A pimple-removing composition acting on biofilm and its preparation method and application

Abstract

The present application relates to the technical field of skin care products, in particular to a kind of acne-removing composition acting on biological membrane and its preparation method and application.By compounding Xikunshi gum and composite fermentation liquor in a certain proportion, synergistic effect can be achieved, on the one hand, the formation of propionibacterium acnes biofilm can be inhibited better, and the already formed propionibacterium acnes biofilm can also be dissolved;On the other hand, acne-removing, soothing and oil control effects can also be achieved;Preferably, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri are selected for composite fermentation, so that the activity of the obtained composition is better, and the effect of inhibiting acne recurrence is more significant;The present application uses reasonable active ingredient collocation and special preparation process, from the dimension of dissolving the already formed propionibacterium acnes biofilm, soothing and oil control, regulating skin microecological balance, and the comprehensive effect of inhibiting acne recurrence can be achieved, and the effect is more significant.

Description

Technical Field

[0001] This invention relates to the field of skincare technology, and in particular to an acne-removing composition that can act on biological membranes, its preparation method, and its application. Background Technology

[0002] Acne is a common dermatological disease, a chronic inflammatory disorder of the hair follicles and sebaceous glands. The main clinical manifestations include comedones, papules, pustules, and cysts. Sebum secretion is also commonly observed. It is most common on the face of adolescent boys and girls, hence the name "acne vulgaris." In reality, many people's acne is often difficult to cure completely, recurring repeatedly, and may even leave behind numerous acne scars and pits that are difficult to remove, severely affecting their appearance, self-confidence, and social life.

[0003] Acne is a chronic inflammatory skin disease that commonly occurs in areas with high density of hair follicles and sebaceous glands. Its pathogenesis is related to multiple factors, among which the proliferation of Propionibacterium acnes is closely related to acne development. Systemic or topical application of antibacterial drugs to inhibit Propionibacterium acnes is an important treatment for acne. In recent years, there has been increasing research on antibiotic resistance caused by bacterial biofilm formation. A bacterial biofilm is a microbial community that forms and adheres to biological or non-biological surfaces and is surrounded by extracellular polymeric substances. Bacteria within biofilms are 1000 times more resistant to antibacterial drugs than planktonic bacteria. Bacteria can form a mature biofilm 24 hours after attaching to a biological or non-biological interface. During acne formation, microorganisms such as Propionibacterium acnes can form biofilms within the hair follicles and sebaceous glands. This biofilm enhances the microorganisms' resistance to antibiotics and the host's immune system. It provides a protective environment for the microorganisms, making them more difficult to eliminate, leading to persistent or recurrent inflammation, thus making acne prone to recurrence.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] To address the above-mentioned problems, the present invention aims to provide an acne-removing composition that can act on biological membranes, its preparation method, and its application.

[0006] The technical content of this invention is as follows:

[0007] This invention provides an acne-removing composition that can act on biological membranes and its preparation method, wherein the composition includes aralia elata gel and a compound fermentation liquid.

[0008] In a specific embodiment of the present invention, the compound fermentation broth is obtained by using red pine leaf extract and spider vine seed extract as raw materials, through anaerobic fermentation of compound bacteria for 2-5 days, sterilization, and centrifugation filtration.

[0009] In a specific embodiment of the present invention, the preparation process of the red pine leaf extract is as follows:

[0010] Step 1. Wash and dry the red pine needles, then mix them with pure water in a 1:1 ratio and grind them into a slurry to obtain slurry one;

[0011] Step 2. Add 0.5-1% cellulase to the slurry obtained in Step 1, and perform enzymatic hydrolysis at 40-50℃ for 1-2 hours. Inactivate the enzyme in a water bath at 80-90℃ for 8-15 minutes. After cooling to room temperature, centrifuge and filter to obtain filter residue and enzymatic hydrolysate.

[0012] Step 3. Add 10-15 times the volume of ethanol aqueous solution to the filter residue obtained in step 2, perform ultrasonic extraction, centrifuge, filter and dry to obtain water-ethanol extract;

[0013] The volume fraction of ethanol is 50-60%, the temperature of ultrasonic extraction is 50-60℃, the working frequency of ultrasonic extraction is 30-40KHz, the ultrasonic power of ultrasonic extraction is 400-600W, and the ultrasonic extraction time is 20-40min.

