Composition capable of regulating and controlling teenager micro acne, application and skin care product

Abstract

The invention belongs to the technical field of skin care, and discloses a composition capable of regulating and controlling teenager micro-acnes, application and a skincare product, the composition capable of regulating and controlling teenager micro-acnes comprises an olive leaf extract, a pinus densiflora leaf extract and adenosine, and the weight ratio of the olive leaf extract to the pinus densiflora leaf extract to the adenosine is (1-3): (0.02-0.1): (0.5-1.5). According to the composition capable of regulating and controlling the micro acnes of the teenagers, the olive leaf extract, the pinus densiflora leaf extract and adenosine are compounded, so that the oil control and antibacterial effects of the composition are remarkably improved.

Description

Technical Field

[0001] The present application relates to the technical field of skin care, and in particular to a composition, use and skin care product capable of regulating microacne in adolescents. Background Art

[0002] As adolescents enter puberty, influenced by adolescent hormones and academic pressures, skin problems are becoming increasingly prominent. More and more teenagers are experiencing skin problems such as oily skin, enlarged pores, and acne. When acne develops, the skin first enters the microcomedones stage, which then gradually develops into pimples. Therefore, controlling microcomedones is particularly important for managing skin problems in adolescents. In the early stages of acne, hyperkeratinization of the hair follicle infundibulum and the resulting blockage of the sebaceous ducts in the follicles form microcomedones. Microcomedones are the foundation of all acne lesions. Invisible to the naked eye, they can be detected in skin biopsies, and their size and number correlate with acne severity and recurrence rate. Therefore, controlling the formation of microcomedones is key to managing and reducing acne recurrences. Microcomedones can further develop into whiteheads, blackheads, papules, nodules, or cysts, and sometimes resolve on their own. Microcomedones can be categorized into prodromal, early, and late stages based on their pathological characteristics. The prodromal stage of micro-acne is characterized by abnormal proliferation of sebaceous gland cells and no obvious keratinization of the hair follicle epithelium; the early stage of micro-acne is characterized by enlargement of the hair follicle cavity, increased sebum secretion, hyperplasia and keratinization of the hair follicle epithelium, and increased reproduction of bacteria, especially Propionibacterium acnes; the late stage of micro-acne is characterized by atrophy of the sebaceous glands, further keratinization of the hair follicle epithelium, resulting in blockage of the hair follicle opening, causing the hair follicle to expand, and massive reproduction of bacteria.

[0003] The mechanism of microcomedones is primarily influenced by several factors, including sebum secretion, keratinization of the hair follicle epithelium, colonization of Propionibacterium acnes, and the release of inflammatory mediators. Hyperkeratinization and abnormal differentiation of the sebaceous duct epithelium in the pilosebaceous follicles result in the retention of overproliferating keratinocytes within the ducts, leading to pore clogging. Studies have shown that ductal hyperkeratinization can be caused by two primary factors: excessive proliferation of ductal keratinocytes and decreased keratinocyte segregation. Multiple factors influence keratinocyte hyperkeratinization, including abnormal sebum secretion and composition, androgens, inflammation, and Propionibacterium acnes. Inflammation and androgens stimulate keratinocyte differentiation, which is mediated by interleukin-1α (IL-1α) and growth factors, leading to hyperkeratinization and comedonal formation. Propionibacterium acnes secretes various substances to form a biofilm, which acts as a physical barrier to protect against the external environment and antimicrobial agents. According to relevant reports, glycocalyx polymers secreted during biofilm formation can be incorporated into sebum, acting as a biological adhesive between keratinocytes and promoting comedonal formation. Desmosomes, as specialized cell junctions, are primarily found in the epidermis, connecting two cell membranes via intervening keratin filaments (also known as tension fibers). Keratinocytes in acne-affected follicles have more desmosomes and tension fibers, resulting in a thicker and more sticky stratum corneum, which prevents keratinocytes from properly shedding from the skin surface. These multiple factors contribute to the formation of microcomedones, further contributing to the development of acne. Controlling the formation of microcomedones can control the progression of comedones and inflammatory acne lesions, making it a key to regulating and treating acne.

