An acne composition and its use on dissolvable microneedles in solid flash release

Abstract

The application discloses a kind of acne-removing compositions and its application on solid-state flash-release soluble microneedle, belong to daily-use cosmetics technical field.The acne-removing composition comprises 10-13 parts of dihydrofarnesic acid, 1-4 parts of spironolactone extract, 0.3-1 parts of yeast / zinc fermentation product and 2.0-2.6 parts of hamamelis virginiana extract by mass fraction, wherein the hamamelis virginiana extract is prepared by compound fermentation of cellulomonas huizhi and bacillus subtilis, secondary fermentation of streptomyces thermophilus and ethanol reflux extraction, and the utilization rate of active ingredients is high;The composition provided by the application can significantly inhibit lipid synthesis through the synergistic effect of multiple components, and effectively reduce the level of inflammatory factors.The composition is loaded in solid-state flash-release soluble microneedle, which can quickly dissolve and release active ingredients when used, achieve the effect of quickly removing acne, anti-inflammatory and detumescence, and has good safety without irritating reaction.

Description

Technical Field

[0001] This invention belongs to the technical field of daily cosmetics, specifically relating to an acne-removing composition and its application on solid-state flash-release soluble microneedles. Background Technology

[0002] Acne is a common skin problem in clinical practice. Its causes are closely related to excessive sebum secretion from the sebaceous glands, abnormal keratinization of the pilosebaceous duct, proliferation of Propionibacterium acnes, and imbalance of inflammatory response. It not only affects skin health but also easily causes adverse effects on appearance. Therefore, it is of great significance to develop an effective and safe oil-controlling and acne-removing skin care product.

[0003] Currently, most acne treatment products on the market are based on traditional formulations such as gels, lotions, and serums. These products suffer from problems such as difficulty in penetrating the stratum corneum and low transdermal absorption efficiency, resulting in insufficient efficacy. Some products rely on high concentrations of acids and antibiotics, which may provide short-term effects, but long-term use can easily lead to side effects such as skin irritation, barrier damage, and flora imbalance, raising concerns about safety. Furthermore, existing acne treatment compositions often suffer from unreasonable ingredient combinations and weak synergistic effects, making it difficult to simultaneously achieve comprehensive effects such as oil control, anti-inflammation, and antibacterial properties. In addition, the preparation processes of some natural extracts are simple and crude, resulting in low utilization rates of active ingredients, further limiting the acne-reducing effects of these products.

[0004] For solid microneedle skincare products, some soluble microneedles in the existing technology have problems such as poor compatibility of the framework material, low loading of active ingredients, and uneven release rate. In addition, there is a lack of precise formula design specifically for acne treatment scenarios, which cannot effectively adapt to the physiological needs of acne-prone skin.

[0005] Therefore, developing an acne-removing composition with strong synergistic effects, high utilization rate of active ingredients, good transdermal absorption, and the advantages of oil control, anti-inflammation, safety and non-irritation, as well as an efficient delivery carrier adapted to the composition, has become an important research direction in the current field of acne-removing skin care. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to: First, provide an acne-removing composition with strong synergistic effects. Through the scientific combination of dihydroroic acid, purslane extract, yeast / zinc fermentation products, and witch hazel extract, it simultaneously achieves comprehensive acne-removing effects of oil control, anti-inflammation, and antibacterial properties, solving the problem of poor effects from single ingredients or unreasonable combinations. Second, the present invention also optimizes the preparation process of witch hazel extract by employing secondary fermentation of specific compound bacteria combined with ethanol reflux extraction technology to improve the utilization rate and bioactivity of natural active ingredients. Combined with a soluble microneedle carrier, it addresses the pain point of low transdermal absorption efficiency of active ingredients in traditional skin care formulations, achieving rapid release and precise delivery of active ingredients, thereby enhancing the acne-removing effect.

[0007] To achieve the above objectives, the present invention discloses the following technical solutions:

[0008] In a first aspect, the present invention provides an acne-removing composition, which, by weight, contains the following components: 10-13 parts of dihydroroic acid, 1-4 parts of purslane extract, 0.3-1 parts of yeast / zinc fermentation product, and 2.0-2.6 parts of witch hazel extract.

[0009] Preferably, the composition contains the following components by mass: 12-13 parts dihydroroic acid, 3-4 parts purslane extract, 0.7-1 parts yeast / zinc fermentation product, and 2.4-2.6 parts witch hazel extract.

[0010] More preferably, the preparation method of the witch hazel extract includes the following steps:

[0011] Step 1: Crush and sieve the dried witch hazel roots to obtain witch hazel powder. Moisten the witch hazel powder with sterile water to 55-60%, and then sterilize to obtain fermentation substrate.

