the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate can be prepared into other pharmaceutically acceptable preparations for therapeutic or cosmetic purposes for skin anti-aging; or the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate can be prepared into anti-aging compositions for skin with other carriers suitable for topical application to the skin.

In a second aspect of the present disclosure, an anti-aging preparation for skin is provided, including a 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or a salt thereof as components.

In the present disclosure, excipients acceptable in the fields of pharmaceutics, or skincare product, cosmetic, and cosmeceutical are included. Specifically, they may be pharmaceutically acceptable carriers and solvents, or additives commonly used in skincare products, cosmetics, or cosmeceuticals, and the like.

In a preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or the salt thereof in the anti-aging preparation for skin is 0.5-1 μM. In a more preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid in the anti-aging preparation for skin is 0.5-1 μM, or, the content of a sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging preparation for skin is 0.5-1 μM. In a further preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyneene]-benzoic acid in the anti-aging preparation for skin is 0.5 μM, or, the content of the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging preparation for skin is 0.5 μM.

In a third aspect of the present disclosure, an anti-aging composition for skin is provided, including a 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or a salt thereof as components.

In a preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or the salt thereof in the anti-aging composition for skin is 0.5-1 μM. In a more preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid in the anti-aging composition for skin is 0.5-1 μM, or, the content of a sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging composition for skin is 0.5-1 μM. In a further preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyneene]-benzoic acid in the anti-aging composition for skin is 0.5 μM, or, the content of the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging composition for skin is 0.5 μM.

In a fourth aspect of the present disclosure, applications of the forgoing anti-aging preparation for skin and anti-aging composition for skin in preparing a drug for promoting the synthesis of type I collagen are provided.

In a fifth aspect of the present disclosure, applications of the forgoing anti-aging preparation for skin and anti-aging composition for skin in preparing an anti-aging drug for skin, an anti-aging skincare product for skin, an anti-aging cosmetic for skin and an anti-aging cosmeceutical for skin are provided.

In a sixth aspect of the present disclosure, an anti-aging drug for skin is provided, including a 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or a salt thereof as components.

In a preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or the salt thereof in the anti-aging drug for skin is 0.5-1 μM. In a more preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid in the anti-aging drug for skin is 0.5-1 μM, or, the content of a sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging drug for skin is 0.5-1 μM. In a further preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid in the anti-aging drug for skin is 0.5 μM, or, the content of the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging drug for skin is 0.5 μM.

In a seventh aspect of the present disclosure, an anti-aging skincare product for skin is provided, including a 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or a salt thereof as components.

In a preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or the salt thereof in the anti-aging skincare product for skin is 0.5-1 μM. In a more preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid in the anti-aging skincare product for skin is 0.5-1 μM, or, the content of a sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging skincare product for skin is 0.5-1 μM. In a further preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid in the anti-aging skincare product for skin is 0.5 μM, or, the content of the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging skincare product for skin is 0.5 μM.

In an eighth aspect of the present disclosure, an anti-aging cosmetic for skin is provided, including a 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or a salt thereof as components.

In a preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or the salt thereof in the anti-aging cosmetic for skin is 0.5-1 μM. In a more preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid in the anti-aging cosmetic for skin is 0.5-1 μM, or, the content of a sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging cosmetic for skin is 0.5-1 μM. In a further preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid in the anti-aging cosmetic for skin is 0.5 μM, or, the content of the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging cosmetic for skin is 0.5 μM.

In a ninth aspect of the present disclosure, an anti-aging cosmeceutical for skin is provided, including a 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or a salt thereof as components.

In a preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or the salt thereof in the anti-aging cosmeceutical for skin is 0.5-1 μM. In a more preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid in the anti-aging cosmeceutical for skin is 0.5-1 μM, or, the content of a sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging cosmeceutical for skin is 0.5-1 μM. In a further preferred embodiment of the present disclosure, the content of the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid in the anti-aging cosmeceutical for skin is 0.5 μM, or, the content of the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the anti-aging cosmeceutical for skin is 0.5 μM.