[0004] Microacne problems during adolescence are significantly age-specific. Epidemiological surveys show that approximately 85% of adolescents aged 12-24 experience varying degrees of microacne, with the incidence rate reaching as high as 73% in those aged 11-15, and the age of onset is declining year by year. Furthermore, adolescents commonly engage in inappropriate behavior in handling microacne. Surveys show that 67% of adolescents squeeze microacne, causing localized epidermal damage, deep spread of bacterial infection, and the formation of depressed scars. Excessive cleansing is also common, with approximately 38% of adolescents with oily skin cleansing more than three times a day. This damages the integrity of the sebum membrane and triggers compensatory sebum secretion, creating a vicious cycle of "the more you wash, the oilier your skin becomes."

[0005] Therefore, developing a composition that can regulate the microacne problem on adolescent skin is of great significance to the adolescent population. Summary of the Invention

[0006] One of the purposes of the present application is to provide a composition that can regulate microacne in adolescents. The composition can effectively inhibit the biofilm of Propionibacterium acnes by combining olive leaf extract, red pine leaf extract and adenosine, and can inhibit excessive sebum secretion and improve abnormal skin keratinization, thereby regulating the problem of microacne.

[0007] Another object of the present application is to provide a composition for regulating microacne in adolescents, wherein the composition is used to prepare skin care products to inhibit the biofilm of Propionibacterium acnes, improve abnormal skin keratinization, and control oil.

[0008] At the same time, the present application also provides a skin care product containing the composition of the present application that can regulate micro-acne in adolescents, which can regulate micro-acne in adolescents.

[0009] To achieve the above objectives, the present application provides a composition for regulating microacne in adolescents, comprising olive leaf extract, red pine leaf extract and adenosine, wherein the weight ratio of the olive leaf extract, red pine leaf extract and adenosine is 5-250:5-250:1-50.

[0010] Olive leaf extract, a natural active ingredient extracted from olive tree leaves, is rich in oleuropein and luteolin. It possesses multiple biological activities, including antioxidant, anti-inflammatory, antibacterial, and repairing properties, and offers potent anti-inflammatory and soothing benefits. Oleuropein inhibits inflammatory factors by blocking the NF-κB and MAPK signaling pathways, reducing the release of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-8. It also reduces histamine release by inhibiting mast cell degranulation, alleviating allergic reactions. Therefore, it can quickly relieve inflammatory reactions such as redness, stinging, and itching. Luteolin possesses anti-inflammatory properties, suppressing inflammation and alleviating symptoms such as redness, swelling, and itching. For sensitive skin, acne-prone skin, and other skin types prone to inflammation, luteolin can help alleviate inflammation and promote skin repair and recovery. Luteolin has inhibitory effects on a variety of bacteria and viruses, helping to protect the skin from external pathogens, maintaining a balanced skin microbiome, and reducing skin problems caused by infections, such as acne and blackheads. Olive leaf extract can also accelerate epidermal cell regeneration, repair damaged skin barrier, and enhance the skin's ability to lock in moisture; mainly by stimulating keratinocyte proliferation: activating the EGFR (epidermal growth factor receptor) pathway to promote wound healing; and by enhancing tight junction proteins: upregulating Claudin-1 and Occludin expression to improve barrier integrity.

[0011] Red pine leaf extract is rich in antioxidants such as polyphenols and flavonoids, which can reduce the damage of oxidative stress to cells and inhibit inflammatory responses. It is rich in terpenoids and polyphenols from pine needles and bark, and has a strong antibacterial effect, especially against the biofilm of Propionibacterium acnes, and can achieve a significant inhibitory effect.

[0012] Adenosine is a naturally occurring nucleoside composed of adenine (a nitrogenous base) and ribose (a five-carbon sugar). It is one of the most important molecules in living organisms. It plays a key role in energy metabolism, signal transduction, and genetic synthesis. In cosmetic applications, it has the following functions: (1) Anti-inflammatory and soothing effects: After the adenosine A2A receptor is activated, it can exert an anti-inflammatory effect by reducing the production of pro-inflammatory cytokines such as IL-6, IL-1β, TNF-α, etc.; in addition, it can also promote the expression of anti-inflammatory cytokine IL-10, regulate the skin's immune response, reduce inflammatory response, and make the skin healthier and more stable.

[0013] (2) Secondly, it also has the function of moisturizing and repairing: by reducing transepidermal water loss (TEWL), it enhances the skin's moisturizing ability, reduces water loss, and maintains the skin's water and oil balance; it promotes skin repair and stimulates the secretion of growth factors, thereby promoting tissue regeneration and wound healing, and accelerating the skin's self-repair process.