[0012] Step 2: Take moist Cellomonas bacteria solution and Bacillus subtilis bacteria solution respectively and mix them at a volume ratio of (2-3):1 to obtain compound fermentation solution;

[0013] Step 3: Inoculate the compound fermentation liquid into the fermentation substrate at an inoculation rate of 10-12wt%, and ferment at 30-35℃ for 48-60h. After fermentation, fermentation material 1 is obtained.

[0014] Step 4: Inoculate the thermophilic Streptomyces bacterial solution into fermentation material 1 at an inoculation rate of 6-8%, and ferment at 25-30℃ for 72-96 hours. After fermentation, fermentation material 2 is obtained.

[0015] Step 5: Mix fermentation material 2 with water, wash and filter, collect the filter residue and filtrate, sterilize the filtrate filter membrane for later use, reflux the filter residue with 80-85 v / v% ethanol aqueous solution to obtain the extract, mix with the above filtrate to obtain the crude extract, concentrate the crude extract under reduced pressure and vacuum dry to the water content ≤8% to obtain the witch hazel extract.

[0016] More preferably, the viable count of the moist Cellulosum bacterial suspension is 10. 8-9 CFU / mL.

[0017] More preferably, the viable count of the Bacillus subtilis bacterial solution is 10. 8-9 CFU / mL.

[0018] More preferably, during the fermentation process in step 3, the air permeability is 0.6-0.7 vvm, and the fermentation substrate is turned over every 30 minutes.

[0019] More preferably, the viable count of the *Streptomyces thermophilus* bacterial solution is 10. 8-9 CFU / mL;

[0020] During the fermentation process in step 4, the air ventilation rate is 1.1-1.3 vvm, and the fermentation substrate is turned over every 15 minutes.

[0021] More preferably, in step 5, the filter residue is mixed with an 80-85% v / v% ethanol aqueous solution at a mass-volume ratio of 1:20-25 g / mL and then refluxed for extraction three times, each time for 30-40 min.

[0022] Secondly, the present invention provides the application of the acne-removing composition described in the first aspect in the preparation of skin care products with oil-controlling and acne-removing effects.

[0023] Thirdly, the present invention provides a solid-state flash-release soluble microneedle containing the acne-removing composition described in the first aspect.

[0024] Fourthly, the present invention provides a method for preparing the soluble microneedles described in the third aspect, the method comprising the following steps:

[0025] Step 1: Mix hyaluronic acid, carboxymethyl cellulose, mannitol and deionized water in a mass ratio of 1:0.1:0.03:98.87, stir evenly and allow to fully swell to obtain a microneedle skeleton solution;

[0026] Step 2: Mix the acne treatment composition and the microneedle skeleton solution at a mass ratio of 1:8 to obtain the microneedle solution;

[0027] Step 3: Inject the microneedle solution into the microneedle mold, centrifuge the microneedle mold containing the microneedle solution at 3000 rpm for 10 min, allow it to stand and dry into an elastic gel after centrifugation, and finally freeze-dry it, slowly cooling it to -25℃ and drying it for 1 h to obtain the solid flash-release soluble microneedles.

[0028] In this invention:

[0029] Dihydroroyal acid, as one of the core active ingredients of the composition, has both strong antibacterial and anti-inflammatory activities. It can specifically inhibit the proliferation of acne-causing bacteria such as Propionibacterium acnes, reduce the inflammatory response caused by bacterial imbalance, and promote fibroblast proliferation, accelerate the healing of skin after acne damage, and achieve a rapid repair effect. Dihydroroyal acid (10-hydroxydecanoic acid) is one of the important hydroxy fatty acids in royal jelly and is also a natural precursor of royal acid (10-hydroxy-2-decenoic acid). Compared with royal acid, dihydroroyal acid has better anti-inflammatory and antioxidant properties and is more chemically stable.

[0030] Purslane extract is rich in flavonoids, polysaccharides and other active substances, which have significant anti-inflammatory and soothing effects. It can target and inhibit the release of inflammatory factors, reduce erythema and swelling symptoms in acne areas, repair damaged skin barriers, enhance the skin's own resistance, reduce the impact of external stimuli on acne-prone skin, and synergistically enhance the anti-inflammatory effect with dihydroroyal acid.

[0031] Yeast / zinc fermentation products combine zinc with yeast metabolites through bio-fermentation technology. Zinc ions can regulate the secretion function of sebaceous glands, reduce excessive oil secretion, and participate in skin keratin metabolism, improving abnormal keratinization of hair follicle ducts. Yeast metabolites can replenish skin nutrients, enhance cell activity, and synergistically enhance oil control and acne removal effects with other ingredients.