[0014] Step 4. Mix the enzymatic hydrolysate obtained in Step 2 and the hydroalcohol extract obtained in Step 3 evenly to obtain the red pine leaf extract.

[0015] In a specific embodiment of the present invention, the weight ratio of the red pine leaf extract and the bird's eye seed extract is 3-5:1-2.

[0016] In a specific embodiment of the present invention, the compound bacteria are Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri with a live bacteria ratio of 1:1:1.

[0017] In a specific embodiment of the present invention, the inoculation ratio of the compound bacteria is 0.5 × 10⁻⁶. 5 -1.2×10 5 cfu / mL.

[0018] In a specific embodiment of the present invention, the fermentation temperature is 28-37℃.

[0019] In a specific embodiment of the present invention, the acne-removing composition that can act on biological membranes further includes solvent A and solvent B. Solvent A comprises water and butanediol in a mass ratio of 5:1, and solvent B comprises 1,2-pentanediol and 1,2-hexanediol in a mass ratio of 2:1.

[0020] In a specific embodiment of the present invention, the preparation method of the acne-removing composition is as follows:

[0021] Dissolve the prickly pear gum in solvent A to obtain mixture 1, dissolve the compound fermentation liquid in solvent B to obtain mixture 2, and mix mixture 1 and mixture 2 evenly to obtain the acne-removing composition.

[0022] In a specific embodiment of the present invention, the ratio of the prickly pear jelly to solvent A is 0.1-1:20-30.

[0023] In a specific embodiment of the present invention, the ratio of the composite fermentation broth to solvent B is 45-70:2-5.

[0024] Another aspect of the present invention provides the application of any of the above-mentioned acne-removing compositions in a serum.

[0025] In a specific embodiment of the present invention, the acne-removing composition is 2-10% by mass in the acne-removing essence.

[0026] In this invention:

[0027] Red pine leaf extract: Red pine is a woody plant belonging to the genus Pinus of the family Pinaceae. Its needles are rich in chlorophyll derivatives, polyisoprene alcohols, carotenoids, terpenes, vitamin E, and phytosterols. It has excellent antioxidant properties and can achieve a certain oil-controlling effect by improving lipid metabolism.

[0028] Bird's eye root seed extract: Bird's eye root is a plant belonging to the genus *Spiraea* in the legume family, also known as five-leaf clover, horn flower, and bird's-spur grass. In my country, bird's eye root mainly grows on moist, slightly alkaline slopes, fields, grasslands, or riverbanks in the northwest, southwest, and upper reaches of the Yangtze River. Bird's eye root is a...

[0029] Traditional Chinese herbal medicines possess properties such as clearing heat and dampness, relieving cough and detoxifying, and promoting blood circulation and removing blood stasis. Analysis of its components has revealed a variety of phytochemicals, including alkaloids, steroids, flavonoids, and phenolic glycosides. Further research has shown that extracts from *Spiraea japonica* have inhibitory effects on Gram-positive and Gram-negative bacteria, including *Propionibacterium acnes*, *Staphylococcus aureus*, *Escherichia coli*, and *Salmonella typhi*.

[0030] The thorn cloud gel has a unique 3D colloidal structure. The polysaccharides in the 3D colloid will competitively attach to microbial lectins, preventing skin glycoproteins from binding with pathogenic bacteria, thereby avoiding colony aggregation and skin inflammation.

[0031] The beneficial effects of this invention are as follows:

[0032] (1) The acne-removing composition provided by the present invention can achieve a synergistic effect by combining the acne-removing gel and the compound fermentation liquid in a certain proportion. On the one hand, it can make the inhibition of the formation of Propionibacterium acnes biofilm better, and at the same time, it can also break down the biofilm of Propionibacterium acnes that has already been generated; on the other hand, it can also achieve the effects of acne removal, soothing and oil control.