[0014] (3) Adenosine also has a strong inhibitory effect on the secretion of oil from the sebaceous glands, which has an oil-control effect.

[0015] The present application significantly improves the oil control and antibacterial effects of the composition by compounding olive leaf extract, red pine leaf extract and adenosine.

[0016] The present application also provides the use of the above-mentioned composition capable of regulating juvenile microacne in preparing skin care products.

[0017] The present application also discloses a skin care product containing 0.01-99.9 wt % of the above-mentioned composition capable of regulating juvenile microacne.

[0018] Beneficial effects Compared with the existing technology, the present application provides a composition that can regulate micro-acne in adolescents. The present application significantly improves the oil control, skin keratinization improvement and antibacterial effects of the composition by compounding olive leaf extract, red pine leaf extract and adenosine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments; Figure 1 The morphological and tissue images of replicate 1 in the BC group in the 3D epidermal skin model and the content of K16 and inflammatory factor IL-1α were obtained. Figure 2 The morphological and tissue images of the BC group were repeated in the 3D epidermal skin model, as well as the K16 and inflammatory factor IL-1α content tests. Figure 3The morphological and tissue images of the BC group were repeated three times in the detection of the tissue morphology of the 3D epidermal skin model and the content of K16 and inflammatory factor IL-1α; Figure 4 The morphological and tissue images of replicate 1 of the NC group in the detection of K16 and inflammatory factor IL-1α content based on the 3D epidermal skin model are shown; Figure 5 The morphological and tissue images of the NC group were repeated in the 3D epidermal skin model, as well as the K16 and inflammatory factor IL-1α content tests. Figure 6 The morphological images of the NC group were repeated 3 times in the detection of tissue morphology of the 3D epidermal skin model and the content of K16 and inflammatory factor IL-1α. Figure 7 The following are images of the tissue morphology of the PC1 group in the 3D epidermal skin model and the content of K16 and inflammatory factor IL-1α. Figure 8 The following are the morphological and tissue images of the PC1 group in the repeated 2 tests based on the tissue morphology of the 3D epidermal skin model and the content of K16 and inflammatory factor IL-1α; Figure 9 The images show the tissue morphology of the PC1 group in the 3D epidermal skin model and the content of K16 and inflammatory factor IL-1α. Figure 10 The morphological and tissue images of Repeat 1 in Example 2 in the detection of the content of K16 and inflammatory factor IL-1α based on the tissue morphology of the 3D epidermal skin model are shown; Figure 11 The morphological and tissue images of Repeat 2 of Example 2 in the detection of the content of K16 and inflammatory factor IL-1α based on the tissue morphology of the 3D epidermal skin model are shown; Figure 12 The morphological and tissue images of the repetition 3 of Example 2 in the detection of the content of K16 and inflammatory factor IL-1α based on the tissue morphology of the 3D epidermal skin model are shown; Figure 13 Fluorescence microscopy images of replicate 1 of the BC group in the 3D epidermal skin model, showing the tissue morphology and the levels of K16 and inflammatory factor IL-1α. Figure 14 Fluorescence microscopy images of replicate 2 of the BC group in the 3D epidermal skin model showing the tissue morphology and the levels of K16 and inflammatory factor IL-1α. Figure 15Fluorescence microscopy images of the BC group in the 3D epidermal skin model showing the tissue morphology and the levels of K16 and inflammatory factor IL-1α. Figure 16 Fluorescence microscopy images of replicate 1 of the NC group in the detection of tissue morphology and K16 and inflammatory factor IL-1α content based on the 3D epidermal skin model; Figure 17 The following are the fluorescence microscopy images of the NC group in the 3D epidermal skin model, showing the tissue morphology and the content of K16 and inflammatory factor IL-1α. Figure 18 The images are repeated 3 fluorescence microscopy images of the NC group in the detection of tissue morphology based on the 3D epidermal skin model and the content of K16 and inflammatory factor IL-1α; Figure 19 Fluorescence microscopy images of replicate 1 of the PC1 group in the 3D epidermal skin model showing the tissue morphology and the levels of K16 and inflammatory factor IL-1α. Figure 20 Fluorescence microscopy images of repeated 2 samples of the PC1 group in the 3D epidermal skin model showing the tissue morphology and the content of K16 and inflammatory factor IL-1α. Figure 21 Fluorescence microscopy images of the PC1 group in the 3D epidermal skin model show the tissue morphology and the levels of K16 and inflammatory factor IL-1α. Figure 22 Fluorescence microscope images of repeat 1 of Example 2 in the detection of the tissue morphology of the 3D epidermal skin model and the content of K16 and inflammatory factor IL-1α; Figure 23 Fluorescence microscope images of repeat 2 of Example 2 in the detection of the tissue morphology of the 3D epidermal skin model and the content of K16 and inflammatory factor IL-1α; Figure 24 Fluorescence microscope images of the 3D epidermal skin model showing the histomorphology and the levels of K16 and inflammatory factor IL-1α in Example 2, repeated 3 times; Figure 25 This is a picture of the biofilm structure morphology of the control group in the biofilm test based on Propionibacterium acnes (300 times magnification); Figure 26 This is a picture of the biofilm structure morphology of the control group in the biofilm test based on Propionibacterium acnes (1000 times magnification); Figure 27 for Figure 26 The red frame shows the biofilm structure and morphology of the control group (5000 times magnification); Figure 28This is a picture of the biofilm structure morphology of Example 2 in the biofilm test based on Propionibacterium acnes (300 times magnification); Figure 29 This is a picture of the biofilm structure morphology of Example 2 in the biofilm test based on Propionibacterium acnes (1000 times magnification); Figure 30 for Figure 29 The red framed portion shows the biofilm structure morphology of Example 2 (5000x magnification). DETAILED DESCRIPTION