[0032] The witch hazel extract, prepared through a secondary fermentation process using specific compound bacteria and ethanol reflux extraction, is rich in active ingredients such as tannins and flavonoids. It has strong astringent, oil-controlling, and anti-inflammatory effects. On the one hand, it can shrink pores and reduce oil residue on the skin surface; on the other hand, it can inhibit the release of inflammatory factors and relieve acne inflammation. The bioavailability of its active ingredients is significantly improved after fermentation, and its oil-controlling and anti-inflammatory effects are superior compared to products obtained through traditional extraction processes.

[0033] This invention constructs a multi-target synergistic system for oil control, antibacterial, anti-inflammatory and repair by precisely proportioning dihydroroic acid, purslane extract, yeast / zinc fermentation product and witch hazel extract, thus avoiding the problem of limited efficacy of single ingredients.

[0034] The beneficial effects of this invention are:

[0035] 1. This invention constructs a multi-target synergistic system for oil control, antibacterial, anti-inflammatory and repair by using a specific ratio of dihydroroic acid, purslane extract, yeast / zinc fermentation product and witch hazel extract. It can inhibit excessive sebum secretion and block inflammatory response, solve the problem of acne formation from the root, achieve comprehensive acne removal effect, and overcome the defects of the limited efficacy of single ingredients.

[0036] 2. The preparation of witch hazel extract in this invention adopts an optimized process of segmented compound solid-state fermentation. Through stepwise fermentation, the plant cell wall is destroyed and new active substances are generated, which greatly improves the oil control and anti-inflammatory activity of the extract. Compared with the products of traditional extraction processes, the efficacy is significantly improved, and the utilization rate of active ingredients is higher and the stability is stronger.

[0037] 3. This invention loads an acne-removing composition onto solid flash-release soluble microneedles. The microneedles can penetrate the stratum corneum of the skin, and rapidly dissolve and release the active ingredients upon contact with the skin. This solves the problem of low transdermal absorption efficiency of traditional skin care products, achieving precise and efficient delivery of active ingredients, significantly improving the acne-removing effect. Moreover, the microneedle carrier is gentle and non-invasive, suitable for a wide range of skin types, and provides acne patients with an efficient and safe skin care solution. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] To further illustrate the present invention, detailed descriptions are provided below through the following embodiments. The raw materials used in the following embodiments and comparative examples of the present invention are all commercially available products; the raw material companies listed below represent one of the ways to purchase the raw materials.

[0040] I. Raw Materials

[0041] Dihydroroic acid: also known as 10-hydroxydecanoic acid, purchased from Shanghai Keqin Technology Co., Ltd.

[0042] Purslane extract: purchased from Green Valley Plant Source (Guangzhou) Biotechnology Co., Ltd.;

[0043] Yeast / zinc fermentation products: purchased from Guangzhou Youran Biotechnology Co., Ltd.;

[0044] Witch hazel: The dried root of the plant *Hamamelis mollis* Oliv., belonging to the family Hamamelidaceae; commercially available.

[0045] Moistened Fibromonas: accession number GDMCC 1.187, purchased from Guangdong Provincial Microbial Culture Collection Center;

[0046] Bacillus subtilis: accession number GDMCC 1.372, purchased from Guangdong Provincial Center for Microbial Culture Collection;

[0047] Streptomyces thermophilus: accession number GDMCC 4.48, purchased from Guangdong Provincial Microbial Culture Collection Center.

[0048] The specific steps for preparing witch hazel extract are as follows:

[0049] Step 1: Take dried witch hazel roots and crush them in a high-speed pulverizer. Pass them through a 60-mesh sieve to obtain witch hazel powder. Use sterile water to moisten the witch hazel powder. Stop moistening when the powder moisture content reaches 55-60%. Sterilize at 121℃ for 20 minutes to obtain fermentation substrate material for later use.

[0050] Step 2: Mix *Cyclomonella hygroscopica* bacterial suspension and *Bacillus subtilis* bacterial suspension at a volume ratio of 3:1 to obtain a compound fermentation broth. The viable count of the *Cyclomonella hygroscopica* bacterial suspension is 10. 9 The viable count of the Bacillus subtilis culture was 10 CFU / mL. 9 CFU / mL;

[0051] Step 3: Transfer the fermentation substrate to the fermentation tank, and spray the compound fermentation liquid evenly onto the fermentation substrate at 11% of the mass of the fermentation substrate. Ferment at 33℃ for 60 hours. The air ventilation rate during the fermentation process is 0.6-0.7 vvm. Turn the fermentation substrate over once every 30 minutes. After the fermentation is completed, fermentation material 1 is obtained.

[0052] Step 4: Take a sample with a live bacteria count of 10. 9 The thermophilic Streptomyces broth at CFU / mL was inoculated into fermentation substrate 1 at 7% of the mass of fermentation substrate 1. Fermentation was carried out at 27℃ for 72h. The air flow rate during fermentation was 1.1-1.3 vvm. The fermentation substrate was turned over every 15min. After fermentation, fermentation substrate 2 was obtained.