In the following examples, experiments for the anti-aging effect on skin are conducted using sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate as an example. The sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate has the following structural formula:

Example 1

The anti-aging effect of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate on skin

(1) Culture of Mouse Skin Fibroblasts

Experimental method: skin tissues were taken from the backs of newborn C57BL/6 mice, and fibroblasts obtained from mice were isolated and purified. The fibroblasts were cultured in a dulbecco's modified eagle medium (DMEM)/F12 medium (hereinafter referred to as “normal medium”) containing 10% (volume fraction) of superior fetal bovine serum (FBS) and 1% (volume fraction) of penicillin-streptomycin, followed by culturing in a 37° C. incubator with 5% (volume fraction) CO2. The third-generation of fibroblasts were taken for experiments.

(2) Determination of Appropriate Drug Concentration

Experimental method: the COL1A2 level was detected using fluorescent quantitative polymerase chain reaction (PCR) method. The experiment was divided into two major groups: a blank control group and a sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group. In the blank control group, the fibroblasts were cultured in a normal medium; and in the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group, the fibroblasts were treated with five media containing different concentrations (0.1 μM, 0.5 M, 1 μM, 3 μM, and 6 μM) of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate (i.e., normal medium added with sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate at the aforementioned concentrations) for 24 h. The specific process was as follows. The third-generation of fibroblasts was uniformly inoculated into a six-well plate. In the blank control group, the fibroblasts were cultured in the normal medium, and in the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group, the fibroblasts were cultured in media containing sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate at the corresponding concentrations. After 24 h of culture, total RNA was extracted from the fibroblasts and subjected to reverse transcription to obtain complementary deoxyribonucleic acid (cDNA), which was then subjected to fluorescent quantitative PCR. After the end of reaction, an amplification curve and a melting curve of the fluorescent quantitative PCR were confirmed, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene. The results were analyzed using the internationally accepted 2−ΔΔCtmethod.

Total RNA of fibroblasts was extracted using standard laboratory RNA extraction method and stored at −80° C. for later use. The extracted total RNA of fibroblasts was used as a template to synthesize cDNA by using the standard laboratory method, and the synthesized cDNA was stored at −20° C. for later use.

Fluorescent quantitative PCR system: 10 μL of qPCR SYBR Green Master Mix, 2 μL of 2 μM upstream primer, 2 μL of 2 μM downstream primer, and 1000 ng of cDNA template, a total volume of 20 μL by supplementing with diethyl pyrocarbonate (DEPC)-treated water. The upstream and downstream primers are listed in Table 1.

Reaction conditions: pre-denaturation at 95° C. for 5 min, followed by 40 cycles of 10 sec at 95° C., 20 sec at 52.9° C., and 20 sec at 72° C. After the end of reaction, the melting curve was analyzed.

TABLE 1Fluorescent quantitative PCR amplification primersGene namePrimer sequenceCOL1A2Upstream (5′-3′)GACAAATGAATGGGGCAAGDownstream (5′-3′)CAATGTCCAGAGGTGCAATGGAPDHUpstream (5′-3′)TGGCCTTCCGTGTTCCTACDownstream (5′-3′)GAGTTGCTGTTGAAGTCGCA

Results: as shown inFIG.1, after the fibroblasts are treated with sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate at a concentration of 0.5 μM for 24 h, the expression of COL1A2 in the fibroblasts is 1.613 times that of the blank control group (0 μM group), with a statistically significant difference compared to the blank control group (P=0.0003). After the fibroblasts are treated with 1 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate for 24 h, the expression of COL1A2 in the fibroblasts is 1.173 times that of the blank control group, showing a lower increase than the 0.5 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group, with a statistical difference compared to the blank control group (P<0.0001). The treatment of fibroblasts with any other concentrations of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate cannot increase the expression of COL1A2 in the fibroblasts, suggesting that the treatment with 0.5-1 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate can increase the expression of COL1A2 in fibroblasts, with 0.5 μM being better than 1 μM.