[0020] The present application is further described below in conjunction with embodiments, but does not constitute any limitation to the present application. Any limited modifications made within the scope of the claims of the present application are still within the scope of the claims of the present application.

[0021] In order to explain the technical content of this application in detail, further explanation is given below in conjunction with the implementation methods.

[0022] In the following examples and comparative examples, the olive leaf extract was purchased from Olive Clear Extract PRO provided by Shanghai Boshuo Industrial Co., Ltd., manufactured by Draco Natural Products, Inc.; the red pine leaf extract was purchased from Xianting (Guangzhou) Trading Co., Ltd., with the trade name: QUORASTOP; and the adenosine was purchased from Hangzhou Weitai Biopharmaceutical Co., Ltd.

[0023] It should be noted that the technical solution of the present application can be obtained through other commercially available products and is not limited to the above raw material sources.

[0024] The following examples and comparative examples are based on the actual usage of olive leaf extract, red pine leaf extract and adenosine. Unless otherwise specified, parts are parts by weight and % are percentages by weight.

[0025] Examples and Comparative Examples The preparation method of the present application is: heating adenosine to 42±2° C., adding olive leaf extract and red pine leaf extract with stirring, and stirring and mixing evenly to obtain a composition.

[0026] The composition was prepared according to the above preparation method and the formula in Table 1 below.

[0027] Table 1 Composition formula Olive Leaf Extract (part) Red pine leaf extract (part) Adenosine (parts) Example 1 1 0.1 1.5 Example 2 2 0.05 1 Example 3 3 0.02 0.5 Comparative Example 1 2.9 0.15 0 Comparative Example 2 0 0.15 1.9 Comparative Example 3 2.03 0 1.02

[0028] Efficacy testing 1. Propionibacterium acnes inhibition rate Test method: 1. Test equipment and reagents Instruments and equipment: anaerobic incubator, vertical high-pressure steam sterilizer; Reagents: phosphate buffered saline, reinforced clostridial medium (RCM), Propionibacterium acnes.

[0029] 2 Experimental methods 2.1 Sample processing Test sample: dilute with water to a composition concentration of 2%; Blank control: phosphate buffer; Control sample group: Propionibacterium acnes suspension + phosphate buffer.

[0030] 2.2 Experimental steps Take a suspension of Propionibacterium acnes and add it to each test sample and control sample, mix thoroughly, and start timing. When the designated exposure time has elapsed, make an appropriate dilution. Take two to three dilutions and place the test sample and control sample onto two separate plates. Pour in Reinforced Clostridium difficile Medium (RCM) and mix thoroughly. After solidification, flip the plates over and incubate in an anaerobic incubator for 48 hours. Count the viable bacteria.

[0031] 3 Calculation formula Inhibition rate = (AB) / A×100%; where: A-average colony count of control sample; B-average colony count of test sample.