[0053] Step 5: Mix fermentation material 2 with sterile water at a mass ratio of 1:1, stir thoroughly at 50 r / min, then separate the residue and filtrate through a 200-mesh filter cloth. Sterilize the filtrate by passing it through a 0.22 μm filter membrane for later use. Mix the residue with an 80 v / v% ethanol aqueous solution at a mass-volume ratio of 1:25 g / mL and reflux extract three times, 35 min each time. Combine the extracts obtained each time into a total extract, mix it with the aforementioned filtrate to obtain a crude extract, concentrate the crude extract under reduced pressure, and vacuum dry it until the water content is ≤8% to obtain the witch hazel extract.

[0054] II. Acne Treatment Combinations

[0055] 1. Composition 1-4

[0056] The raw materials were mixed according to the mass ratio in Table 1 to obtain acne-removing compositions 1-4.

[0057] Table 1. Mass ratio of raw materials for the composition

[0058] Raw material name Composition 1 Composition 2 Composition 3 Composition 4 Dihydroroyalic acid 10 11 12 13 Purslane extract 1 2 3 4 Yeast / Zinc Fermentation Products 0.3 0.5 0.7 1 witch hazel extract 2 2.2 2.4 2.6

[0059] 2. Comparative compositions 1-4

[0060] Based on the formulation of composition 3, the raw materials were omitted, and the raw materials were compounded and mixed according to the mass ratio of the raw materials in Table 2 to obtain comparative compositions 1-4.

[0061] Table 2 Comparative Compositions 1-4 Raw Material Mass Ratio

[0062] Raw material name Comparative composition 1 Comparative composition 2 Comparative composition 3 Comparative composition 4 Dihydroroyalic acid 15 12.7 14.4 4.525 Purslane extract / 3 3 4.525 Yeast / Zinc Fermentation Products 0.7 / 0.7 4.525 witch hazel extract 2.4 2.4 / 4.525

[0063] Note: " / " in the table indicates no addition.

[0064] 3. Comparative composition 5-9

[0065] Comparative Composition 5: Based on the formulation of Composition 3, witch hazel extract was replaced with witch hazel extract ① to obtain Comparative Composition 5. The preparation method of witch hazel extract ① is the same as that of witch hazel extract. The difference is that the compound fermentation liquid is replaced with a separate moist cellomonas liquid for fermentation in "step 3". The rest remains unchanged.

[0066] Comparative Composition 6: Based on the formulation of Composition 3, witch hazel extract was replaced with witch hazel extract ② to obtain Comparative Composition 6. The preparation method of witch hazel extract ② is the same as that of witch hazel extract. The difference is that the compound fermentation liquid is replaced with a separate Bacillus subtilis liquid for fermentation in "Step 3". Everything else remains the same.

[0067] Comparative Composition 7: Based on the formulation of Composition 3, witch hazel extract was replaced with witch hazel extract ③ to obtain Comparative Composition 7. The preparation method of witch hazel extract ③ is the same as that of witch hazel extract. The difference is that the volume ratio of moist cellomonas bacteria liquid to Bacillus subtilis bacteria liquid in the compound fermentation liquid is adjusted to 1:3. Then, the fermentation of "step 3" is carried out. The rest remains unchanged.

[0068] Comparative Composition 8: Based on the formulation of Composition 3, witch hazel extract was replaced with witch hazel extract ④ to obtain Comparative Composition 8. The preparation method of witch hazel extract ④ is the same as that of witch hazel extract, except that the fermentation of thermophilic Streptomyces in "Step 4" is skipped, and the extraction in "Step 5" is carried out directly after the fermentation in "Step 3". The rest remains unchanged.

[0069] Comparative Composition 9: Based on the formulation of Composition 3, witch hazel extract was replaced with witch hazel extract ⑤ to obtain Comparative Composition 9. The preparation method of witch hazel extract ⑤ is as follows: dried witch hazel was pulverized and passed through a 60-mesh sieve to obtain witch hazel powder. The witch hazel powder was mixed with an 80 v / v% ethanol aqueous solution at a mass-volume ratio of 1:25 g / mL and then refluxed for extraction 3 times, 35 min each time. The extracts obtained each time were combined into a total extract. The extract was concentrated under reduced pressure and vacuum dried until the water content was ≤8% to obtain the witch hazel extract ⑤.

[0070] The specific proportions of the above comparative compositions 5-9 are detailed in Table 3;

[0071] Table 3 Comparative Compositions 5-9 Raw Material Mass Ratio

[0072] Raw material name Comparative composition 5 Comparative composition 6 Comparative composition 7 Comparative composition 8 Comparative composition 9 Dihydroroyalic acid 12 12 12 12 12 Purslane extract 3 3 3 3 3 Yeast / Zinc Fermentation Products 0.7 0.7 0.7 0.7 0.7 Witch hazel extract① 2.4 / / / / Witch hazel extract ② / 2.4 / / / Witch hazel extract ③ / / 2.4 / / Witch hazel extract ④ / / / 2.4 / Witch hazel extract ⑤ / / / / 2.4

[0073] Note: " / " in the table indicates no addition.