(3) Determination of Appropriate Administration Frequency

Experimental method: the COL1A2 level was detected using fluorescent quantitative PCR method. The experiment was divided into two major groups: a blank control group and a sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group. In the blank control group, the fibroblasts were cultured in a normal medium; and in the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group, the fibroblasts were cultured in a medium containing 0.5 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate, with incubation times of 24 h, 48 h, and 72 h. The specific process was as follows. The third-generation of fibroblasts was uniformly inoculated into a six-well plate. In the blank control group, the fibroblasts were cultured in a normal medium, and in the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group, the fibroblasts were cultured in a medium containing 0.5 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate (i.e., normal medium containing 0.5μ M of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate). After culturing for 24 h, 48 h, and 72 h, total RNA was extracted from the fibroblasts and subjected to reverse transcription to obtain cDNA, which was then subjected to fluorescent quantitative PCR. After the end of reaction, an amplification curve and a melting curve of the fluorescent quantitative PCR were confirmed, with GAPDH as an internal reference gene. The results were analyzed using the internationally accepted 2−ΔΔCtmethod.

The method for extracting total RNA of fibroblasts, the method for synthesizing cDNA, the fluorescent quantitative PCR system, the reaction conditions, and the upstream and downstream primer sequences were all the same as those described in “(2) of Example 1”.

Results: as shown inFIG.2, with the extension of incubation time from 24 h to 72 h, the expression of COL1A2 in fibroblasts decreases. At the incubation time of 72 h, there is no difference in the expression of COL1A2 in fibroblasts compared to the blank control group, suggesting that an administration frequency of once every 24-48 h is appropriate, with once every 24 h being optimal.

(4) Detection of Cell Viability after Administration

Experimental method: the third-generation of fibroblasts was uniformly inoculated into a six-well plate. In the blank control group, the fibroblasts were cultured in a normal medium, and in the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group, the fibroblasts were cultured in a medium containing 0.5 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate (i.e., normal medium containing 0.5 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate). After culturing for 24 h and 48 h, respectively, the status of fibroblasts was photographed under a microscope.

Results: as shown inFIG.3, “−” represents the blank control group, and “+” represents the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group. After the fibroblasts with 0.5 of are treated sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate for 24 h and 48 h, there is no obvious and visual changes in the status of fibroblasts, suggesting that 0.5 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate has no adverse effects on viability of fibroblasts.

Example 2

Anti-aging preparation for skin containing sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate

Sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate was dissolved in water to prepare an aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate (i.e., an anti-aging preparation for skin). Six healthy C57BL/6 mice aged 6-8 weeks with similar body sizes were selected, and the hair on the backs of the mice was removed gently and harmlessly to expose a similar area of skin on the back of each mouse. The six mice were divided into two groups equally. The same volume of pure water and aqueous solutions containing 0.5 of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate were applied, respectively, to the hair-removed skin on the backs of the mice once a day for 30 consecutive days.

(1) Detection of COL1A2 Content in Skin

Experimental method: after continuously applying the same volume of aqueous solutions containing 0.5 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate or pure water as a blank control to the backs of mice for 30 days, the mice were euthanized for cutting the skin from the treated areas. The Westernblot method was employed to detect the protein level of COL1A2, and the fluorescent quantitative PCR method was employed to detect the mRNA level of COL1A2.

The specific process for the Westernblot experiment was as follows. Total protein was extracted from skin tissues, and an equal mass of the collected protein samples were loaded into wells of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by electrophoresis, transmembrane, blocking, and incubation of primary antibodies (COL1A2 antibody and GAPDH antibody). After fluorescent secondary antibodies were incubated, the samples were visualized using an infrared fluorescence scanning imaging system. The visualized images were analyzed for grayscale of band using Image J software on a gel imaging system, and the relative expression of the target protein was calculated using internal reference protein GAPDH as correction.

Results: as shown inFIG.4AandFIG.4B,FIG.4Ashows gel images, andFIG.4Bshows a quantitative analysis bar graph. “−” represents the pure water blank control group, and “+” represents the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group. Compared to the pure water blank control group, the protein level of COL1A2 in the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group increases by 4.024 times, with a statistical difference compared to the pure water blank control group (P=0.0378), suggesting that applying the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate to skin can increase the protein content of COL1A2 in the skin.