[0032] This application commissioned Guangzhou Huamiao Testing Service Co., Ltd. to test the 10-minute and 30-minute antibacterial rates of the compositions of Examples 1-3 and Comparative Examples 1-3 according to the above method, and issued a test report on the 10-minute antibacterial rate. The results are shown in Table 2. Among them, the test report number of Example 1 is 2505WH033-07A; the test report number of Example 2 is 2505WH033-04A; the test report number of Example 3 is 2505WH033-05A; the test report number of Comparative Example 1 is 2505WH033-02A; the test report number of Comparative Example 2 is 2505WH033-01A; and the test report number of Comparative Example 3 is 2505WH033-03A.

[0033] Table 2 Antibacterial effect of the compositions of Examples 1-3 and Comparative Examples 1-3 on Propionibacterium acnes 10-minute antibacterial rate (%) 30-minute antibacterial rate (%) Example 1 40.91 32.86 Example 2 47.73 33.57 Example 3 39.77 31.43 Comparative Example 1 18.18 14.29 Comparative Example 2 34.09 21.43 Comparative Example 3 14.77 21.43

[0034] According to the results in Table 2, we can see that: According to the data of Examples 1-3, the composition of the present application has a high antibacterial rate, especially the antibacterial effect is more obvious within 10 minutes, while the antibacterial rate shows a downward trend during the period of 10-30 minutes.

[0035] According to the data of Comparative Examples 1-3, it can be seen that when the technical solution of the present application lacks any one of olive leaf extract, red pine leaf extract, and adenosine, under the condition that the total amount of active ingredients is the same, the combination of the other two shows a significant decrease in antibacterial rate compared to the combination of olive leaf extract, red pine leaf extract and adenosine used in the present application. It is speculated that the technical solution of the present application has the effect of synergistically enhancing the antibacterial effect of Propionibacterium acnes.

[0036] Among them, the antibacterial effect of Comparative Example 2 in the first 10 minutes was significantly better than that of Comparative Example 1 and Comparative Example 3. However, after 30 minutes, the antibacterial rate of Propionibacterium acnes in Comparative Example 3 showed an upward trend, and the antibacterial rate was even consistent with that of Comparative Example 2. It is speculated that the possible reasons are: (1) The red pine leaf extract and adenosine have a certain synergistic effect, which can enhance the antibacterial efficacy of the composition against Propionibacterium acnes in a short period of time; (2) When olive leaf extract is combined with adenosine, it can produce a long-lasting antibacterial effect and can continuously inhibit bacteria.

[0037] When red pine leaf extract, olive leaf extract and adenosine are present at the same time, the present application can produce a further synergistic effect, and the inhibition rate of Propionibacterium acnes produced at 10 minutes and 30 minutes is significantly improved.

[0038] 2. 5α-reductase inhibition rate Test method: 1. Experimental Purpose and Principle Oily skin has more active sebaceous glands, which makes the face feel greasy, difficult to clean, and has an oily sheen that affects appearance. At the same time, it is also prone to some corresponding skin diseases, such as acne vulgaris and seborrheic dermatitis. Given the relationship between 5α-reductase and sebaceous glands, the inhibitory effect of test samples on 5α-reductase is usually tested to reflect the sebum regulation effect and to characterize the oil control efficacy of the test samples.

[0039] 2 Test indicators 5α-reductase inhibition rate determination standard: If the 5α-reductase inhibition rate of the sample is higher than that of the negative control and there is a significant difference, it can be considered that the test sample has a certain oil control effect.

[0040] 3 Test materials Testosterone (≥98%), NADPH (90%), methanol (HPLC grade), finasteride (≥98%), buffer: PBS (pH=5.5).

[0041] 3.1 Test method (1) Treatment of control and test samples Sample group: Diluted with buffer to a concentration of 1% for the composition samples of Examples 2-3 and Comparative Examples 1-3; Positive control (finasteride, purity ≥98%): Dissolve in methanol first, then dilute with buffer to a finasteride test concentration of 400 nM; Negative control: PBS buffer.

[0042] (2) Test operation steps Set up a sample group, a positive control group, a negative control group and a blank group. Each group needs to set up two parallels. Add different reagent solutions to the three groups respectively, shake well, and perform HPLC analysis on each group to determine the testosterone content in each tube.

[0043] (3) Calculation formula .

[0044] This application commissioned Guangzhou Huamiao Testing Service Co., Ltd. to test the compositions of Examples 2-3 and Comparative Examples 1-3 according to the above method and issue a test report. The results are shown in Table 3. Among them, the test report number of Example 2 is: 2505WH133-05AR1; the test report number of Example 3 is: 2505WH133-03AR1; the test report number of Comparative Example 1 is: 2506WH030-02AR1; the test report number of Comparative Example 2 is: 2506WH030-01AR1; the test report number of Comparative Example 3 is: 2506WH030-03AR1.