[0074] III. Performance Testing

[0075] 1. Test on the effect of lipid synthesis

[0076] 1.1 Experimental Materials

[0077] Cell line: Human sebaceous gland cells SZ95.

[0078] Experimental samples: compositions 1-4, control compositions 1-9.

[0079] Reagents: Linoleic acid, isotretinoin (positive control), CCK-8 reagent, Nile red, fluorescein diacetate, DMEM medium, fetal bovine serum, penicillin-streptomycin solution, DMSO.

[0080] Sample dilution preparation: Dissolve each experimental sample (compositions 1-4, control compositions 1-9) in DMSO to prepare a high-concentration stock solution, and then dilute it in DMEM medium in a gradient to the required concentration for the experiment. It is necessary to ensure that the DMSO content in each system is <0.1%.

[0081] 1.2 Experimental Apparatus

[0082] Carbon dioxide incubator, ELISA reader, fluorescence microscope, centrifuge, biosafety cabinet, 96-well plate, pipette, electronic balance.

[0083] 1.3 Experimental Methods

[0084] 1.3.1 Preliminary Cytotoxicity Assay (CCK-8 assay)

[0085] Cell preparation: 2 × 10 4SZ95 cells were seeded into 96-well plates at a density of cells / well and cultured for 24 hours. The old culture medium was then discarded.

[0086] Experimental sample gradient settings: The stock solutions of each experimental sample were diluted with culture medium to six gradients, where the mass fractions of each composition (compositions 1-4 and control compositions 1-9) in their respective experimental sample systems were 0.001%, 0.005%, 0.01%, 0.025%, 0.05%, and 0.1%, respectively.

[0087] Grouping: blank group (culture medium only), solvent group (culture medium + 0.1% DMSO), and sample group (each experimental sample corresponding to 6 concentration gradients), with 3 replicates per group;

[0088] Grouped drug administration: Add 200 μL of the corresponding system to each well, continue culturing for 24 h, add 10 μL of CCK-8 solution to each well, incubate at 37℃ and 5% CO2 for 3 h, and measure the OD value at 450 nm using a microplate reader. Calculate cell viability: Cell viability = (OD of test group - OD of blank group) / (OD of solvent group - OD of blank group) × 100%;

[0089] Final concentration determination: Cell viability of all experimental samples was >90% within the concentration range of ≤0.05%, which met the safety requirements. Therefore, 0.05% was determined as the working concentration for subsequent oil control tests of each experimental sample.

[0090] 1.3.2. Lipid Synthesis Inhibition Experiment

[0091] Grouping: blank group (culture medium), model group (100 μmol / L linoleic acid), negative group (100 μmol / L linoleic acid + 0.1% DMSO), positive group (100 μmol / L linoleic acid + 0.01 mmol / L isotretinoin), and sample group (100 μmol / L linoleic acid + 0.05% of each experimental sample), with 3 replicates per group;

[0092] Grouped administration: After cell seeding and culture to 60% confluence, cells were added to the corresponding system according to the group and cultured for 24 h. The supernatant was discarded, and the cells were washed twice with PBS. Nile red (10 μg / mL) + fluorescein diacetate (15 μg / mL) were added, and the cells were stained at 37℃ in the dark for 10 min. After washing with PBS, the cells were observed and photographed under a fluorescence microscope. The fluorescence intensity was measured by an ELISA reader under Ex485nm / Em565nm (neutral lipids) and Ex494nm / Em523nm (polar lipids), respectively. The lipid synthesis inhibition rate was calculated as follows: Inhibition rate = (1 - mean lipid content of sample group / mean lipid content of model group) × 100%.

[0093] 1.4 Test Results

[0094] Table 4 Results of lipid synthesis inhibition rate

[0095] Sample group concentration Neutral oil inhibition rate (%) Polar oil inhibition rate (%) Positive group (isotretinoin) 0.01 mmol / L 13.4 18.3 Composition 1 0.05% 17.2 23.1 Composition 2 0.05% 19.0 24.7 Composition 3 0.05% 22.0 28.5 Composition 4 0.05% 20.3 26.0 Comparative composition 1 0.05% 9.1 12.3 Comparative composition 2 0.05% 10.0 13.7 Comparative composition 3 0.05% 8.5 11.6 Comparative composition 4 0.05% 10.8 14.5 Comparative composition 5 0.05% 13.5 17.1 Comparative composition 6 0.05% 12.9 17.0 Comparative composition 7 0.05% 15.2 18.8 Comparative composition 8 0.05% 11.7 16.2 Comparative composition 9 0.05% 9.0 12.0

[0096] 1.5 Results Analysis

[0097] As shown in Table 4, compositions 1-4 provided by this invention have a significant inhibitory effect on the synthesis of oils and fats, and the inhibitory effect is better than that of the positive control. Among them, composition 3 has the best inhibitory effect, with an inhibition rate of 22.0% for neutral oils and 28.5% for polar oils, indicating that the components form a highly efficient and synergistic oil control system under specific ratios.