Fluorescence quantitative PCR experiment was performed specifically as follows. Total RNA was extracted from skin tissues, and subjected to reverse transcription to obtain cDNA, which was then subjected to fluorescence quantitative PCR. After the end of reaction, an amplification curve and a melting curve of the fluorescent quantitative PCR were confirmed, with GAPDH as an internal reference gene. The results were analyzed using the internationally accepted 2−ΔΔCtmethod.

Total RNA of tissues was extracted using standard laboratory RNA extraction method and stored at −80° C. for later use. The extracted total RNA of tissues was used as a template to synthesize cDNA by using the standard laboratory method, and the synthesized cDNA was stored at −20° C. for later use.

The fluorescence quantitative PCR system, the upstream and downstream primers, and the reaction conditions were the same as those in “(2) of Example 1”.

Results: as shown inFIG.5, “−” represents the pure water blank control group, and “+” represents the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group. Compared to the pure water blank control group, the mRNA level of COL1A2 in the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group increases by 2.681 times, with a statistical difference compared to the pure water blank control group (P=0.0178), suggesting that applying the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate to skin can increase the mRNA content of COL1A2 in the skin.

(2) Detection of Collagen Fiber Content in Skin

Experimental method: after continuously applying an aqueous solution containing 0.5 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate or pure water as a blank control to the backs of mice for 30 days, the mice were euthanized for cutting the skin from the treated areas, and the Masson staining method was used for detecting the collagen fiber content in the skin. Specifically, tissues were fixed, dehydrated with a sucrose solution, embedded in an optimal cutting temperature (OCT), sliced, stained with hematoxylin, differentiated with acidic ethanol, counterstained blue with a Masson's blueing solution, stained with a Ponceau-Fuchsin staining solution, stained with an aniline blue staining solution, rinsed with a weak acid working solution, dehydrated with 95% ethanol (volume fraction), dehydrated with absolute ethanol, cleared with xylene, and mounted with neutral gum.

Results: as shown inFIG.6AandFIG.6B,FIG.6Ashows staining result images; andFIG.6Bis a quantitative analysis bar graph. “−” represents the pure water blank control group, and “+” represents the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group. Compared to the pure water blank control group, the collagen fiber content in the skin of the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group increases by 3.3904 times, with a statistical difference compared to the pure water blank control group (P=0.0010), suggesting that applying the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate to skin can increase the collagen fiber content in the skin.

(3) Detection of Dermal Thickness in Skin

Experimental method: after continuously applying an aqueous solution containing 0.5 μM of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate or pure water as a blank control to the backs of mice for 30 days, the mice were euthanized for cutting the skin from the treated areas, and hematoxylin-eosin (HE) staining method was used for detecting the dermal thickness in the skin. Specifically, the tissues were fixed, dehydrated with a sucrose solution, embedded in OCT, sliced, stained with hematoxylin, counterstained blue with tap water, stained with eosin, dehydrated and cleared, and then mounted.

Results: as shown inFIG.7AandFIG.7B,FIG.7Ashows staining result images, where the range marked by the line segments represents the range of dermal thickness; andFIG.7Bis a quantitative analysis bar graph. “−” represents the pure water blank control group, and “+” represents the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group. Compared to the pure water blank control group, the dermal thickness in the skin of the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group increases by 2.9101 times, with a statistical difference compared to the pure water blank control group (P=0.0004), suggesting that applying the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate to skin can increase the dermal thickness in the skin.

(4) Detection of MMP-13 Content in Skin

Experimental method: after continuously applying an aqueous solution containing 0.5 M of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate or pure water as a blank control to the backs of mice for 30 days, the mice were euthanized for cutting the skin from the treated areas, and the fluorescent quantitative PCR method was used for detecting the mRNA level of MMP-13 in skin tissue. Specifically, total RNA was extracted from skin tissues, and subjected to reverse transcription to obtain cDNA, which was then subjected to fluorescence quantitative PCR. After the end of reaction, an amplification curve and a melting curve of the fluorescent quantitative PCR were confirmed, with GAPDH as an internal reference gene. The results were analyzed using the internationally accepted 2−ΔΔCtmethod.