[0045] Table 3 5α-reductase inhibition results of the compositions of Examples 2-3 and Comparative Examples 1-3 5α-reductase inhibition rate (%) Example 2 64.29 Example 3 50.86 Comparative Example 1 29.12 Comparative Example 2 26.20 Comparative Example 3 29.28

[0046] According to the results in Table 3: According to the data of Examples 2-3, the present invention has a good oil control effect by compounding olive leaf extract, red pine leaf extract and adenosine, and the 5α-reductase inhibition rate is as high as more than 50%.

[0047] According to the data comparison of Example 2 and Comparative Examples 1-3, it can be seen that when the technical solution of the present application lacks any one of olive leaf extract, red pine leaf extract, and adenosine, under the condition that the total amount of active ingredients is the same, the combination of the other two shows a significant decrease in 5α-reductase inhibition rate compared to the combination of olive leaf extract, red pine leaf extract and adenosine used in the present application; it is speculated that the technical solution of the present application has the effect of synergistically enhancing the 5α-reductase inhibition effect.

[0048] In order to further verify whether the composition of the present application has the effect of regulating acne, the present application further conducted the following efficacy verification on Example 2: 3. Detection of tissue morphology and K16 and inflammatory factor IL-1α content based on the 3D epidermal skin model (EpiKutis®) 1Test basis Testing was conducted based on the "Method for Detecting the Tissue Morphology and Content of K16 and Inflammatory Factor IL-1α Based on the 3D Epidermal Skin Model (EpiKutis®)," the "Biochemical Experimental Method for 5α-Reductase Inhibition Rate," and the "Method for Detecting the Biofilm Structure Morphology Based on Propionibacterium acnes."

[0049] 2. Test Purpose This test consists of three parts. The first part, based on an oleic acid-stimulated 3D epidermal skin model (EpiKutis®), examines tissue morphology and changes in K16 and IL-1α levels. The second part measures the sample's 5α-reductase inhibition rate. The third part, based on Propionibacterium acnes, uses scanning electron microscopy to observe the biofilm structure and morphology. This comprehensive evaluation evaluates the sample's ability to regulate microcomedone formation.

[0050] 3 Test materials 3.1 Test System The model used in this test is a 3D epidermal skin model (EpiKutis), batch number ES250501, provided by Guangdong Boxi Shaanxi Branch; The strain used in this test was Propionibacterium acnes (ATCC6919).

[0051] 3.2 Sample information: Example 2 sample.

[0052] 3.3 Main reagents EpiGrowth culture medium (Guangdong Boxi Shaanxi Branch), PBS (Solaibao), oleic acid (Sinopharm), vitamin E (VE, Sigma), dexamethasone (China Food and Drug Administration), hematoxylin (Biyuntian), eosin (Biyuntian), xylene (Sinopharm), paraformaldehyde (Biosharp), K16 antibody (Abcam), IL-1α ELISA kit (Abcam), 5α-reductase (Guangdong Pharmaceutical University), dutasteride (McLean), testosterone (Beijing Wokai), and reduced coenzyme II tetrasodium salt (NADPH) (McLean).

[0053] 3.4 Main equipment CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-2F), fluorescence microscope (Olympus, BX43), upright microscope (Olympus, BX53), microplate reader (BioTek, Epoch), biological safety cabinet (Sujing Antai, BSC-1304IIA2), biochemical incubator (Shanghai Hengyi, SHP-250), high-pressure steam sterilizer (Shanghai Shen'an, 50GS180371), water-insulated constant temperature incubator (Shanghai Yiheng, Model 9050), high-speed desktop refrigerated centrifuge (Hunan Xiangyi, H1850R), scanning electron microscope (Hitachi).

[0054] 4.1 Test Method 1) Test groups are divided as described in Table 4; Table 4 Test groups

[0055] Note: PC1 (VE) is used for tissue morphology and K16 detection; PC2 (dexamethasone) is used for IL-1α detection.

[0056] 2) Pre-test Preparation: Transfer the model to a 6-well plate and add 3.7 mL of model culture medium to each well. Randomly divide the model into a blank control (BC), a negative control (NC), a positive control (PC1), a positive control (PC2), and a sample group, with three replicates per group.