[0098] Compared with compositions 1-4, which lacked a single component or had their component ratios adjusted, the oil control effect of compositions 1-4 was significantly reduced, far lower than that of composition 3. This proves that dihydroroic acid, purslane extract, yeast / zinc fermentation product and witch hazel extract are indispensable, and their synergistic effect is the key to achieving efficient oil control.

[0099] Compared with the witch hazel extracts prepared by different processes using compositions 5-9, their oil-controlling effects were all weaker than those of composition 3. This verifies that the compound bacterial fermentation combined with the thermophilic streptomyces secondary fermentation process of the present invention can significantly improve the oil-controlling activity of the extract, thereby enhancing the overall efficacy of the composition.

[0100] 2. Inflammatory factor influence test

[0101] 2.1 Experimental Materials

[0102] Cell line: mouse macrophage RAW264.7.

[0103] Experimental samples: compositions 1-4, control compositions 1-9.

[0104] Reagents: Bacterial lipopolysaccharide (LPS), dexamethasone sodium phosphate, CCK-8 reagent, ELISA kit for IL-6 and NO, DMEM medium, fetal bovine serum, penicillin-streptomycin solution, DMSO.

[0105] Sample dilution preparation: Dissolve each experimental sample (compositions 1-4, control compositions 1-9) in DMSO to prepare a high-concentration stock solution, and then dilute it in DMEM medium in a gradient to the required concentration for the experiment. It is necessary to ensure that the DMSO content in each system is <0.1%.

[0106] 2.2 Experimental Apparatus

[0107] CO2 incubator, ELISA reader, biosafety cabinet, 96-well plate, pipette, centrifuge, electronic balance.

[0108] 2.3 Experimental Methods

[0109] 2.3.1 Preliminary Cytotoxicity Assay (CCK-8 assay)

[0110] Cell preparation: Seed RAW264.7 cells into 96-well plates (1×10⁻⁶ cells / well). 4 (number per well), after culturing for 24 hours, discard the old culture medium;

[0111] Concentration gradient settings: Six gradients were set: 0.005%, 0.01%, 0.025%, 0.05%, 0.1%, and 0.2%. The six concentrations were the mass fractions of each composition (compositions 1-4 and control compositions 1-9) in the experimental sample system.

[0112] Grouping: blank group (culture medium only), solvent group (culture medium + 0.1% DMSO), and sample group (each experimental sample corresponding to 6 concentration gradients), with 3 replicates per group;

[0113] Grouped drug administration: Add 200 μL of the corresponding system to each well, continue culturing for 24 h, add 10 μL of CCK-8 solution to each well, incubate at 37℃ and 5% CO2 for 3 h, measure the OD value at 450 nm with a microplate reader, and calculate cell viability: Cell viability = (OD of test group - OD of blank group) / (OD of solvent group - OD of blank group) × 100%;

[0114] Final concentration determination: Cell viability of all experimental samples was >90% within the concentration range of ≤0.1%, which met the safety requirements. Therefore, 0.1% was determined as the working concentration for subsequent inflammatory factor inhibition experiments.

[0115] 2.3.2 Inflammatory factor inhibition experiment

[0116] Grouping: blank group (culture medium), model group (1 μg / mL LPS), negative group (1 μg / mL LPS + 0.1% DMSO), positive group (1 μg / mL LPS + 100 μg / mL dexamethasone sodium phosphate), and sample group (1 μg / mL LPS + 0.1% of each experimental sample), with 3 replicates per group;

[0117] Grouped administration: After the cells were seeded and cultured to a confluence of 60%, they were added to the corresponding system according to the group and cultured for 24 hours. The supernatant of each well was collected, and a standard curve was plotted according to the instructions of the ELISA detection kit for IL-6 and NO. The concentration of inflammatory factors in the supernatant was measured, and the inhibition rate of inflammatory factors was calculated: Inhibition rate = (1 - mean concentration of inflammatory factors in the sample group / mean concentration of inflammatory factors in the model group) × 100%.