[0033] (2) The compound fermentation liquid in the acne-removing composition provided by the present invention is preferably compound fermented by specifically selecting Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri, so that the resulting composition has better activity and more significant effect in inhibiting the recurrence of acne.

[0034] (3) This invention uses a reasonable combination of active ingredients and a special preparation process to inhibit the formation of Propionibacterium acnes biofilm. It works by dismantling the biofilm of already generated Propionibacterium acnes, soothing and controlling oil, and regulating the skin's microecological balance. This can achieve the effect of comprehensively inhibiting the recurrence of acne, and the effect is more significant. Attached Figure Description

[0035] Figure 1 This is a scanning electron microscope image of the biofilm breakdown by the essence of this invention;

[0036] Figure 2 The diagram shows the alpha diversity analysis (A. Ace index, B. Chaos index, C. Sobs index, D. Coverage index).

[0037] Figure 3 Venn diagram for species;

[0038] Figure 4 A graph showing the differences in bacterial community composition at the phylum level;

[0039] Figure 5 This is a graph showing the differences in bacterial community composition at the genus level. Detailed Implementation

[0040] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0041] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0042] Example 1

[0043] The preparation of an acne-removing composition that can act on biological membranes includes the following steps:

[0044] S1: Dissolve 0.2 parts of *Ligustrum lucidum* gum in 5 parts of deionized water and 1 part of butanediol to obtain mixture 1.

[0045] S2: Mix 60 parts of red pine leaf extract and 20 parts of coix seed extract evenly to obtain a compound extract.

[0046] S3: Add 0.8 × 10 to the compound extract. 5 A compound culture of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri* was fermented at a constant temperature of 30°C for 3 days, sterilized, and centrifuged to obtain the compound fermentation broth. The compound culture consisted of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri* in a live bacteria ratio of 1:

[0047] S4: Dissolve 50 parts of the compound fermentation broth in 3 parts of 1,2-pentanediol and 1.5 parts of 1,2-hexanediol to obtain mixture 2.

[0048] S5: Mix mixture 1 and mixture 2 evenly to obtain the acne-removing composition.

[0049] The preparation method of the red pine leaf extract is as follows:

[0050] 1) Wash and dry the red pine needles, then mix them with pure water in a 1:1 ratio and grind them into a slurry to obtain slurry one;

[0051] 2) Add 0.6% cellulase to the slurry obtained in step 1, perform enzymatic hydrolysis at 45℃ for 1.5h, inactivate it in an 80℃ water bath for 10min, cool to room temperature, centrifuge and filter to obtain filter residue and enzymatic hydrolysate.

[0052] 3) Add 12 times the amount of ethanol aqueous solution to the filter residue obtained in step 2, perform ultrasonic extraction, centrifuge, filter and dry to obtain water-ethanol extract.

[0053] The volume fraction of ethanol is 55%, the ultrasonic extraction temperature is 60°C, the ultrasonic extraction frequency is 33 kHz, the ultrasonic extraction power is 500 W, and the ultrasonic extraction time is 30 min.

[0054] 4) Mix the enzymatic hydrolysate obtained in step 2 and the hydroalcohol extract obtained in step 3 evenly to obtain the red pine leaf extract.

[0055] The aforementioned *Polygonum multiflorum* seed extract was purchased from Shanghai Jiakai Biotechnology Co., Ltd.

[0056] The *Lactobacillus plantarum* was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0057] The Lactobacillus acidophilus mentioned was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0058] The Lactobacillus reuteri mentioned was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0059] This embodiment provides a serum comprising the following components by weight percentage: allantoin 0.1%, sodium EDTA-2 0.02%, glycerin 2%, 1,3-propylene glycol 3%, carbomer 0.15%, arginine 0.15%, 1,2-hexanediol 0.5%, p-hydroxyphenylethanol 0.5%, acne-fighting composition 2%, and water as the balance.

[0060] The preparation method of the serum is as follows: Allantoin, sodium EDTA-2, glycerin, 1,3-propylene glycol, carbomer and water are mixed evenly in proportion. The resulting mixture is kept at 85°C for 20 minutes. When the temperature drops to 70°C, arginine, 1,2-ethylene glycol and p-hydroxyphenylethanol are added and stirred evenly. When the temperature drops to 45°C, the acne-removing composition is added and stirred evenly to obtain the serum.