[0057] 3) Model Stimulation and Dosing: The NC, PC1, PC2, and sample groups were stimulated with oleic acid at a dose of 1 mg / mL and incubated in a CO2 incubator (37°C, 5% CO2). Topical administration of the sample was performed using gentle circular movements. Oleic acid stimulation lasted for three days, with dosing occurring once daily for a total of three times. After the final stimulation and dosing, the model was incubated for an additional 24 hours. After incubation, any residual test substance on the surface of the model was washed with a sterile PBS solution in a wash bottle, and any residual liquid inside and outside the model was gently wiped away with a sterile cotton swab.

[0058] 4) Histomorphological examination: The models used for histomorphological examination were fixed with 4% paraformaldehyde for 24 hours and then stained with H&E. The samples were photographed under a microscope and analyzed.

[0059] 5) Immunofluorescence test: The model to be tested was fixed with 4% paraformaldehyde for 24 hours, and then immunofluorescence testing was performed. The images were observed under a microscope, collected, and analyzed.

[0060] 6) ELISA test: After incubation, collect the culture medium into a centrifuge tube. After collection, store the sample for ELISA test in a -80℃ freezer and perform the test and analysis according to the operating instructions of the ELISA kit.

[0061] 7) Calculation of down-regulation rate and inhibition rate: ; .

[0062] This application entrusts Shaanxi Boxi General Testing Technology Co., Ltd. to conduct the test and issue a test report with report number G3414-2506103. The test results are as follows: The tissue morphology results are shown in Figure 1-12 ,in: Figure 1-3 These are the morphological and tissue images of replicate 1, replicate 2, and replicate 3 of the BC group; Figure 4-6 These are the morphological and tissue images of replicate 1, replicate 2, and replicate 3 of the NC group; Figure 7-9 These are the morphological and tissue images of replicate 1, replicate 2, and replicate 3 of the PC1 group; Figure 10-12 These are the morphological and organizational pictures of Repetition 1, Repetition 2 and Repetition 3 of Example 2, respectively.

[0063] Note: The above images were taken with an upright microscope (Olympus, BX53) at a magnification of 400×, and the blue represents the cell nucleus.

[0064] according to Figure 1-12 The results of the stratum corneum thickness obtained by analysis are shown in Table 5; Table 5 Summary of stratum corneum thickness data Group Average thickness of stratum corneum (μm) SD Downgrade rate (vs NC) BC 42.16 7.29 / NC 67.71 6.06 / PC1 46.28 7.15 31.65% Example 2 43.39 3.02 35.92%

[0065] Compared with the BC group, the stratum corneum thickness of the NC group was significantly increased, indicating that the stimulation conditions of this test were effective.

[0066] Compared with the NC group, the stratum corneum thickness of the PC1 group was significantly reduced, indicating that the positive control in this test was effective.

[0067] Compared with the NC group, the stratum corneum thickness of Example 2 was significantly reduced, with a downregulation rate of 35.92%.

[0068] K16 immunofluorescence results are shown in Figure 13-24 ,in: Figure 13-15 Fluorescence microscopy images of replicate 1, replicate 2, and replicate 3 of the BC group, respectively; Figure 16-18Fluorescence microscopy images of replicate 1, replicate 2, and replicate 3 of the NC group, respectively; Figure 19-21 Fluorescence microscopy images of replicate 1, replicate 2, and replicate 3 of the PC1 group, respectively; Figure 22-24 These are fluorescence microscope images of repeat 1, repeat 2 and repeat 3 of Example 2, respectively.

[0069] Note: The above images were taken with a fluorescence microscope (Olympus, BX43) at a magnification of 200×. Blue fluorescence represents the cell nucleus, and green fluorescence represents K16. The stronger the green fluorescence intensity, the higher the K16 content.

[0070] according to Figure 13-24 The results of K16 immunofluorescence analysis are shown in Table 6; Table 6 Summary of K16 immunofluorescence analysis results Group Relative IOD average SD Inhibition rate (vs NC) BC 1.00 0.09 / NC 3.60 0.18 / PC1 1.55 0.09 56.94% Example 2 1.53 0.07 57.50%

[0071] Note: The relative integrated optical density (IOD) average value reflects the content of K16.

[0072] Compared with the BC group, the K16 content in the NC group was significantly increased, indicating that the stimulation conditions of this test were effective.

[0073] Compared with the NC group, the K16 content in the PC1 group was significantly reduced, indicating that the positive control in this test was effective.