Total RNA of tissues was extracted using standard laboratory RNA extraction method and stored at −80° C. for later use. The extracted total RNA of tissue was used as a template to synthesize cDNA by using the standard laboratory method, and the synthesized cDNA was stored at −20° C. for later use.

Fluorescent quantitative PCR system: 10 μL of qPCR SYBR Green Master Mix, 2 μL of 2 μM upstream primer, 2 μL of 2 μM downstream primer, and 1000 ng of cDNA template, a total volume of 20 μL by supplementing with DEPC-treated water. The upstream and downstream primers are listed in Table 2.

Reaction conditions: pre-denaturation at 95° C. for 5 min, followed by 40 cycles of 10 sec at 95° C., 20 sec at 55.7° C., and 20 sec at 72° C. After the end of reaction, the melting curve was analyzed.

TABLE 2Fluorescent quantitative PCR amplification primersGene namePrimer sequenceMMP-13Upstream (5′-3′)CTTCTTCTTGTTGAGCTGGACTC(SEQ ID NO. 1)Downstream (5′-3′)CTGTGGAGGTCACTGTAGACT(SEQ ID NO. 2)GAPDHUpstream (5′-3′)TGGCCTTCCGTGTTCCTAC(SEQ ID NO. 3)Downstream (5′-3′)GAGTTGCTGTTGAAGTCGCA(SEQ ID NO. 4)

Results: as shown inFIG.8, “−” represents the pure water blank control group, and “+” represents the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group. Compared to the pure water blank control group, the mRNA level of MMP-13 in the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate treatment group decreases by 0.5124 times, with a statistical difference compared to the pure water blank control group (P=0.0171), suggesting that applying the aqueous solution of sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate to skin can decrease the MMP-13 content in the skin.

It can be seen that long-term application of preparations containing sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate can decrease the clinical symptoms of skin aging, increase the collagen fiber content and the dermal thickness in the skin, lighten wrinkles, and make the skin firmer and more delicate.

In the present disclosure, the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid underwent the same effect verification as sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in the aforementioned Examples 1 and 2. The results show that 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid has equivalent effects to the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate in increasing the expression of type I collagen (COL1A2) in cells, and the collagen fiber content and the dermal thickness in the skin.

It is to be noted that a person skilled in the art can understand that, in view of the anti-aging effect of 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate on skin, the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate can be easily added into skincare products, cosmeceuticals, or other cosmetics to prepare anti-aging skincare products, cosmeceuticals, or other cosmetics for skin containing 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate.

In this example, the sodium 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoate is prepared into an aqueous solution, which has a good anti-aging effect on skin. Those skilled in the art can understand that the dosage forms of the aforementioned solvents, carriers, or 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid (or the salt thereof, such as sodium salt and potassium salt, which will all dissociate

in solution, and this is the main component for the anti-aging effect on skin; and therefore, 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and the salt thereof can achieve the same anti-aging effect) are not intended to limit the present disclosure. Those skilled can mix the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or the salt thereof with moisturizing components such as glycerin, butylene glycol, and sodium hyaluronate to prepare an anti-aging moisturizer for skin. They can also mix the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or the salt thereof with emulsifiers such as stearyl alcohol and glyceryl stearate to prepare a lightweight, stable and sprayable oil-in-water anti-aging emulsion for skin. Furthermore, they can mix the 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or the salt thereof with emulsifiers such as PEG-20 methyl glucose sesquistearate and steareth-21 to prepare a thicker oil-in-water anti-aging cream for skin. In addition, without departing from the basic concept of the present disclosure, multiple compositions containing 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]alkyne]-benzoic acid and/or the salt thereof can be prepared, and as long as the composition is used in beauty or therapeutic treatments for skin anti-aging, it falls within the scope of protection of the present disclosure.

The above-mentioned examples are merely used for describing the preferred embodiment of the present disclosure, rather than limiting the scope of the present disclosure. Without departing from the design spirit of the present disclosure, various modifications and improvements made by those ordinary skilled in the art to the technical solutions of the present disclosure are included in the protection scope defined by the claims of the present disclosure.