[0061] Example 2

[0062] The preparation of an acne-removing composition that can act on biological membranes includes the following steps:

[0063] S1: Dissolve 0.3 parts of *Ligustrum lucidum* gum in 6.25 parts of deionized water and 1.25 parts of butanediol to obtain mixture 1.

[0064] S2: Mix 50 parts of red pine leaf extract and 10 parts of coix seed extract evenly to obtain a compound extract.

[0065] S3: Add 1.2 × 10⁻⁶ to the compound extract. 5 A compound culture of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri* was fermented at a constant temperature of 33°C for 2 days, sterilized, and centrifuged to obtain the compound fermentation broth. The compound culture consisted of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri* in a live bacteria ratio of 1:

[0066] S4: Dissolve 50 parts of the compound fermentation broth in 3 parts of 1,2-pentanediol and 1.5 parts of 1,2-hexanediol to obtain mixture 2.

[0067] S5: Mix mixture 1 and mixture 2 evenly to obtain the acne-removing composition.

[0068] The preparation method of the red pine leaf extract is as follows:

[0069] 1) Wash and dry the red pine needles, then mix them with pure water in a 1:1 ratio and grind them into a slurry to obtain slurry one;

[0070] 2) Add 0.7% cellulase to the slurry obtained in step 1, perform enzymatic hydrolysis at 40℃ for 2 hours, inactivate it in a water bath at 85℃ for 8 minutes, cool to room temperature, centrifuge and filter to obtain filter residue and enzymatic hydrolysate.

[0071] 3) Add 13 times the amount of ethanol aqueous solution to the filter residue obtained in step 2, perform ultrasonic extraction, centrifuge, filter and dry to obtain water-ethanol extract.

[0072] The volume fraction of ethanol is 58%, the ultrasonic extraction temperature is 50°C, the ultrasonic extraction frequency is 35 kHz, the ultrasonic extraction power is 400 W, and the ultrasonic extraction time is 20 min.

[0073] 4) Mix the enzymatic hydrolysate obtained in step 2 and the hydroalcohol extract obtained in step 3 evenly to obtain the red pine leaf extract.

[0074] The aforementioned *Polygonum multiflorum* seed extract was purchased from Shanghai Jiakai Biotechnology Co., Ltd.

[0075] The *Lactobacillus plantarum* was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0076] The Lactobacillus acidophilus mentioned was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0077] The Lactobacillus reuteri mentioned was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0078] The acne-removing composition of this embodiment was used to prepare the serum of this embodiment according to the composition and preparation method of the serum in Example 1.

[0079] Example 3

[0080] The preparation of an acne-removing composition that can act on biological membranes includes the following steps:

[0081] S1: Dissolve 0.5 parts of *Ligustrum lucidum* gum in 10 parts of deionized water and 2 parts of butanediol to obtain mixture 1.

[0082] S2: Mix 65 parts of red pine leaf extract and 18 parts of coix seed extract evenly to obtain a compound extract.

[0083] S3: Add 1.1 × 10⁻⁶ to the compound extract. 5 A compound culture of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri* was fermented at a constant temperature of 35°C for 4 days, sterilized, and centrifuged to obtain the compound fermentation broth. The compound culture consisted of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri* in a live bacteria ratio of 1:

[0084] S4: Dissolve 70 parts of the compound fermentation broth in 2.5 parts of 1,2-pentanediol and 1.25 parts of 1,2-hexanediol to obtain mixture 2.

[0085] S5: Mix mixture 1 and mixture 2 evenly to obtain the acne-removing composition.

[0086] The preparation method of the red pine leaf extract is as follows:

[0087] 1) Wash and dry the red pine needles, then mix them with pure water in a 1:1 ratio and grind them into a slurry to obtain slurry one;

[0088] 2) Add 0.8% cellulase to the slurry obtained in step 1, perform enzymatic hydrolysis at 40℃ for 1 hour, inactivate it in a water bath at 88℃ for 5 minutes, cool to room temperature, centrifuge and filter to obtain filter residue and enzymatic hydrolysate.