[0074] Compared with the NC group, the K16 content in Example 2 was significantly reduced, with an inhibition rate of 57.50%.

[0075] The results of IL-1α content are shown in Table 7; Table 7 Summary of IL-1α content results Group Average concentration (pg / mL) SD Inhibition rate (vs NC) BC 0.88 0.06 / NC 4.20 0.25 / PC2 3.03 0.27 27.86% Example 2 3.10 0.18 26.19%

[0076] Compared with the BC group, the IL-1α content in the NC group was significantly increased, indicating that the stimulation conditions of this test were effective.

[0077] Compared with the NC group, the IL-1α content in the PC2 group was significantly decreased, indicating that the positive control in this test was effective.

[0078] Compared with the NC group, the IL-1α content in Example 2 was significantly reduced, with an inhibition rate of 26.19%.

[0079] 4. Biofilm test based on Propionibacterium acnes 1 Test Method 1) Test groups are divided according to Table 8: Table 8 Test Groups

[0080] 2) Preparation of experimental bacterial suspension: Take the activated bacteria and add 2 mL of BHI medium to make a bacterial suspension. The initial bacterial concentration is adjusted to 1.0 (OD600) for later use.

[0081] 3) Experimental steps: Bacteria were inoculated onto a 6-well plate slide and incubated anaerobically at 37°C for 24 hours. Sample solution (2 mL) was added to the sample group, while fresh culture medium (2 mL) was replaced in the control group and incubated anaerobically at 37°C for 24 hours. After incubation, the samples were washed three times with 2 mL of PBS. Fixation was performed with 2.5% glutaraldehyde solution for 4 hours, followed by three washes with PBS, each for 20 minutes. After fixation, the samples were dehydrated using a gradient of 30%, 50%, 70%, 90%, and 95% ethanol. The samples were dried using a critical point dryer. The samples were gold-sprayed, and bacterial morphology was observed using a scanning electron microscope.

[0082] Test results are shown in Figure 25-30 ,in Figures 25-26 This is the picture of the biofilm structure morphology of the control group. Figure 25 This is a 300-fold magnification image. Figure 26 The image is 1000 times magnified; Figures 28-30 This is a picture of the biofilm structure morphology in Example 2. Figure 28 This is a 300-fold magnification image. Figure 29 This is a 1000-fold magnification image; it should be noted that Figure 27 for Figure 26 5000 times magnified image of the red frame. Figure 30 for Figure 29 The red frame is a 5000 times magnified image. Figure 25-30 Analysis showed that, compared with the control group, the biofilm morphology under electron microscopy changed from "large and dense pieces" to "small and scattered pieces", indicating that Example 2 interfered with the structural stability of the biofilm.

[0083] Based on the 3D epidermal skin model (EpiKutis), compared with the control group, the stratum corneum thickness of Example 2 was significantly reduced, with a downregulation rate of 35.92%, and the K16 content and inflammatory factor IL-1α content were significantly reduced, with inhibition rates of 57.50% and 26.19%, respectively.

[0084] Based on Propionibacterium acnes, compared with the control group, the change in biofilm morphology under electron microscopy from "large and dense pieces" to "small and scattered pieces" indicated that Example 2 interfered with the structural stability of the biofilm.

[0085] In summary, Example 2 can reduce the thickness of the stratum corneum, the content of K16 and the inflammatory factor IL-1α, and has a significant inhibitory effect on the activity of 5α-reductase, can interfere with the structural stability of the biofilm, and has the effect of regulating the formation of microcomedones.

[0086] It can be seen that the technical solution of the present application can regulate the formation of micro-acne and effectively prevent micro-acne.

[0087] The embodiments presented herein are merely embodiments selected from a combination of all possible embodiments. The appended claims should not be limited by the embodiments described herein. Some numerical ranges used in the claims include subranges therein, and variations in these ranges should also be covered by the appended claims.

Claims

1. A composition for regulating microacne in adolescents, characterized in that: The invention comprises olive leaf extract, red pine leaf extract and adenosine, wherein the weight ratio of the olive leaf extract, red pine leaf extract and adenosine is 2:0.05:

1.

2. Use of the composition for regulating juvenile microacne as claimed in claim 1 in preparing skin care products.

3. A skin care product, characterized in that: Contains 0.01-99.9 wt % of the composition for regulating juvenile microacne as claimed in claim 1.

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