[0118] 2.4 Test Results

[0119] Table 5 Results of Inflammatory Factor Inhibition Rate

[0120] Sample group IL-6 inhibition rate (%) NO inhibition rate (%) Positive group (dexamethasone) 78.4 27.2 Composition 1 36.2 18.7 Composition 2 38.5 19.9 Composition 3 43.7 24.2 Composition 4 39.8 21.3 Comparative composition 1 13.6 8.2 Comparative composition 2 15.3 9.5 Comparative composition 3 12.4 7.6 Comparative composition 4 16.8 10.1 Comparative composition 5 28.5 14.8 Comparative composition 6 22.6 11.8 Comparative composition 7 29.7 15.5 Comparative composition 8 20.3 10.7 Comparative composition 9 11.9 7.3

[0121] 2.5 Results Analysis

[0122] Table 5 shows that compositions 1-4 have significant inhibitory effects on both inflammatory factors IL-6 and NO. Among them, composition 3 has an IL-6 inhibition rate of 43.7% and a NO inhibition rate of 24.2%. Although these are lower than the positive control dexamethasone, they are superior among natural ingredient compositions and have better safety.

[0123] Compared with compositions 1-4, which lack any component, the inhibition rate of inflammatory factors was significantly reduced, indicating that the synergistic effect of the four components can effectively block the inflammatory response pathway. Compared with compositions 5-9, the inhibition rate of IL-6 was only 11.9%-29.7%, and the inhibition rate of NO was 7.3%-15.5%, further proving that the specific fermentation process of the witch hazel extract of the present invention can enhance its anti-inflammatory activity and achieve a highly efficient anti-inflammatory effect in synergy with other components.

[0124] IV. Solid-state flash-release microneedles

[0125] A solid-state flash-release microneedle containing an acne-fighting composition is prepared as follows:

[0126] Step 1: Mix hyaluronic acid, carboxymethyl cellulose, mannitol and deionized water in a mass ratio of 1:0.1:0.03:98.87, stir evenly and allow to fully swell to obtain a microneedle skeleton solution;

[0127] Step 2: Take the acne treatment composition 3 prepared above and mix it with the microneedle matrix solution at a mass ratio of 1:8 to obtain microneedle solution;

[0128] Step 3: Inject the microneedle solution into the microneedle mold, centrifuge the microneedle mold containing the microneedle solution at 3000 rpm for 10 min, and let it stand and dry into an elastic gel after centrifugation. Finally, freeze dry it, slowly cool it to -25℃, and dry it for 1 h to obtain solid flash release microneedles with a length of 400 μm.

[0129] V. Human Efficacy Testing

[0130] 1. Human safety evaluation

[0131] The tests were conducted using human skin trials. The specific test methods are in accordance with the "Cosmetic Safety Technical Specifications" (2015 edition), as detailed in the table below.

[0132] Table 6 Skin Reaction Score

[0133] Classification Skin reaction 0 No response 1 faint erythema 2 Erythema, infiltration, and visible papules 3 Erythema, infiltration, edema, papules, vesicles 4 Erythema, edema, bullae

[0134] A total of 60 subjects, aged 25-45 years, were included in the study. Solid-state flash-release microneedles / negative control (sterile water) were applied to the outer forearm for 0.5 hours once a day, and followed up for 4 weeks.

[0135] The test results (see Table 7) show a negative reaction (grade 0) and no erythema reaction was caused on the skin.

[0136] Table 7 Evaluation Results

[0137]

[0138] 2. Acne-removing efficacy test

[0139] 2.1 Inclusion Criteria

[0140] Healthy volunteers, regardless of gender, aged 25-45, with facial acne, assessed by a dermatologist as having acne grade 1-2 (referring to "T / CNMIA 0012-2020 Clinical Evaluation Standards for Acne-Removing Efficacy Skincare Products"), who voluntarily sign an informed consent form, can cooperate to complete the entire testing cycle, maintain a regular lifestyle, and agree not to use any cosmetics, drugs, or health products that may affect the results during the testing period.

[0141] 2.2 Exclusion Criteria

[0142] ① Individuals with severe skin diseases (such as eczema, psoriasis, etc.) or severe facial inflammation that may interfere with the interpretation of test results; ② Individuals with highly allergic constitutions or allergies to the test sample; ③ Women who are pregnant, breastfeeding, or planning to become pregnant during the testing period; ④ Individuals with severe heart, liver, or kidney dysfunction and weakened immune function; ⑤ Individuals who have participated in other skin-related tests within 30 days or have taken orally / topically used products that affect acne within 2 weeks; ⑥ Individuals deemed unsuitable for this test by the researchers.

[0143] 2.3 Test Method

[0144] (1) Grouping

[0145] Test group: solid flash-release microneedles containing composition 3; control group: blank solid flash-release microneedles (containing only microneedle framework components, without acne-removing composition); a randomized double-blind method was used to divide eligible volunteers into test group and control group, with 30 volunteers in each group, and sample group information was concealed.

[0146] (2) Sample usage method

[0147] After cleansing and drying your face, apply the microneedle patch to the acne area, press and hold for 10 minutes, then remove it. Avoid rubbing the application area vigorously. Use once a day for 14 consecutive days.