[0089] 3) Add 15 times the amount of ethanol aqueous solution to the filter residue obtained in step 2, perform ultrasonic extraction, centrifuge, filter and dry to obtain water-ethanol extract.

[0090] The volume fraction of ethanol is 60%, the ultrasonic extraction temperature is 55°C, the ultrasonic extraction frequency is 40 kHz, the ultrasonic extraction power is 550 W, and the ultrasonic extraction time is 25 min.

[0091] 4) Mix the enzymatic hydrolysate obtained in step 2 and the hydroalcohol extract obtained in step 3 evenly to obtain the red pine leaf extract.

[0092] The aforementioned *Polygonum multiflorum* seed extract was purchased from Shanghai Jiakai Biotechnology Co., Ltd.

[0093] The *Lactobacillus plantarum* was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0094] The Lactobacillus acidophilus mentioned was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0095] The Lactobacillus reuteri mentioned was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0096] The acne-removing composition of this embodiment was used to prepare the serum of this embodiment according to the composition and preparation method of the serum in Example 1.

[0097] Comparative Example 1

[0098] An acne-removing composition that can act on biofilms and its preparation method, differing from Example 3 only in that the composition does not contain safflower gum.

[0099] The serum of this comparative example was prepared using the acne-removing composition according to the composition and preparation method of the serum in Example 1.

[0100] Comparative Example 2

[0101] An acne-removing composition that can act on biological membranes and its preparation method, differing from Example 3 only in that the red pine leaf extract in the composite extract is only a water-alcohol extract.

[0102] The serum of this comparative example was prepared using the acne-removing composition according to the composition and preparation method of the serum in Example 1.

[0103] Comparative Example 3

[0104] An acne-removing composition that can act on biological membranes and its preparation method, differing from Example 3 only in that the compound extract is a single red pine leaf extract.

[0105] The serum of this comparative example was prepared using the acne-removing composition according to the composition and preparation method of the serum in Example 1.

[0106] Comparative Example 4

[0107] An acne-removing composition that can act on biological membranes and its preparation method, differing from Example 3 only in that the compound extract is a single extract of *Polygonum multiflorum* seeds.

[0108] The serum of this comparative example was prepared using the acne-removing composition according to the composition and preparation method of the serum in Example 1.

[0109] Comparative Example 5

[0110] An acne-removing composition that can act on biological membranes and its preparation method, differing from Example 3 only in that the compound extract is pine leaf extract and fucus vesiculosus extract.

[0111] The extract of *Fucus vesiculosus* was purchased from Shanghai Jiakai Biotechnology Co., Ltd.

[0112] The serum of this comparative example was prepared using the acne-removing composition according to the composition and preparation method of the serum in Example 1.

[0113] Comparative Example 6

[0114] An acne-removing composition that can act on biofilms and its preparation method, differing from Example 3 only in that the fermentation process is inoculated with only *Lactobacillus plantarum*.

[0115] The serum of this comparative example was prepared using the acne-removing composition according to the composition and preparation method of the serum in Example 1.

[0116] Comparative Example 7

[0117] An acne-removing composition that can act on biofilms and its preparation method, differing from Example 3 only in that the fermentation process is inoculated only with Lactobacillus acidophilus.

[0118] The serum of this comparative example was prepared using the acne-removing composition according to the composition and preparation method of the serum in Example 1.

[0119] Comparative Example 8

[0120] An acne-removing composition that can act on biofilms and its preparation method, differing from Example 3 only in that the fermentation process is inoculated only with Lactobacillus reuteri.

[0121] The serum of this comparative example was prepared using the acne-removing composition according to the composition and preparation method of the serum in Example 1.

[0122] Effect verification

[0123] 1. Inhibits the formation of acne propionate biofilm.

[0124] Step 1: Blank control group: Weigh 2.5g of sterile water and add it to 97.5g of TSB culture medium, and mix well.

[0125] Experimental group: Weigh 2.5g of each of the acne-removing compositions prepared in Examples 1-3 and Comparative Examples 1-8, add them to 97.5g of TSB culture medium, and mix well.

[0126] Step 2: Take a fresh culture of Propionibacterium acnes within 5 generations, and use a pipette to draw an appropriate amount of 0.03 mol / L phosphate buffer solution, repeatedly rinsing to remove the bacterial colony. Mix with an electric mixer for 20 seconds to ensure the bacteria are evenly suspended. Inoculate the bacterial suspension into the culture medium prepared in Step 1, ensuring the initial concentration of Propionibacterium acnes in the culture medium is approximately 10. 8 CFU / ml.

[0127] Step 3: Inoculate the culture medium containing Propionibacterium acnes suspension into 6-well plates, 5 ml per well, with 3 wells in parallel. Incubate at 37°C for 48 hours. After 48 hours of incubation, discard the bacterial culture, add PBS to wash away any unattached bacteria, and repeat the washing process 3 times.

[0128] Step 4: Add 99% methanol to the prepared 6-well plate for fixation for 15 min, discard the liquid, and dry at room temperature. Add 1% crystal violet for staining for 8 min, rinse with sterile water until the water is colorless, and dry at room temperature.

[0129] 10% acetic acid was dissolved in the dissolved biofilm. After the crystals bound to the biofilm were fully dissolved, the absorbance (A) at 570 nm was measured using a UV spectrophotometer.

[0130] Table 1 Absorbance (A) values

[0131] Group Absorbance A Group Absorbance A Blank control group 1.324 Comparative Example 3 0.955 Example 1 0.742 Comparative Example 4 1.184 Example 2 0.725 Comparative Example 5 1.231 Example 3 0.708 Comparative Example 6 1.153 Comparative Example 1 1.082 Comparative Example 7 1.216 Comparative Example 2 1.133 Comparative Example 8 1.163

[0132] As shown in Table 1, compared with the blank control group, Examples 1-3 showed a significant inhibitory effect on the formation of Propionibacterium acnes biofilm, while Comparative Examples 1-8 showed a weaker inhibitory effect on the formation of Propionibacterium acnes biofilm. This indicates that the combination of aralia elata and the compound fermentation liquid has a synergistic effect on inhibiting the formation of Propionibacterium acnes biofilm. At the same time, the combination of different extracts, different extraction processes, and different fermentation strain ratios during compound fermentation all affect the acne-removing composition's ability to inhibit the formation of Propionibacterium acnes biofilm.

[0133] 2. Human efficacy experiments

[0134] The testing method is as follows: 176 volunteers with grade 1-2 acne were recruited, with 16 volunteers in each group. The serum in the control group did not contain the acne-reducing composition, while the composition and content of the remaining components were the same as the serum in Example 1. The experimental groups used the serums obtained in Examples 1-3 and Comparative Examples 1-8 for a total of 28 days. At days 0, 14, and 28, the number of acne lesions was clinically assessed, and the skin's α-value (α-value, which is the redness content of the skin, representing erythema in the dermis and indicating the state of skin inflammation) and sebum secretion in the acne-prone areas were measured using a VISA instrument. The improvement rate was calculated by comparing the data with that of day 0. Volunteers were followed up 56 days after the product was used to inquire whether they had experienced a recurrence of acne. The test results are shown in Table 2.

[0135] Table 2 Skin Effects

[0136]

[0137] The results above show that the acne-removing essences in Examples 1-3 have significant acne-removing effects, and the combination of *Pinus thunbergii* seed extract and compound fermentation liquid has a good effect in inhibiting acne recurrence and has a good soothing and oil-controlling effect. The comparative examples show that the enzymatic hydrolysis of *Pinus thunbergii* leaves before fermentation enhances its soothing and oil-controlling effects, reducing the possibility of acne recurrence; the combination of *Lactobacillus plantarum* seed extract during fermentation has a good promoting effect on acne removal; and the reasonable combination of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri* can fundamentally improve skin condition and make acne less likely to recur.

[0138] 3. Security Testing

[0139] Human skin patch test, the trial method is based on: "Cosmetic Safety Technical Specifications" (2015 edition).