[0148] 2.4 Data Acquisition and Processing

[0149] D0: Data collected before use, without cleansing the face; D7: Data collected under the same conditions after 7 consecutive days of use; D14: Data collected under the same conditions after 14 consecutive days of use.

[0150] At each time point, three experienced dermatologists observed the volunteers on-site or counted the number of pimples, papules, and pustules on their faces based on photos. The average of the three doctors' counts / tests was taken as the skin lesion data for the volunteer at the corresponding time point.

[0151] Skin lesion regression rate calculation: The regression rate of each skin lesion type is calculated according to the following formula: Skin lesion regression rate = (1 - number of skin lesions after test / number of skin lesions before test) × 100%.

[0152] 2.5 Test Results

[0153] Table 8. Number and regression rate of acne lesions in the test group and control group

[0154]

[0155] 2.6 Results Analysis

[0156] As the usage time increased, the number of pimples, papules, and pustules in the test group decreased significantly. On day 14, the regression rates of pimples, papules, and pustules reached 35.47%, 44.76%, and 59.52%, respectively, which were far superior to those in the control group. This indicates that the acne-removing composition provided by the present invention exhibits excellent acne-removing effects.

[0157] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An acne-removing composition, characterized in that, The composition comprises, by weight, 12-13 parts of dihydroroic acid, 3-4 parts of purslane extract, 0.7-1 parts of yeast / zinc fermentation product, and 2.4-2.6 parts of witch hazel extract; The preparation method of the witch hazel extract includes the following steps: Step 1: Crush and sieve the dried witch hazel roots to obtain witch hazel powder. Moisten the witch hazel powder with sterile water to a moisture content of 55-60%, and then sterilize to obtain fermentation substrate. Step 2: Take moist Cellomonas bacteria solution and Bacillus subtilis bacteria solution respectively and mix them at a volume ratio of (2-3):1 to obtain compound fermentation solution; Step 3: Inoculate the compound fermentation liquid into the fermentation substrate at an inoculation rate of 10-12wt%, and ferment at 30-35℃ for 48-60h. After fermentation, fermentation material 1 is obtained. Step 4: Inoculate the thermophilic Streptomyces bacterial solution into fermentation material 1 at an inoculation rate of 6-8%, and ferment at 25-30℃ for 72-96 hours. After fermentation, fermentation material 2 is obtained. Step 5: Mix fermentation material 2 with water, wash and filter, collect the filter residue and filtrate, sterilize the filter membrane of the filtrate for later use, reflux the filter residue with 80-85 v / v% ethanol aqueous solution to obtain the extract, mix with the above filtrate to obtain the crude extract, concentrate the crude extract under reduced pressure and vacuum dry to the water content ≤8% to obtain the witch hazel extract. The viable count of the *Cellulosum humidum* bacterial suspension was 10. 8-9 CFU / mL; The viable count of the Bacillus subtilis culture was 10. 8-9 CFU / mL; The viable count of the *Streptomyces thermophilus* bacterial solution was 10. 8-9 CFU / mL.

2. The acne-removing composition according to claim 1, characterized in that, During the fermentation process in step 3, the air ventilation rate is 0.6-0.7 vvm, and the fermentation substrate is turned over every 30 minutes.

3. The acne-removing composition according to claim 1, characterized in that, During the fermentation process in step 4, the air ventilation rate is 1.1-1.3 vvm, and the fermentation substrate is turned over every 15 minutes.

4. The acne-removing composition according to claim 1, characterized in that, In step 5, the filter residue is mixed with an 80-85% v / v% ethanol aqueous solution at a mass-volume ratio of 1:20-25 g / mL and then refluxed for extraction three times, each time for 30-40 min.

5. The use of the acne-removing composition according to any one of claims 1-4 in the preparation of skin care products with oil-controlling and acne-removing effects.

6. A solid-state flash-release soluble microneedle, characterized in that, The soluble microneedles contain the acne-removing composition according to any one of claims 1-4.

7. A method for preparing the soluble microneedles according to claim 6, characterized in that, The preparation method includes the following steps: Step 1: Mix hyaluronic acid, carboxymethyl cellulose, mannitol and deionized water in a mass ratio of 1:0.1:0.03:98.87, stir evenly and allow to fully swell to obtain a microneedle skeleton solution; Step 2: Mix the acne treatment composition and the microneedle skeleton solution at a mass ratio of 1:8 to obtain the microneedle solution; Step 3: Inject the microneedle solution into the microneedle mold, centrifuge the microneedle mold containing the microneedle solution at 3000 rpm for 10 min, allow it to stand and dry into an elastic gel after centrifugation, and finally freeze-dry it, slowly cooling it to -25℃ and drying it for 1 h to obtain the solid flash-release soluble microneedles.

Images