[0140] General testing method: Select a suitable patch tester and use a closed patch test method. Select at least 30 qualified volunteers to participate in the test. Add the test substance and control sample to the square chamber of the patch tester. Apply the patch tester containing the test substance and control sample to the inner forearm of the subject, and gently press it with the palm of your hand to make it evenly applied to the skin. After 24 hours, remove the patch tester and observe the skin reaction according to the standards in Table 3 at 30 minutes (after the indentation disappears), 24 hours, and 48 hours, and record the observation results.

[0141] Following the general method described above, 34 qualified volunteers were selected to participate in the test. The test samples prepared from the acne-removing compositions of each embodiment were used as test substances, and sterile deionized water was used as the control sample. The patch test apparatus containing the test substance and the control sample was applied to the inner forearm of the subject and removed after 24 hours. The skin reaction was observed 30 minutes, 24 hours, and 48 hours after the patch test apparatus was removed, according to the grading criteria for skin reaction in closed patch tests in Table 3, and the observation results were recorded as shown in Table 4.

[0142] Table 3. Grading Criteria for Skin Reactions in Occlusive Patch Tests

[0143]

[0144] Table 4 Results of the patch test

[0145]

[0146] As shown in Table 4, the patch test results of the compositions prepared in Examples 1-3 and the control group were all negative, indicating that the acne-removing compositions prepared by the acne-removing gel and compound fermentation liquid in this invention are mild and non-irritating.

[0147] 4. Experiment on the Disintegration of Biomembranes

[0148] Control group: Serum without acne-fighting ingredients

[0149] Experimental group: The serum prepared in Example 3

[0150] Activated Propionibacterium acnes was added to 20 ml of BHI medium to prepare a bacterial suspension, and the initial bacterial concentration was adjusted to 1.0 (OD). 600Prepare for use. Inoculate bacterial culture onto 6-well cell slides and anaerobic culture at 37°C for 5 days, changing the culture medium daily. After 5 days, remove the supernatant and wash once with PBS (2 ml). Add sample solution (2 ml) as the sample group and PBS (2 ml) as the control group, and incubate anaerobically at 37°C for 12 h. After incubation, wash three times with PBS (2 ml). Fix with 2.5% glutaraldehyde fixative for 4 h, then wash three times with PBS, allowing to stand for 20 min each time. Spray the samples with gold and observe bacterial morphological changes using field emission scanning electron microscopy.

[0151] Depend on Figure 1 It can be seen that the essence prepared in Example 3 can effectively break down the already formed Propionibacterium acnes biofilm.

[0152] 5. Analysis of the diversity of facial skin microecological community

[0153] Test sample: The test sample was the serum prepared in Example 3.

[0154] How to use: Apply to the entire face, once in the morning and once in the evening. Apply 3-5 drops to the face each time and massage until absorbed.

[0155] All volunteers signed informed consent forms during their initial visit and were then screened according to inclusion and exclusion criteria. The day of the formal trial is designated as day 0. Qualified participants arrived at the visit center, where a 4*4cm² skin swab sample was taken from the volunteers' acne-affected areas for microbiome-related testing. The test sample was then used immediately. A follow-up visit was scheduled 4 weeks later, designated day 28, with the same sampling and testing procedures as day 0. Before each skin sampling, participants were advised not to wash their face in the morning to ensure sufficient biological sample.

[0156] All tests were conducted in a laboratory with a controlled temperature of 20-22℃ and a relative humidity of 40-60%. Sixty-four valid samples were collected, and the D0 (before use of the test substance) and D28 (after 28 days of continuous use of the test substance) groups were designated as D0 and D28 groups, respectively, for 32 volunteers.

[0157] Alpha diversity is mainly used to study the community diversity within a habitat (or sample). It can be assessed by evaluating a series of indices to obtain information such as the richness and diversity of species in an environmental community.

[0158] Indices reflecting community richness include: sobs, chao, and ace.

[0159] The indices reflecting community coverage are: coverage; the higher its value, the higher the probability that the sequences in the sample are detected, and the lower the probability of not being detected. This index reflects whether the sequencing results represent the true situation of the microorganisms in the sample.

[0160] Through the analysis of Alpha diversity indices, information such as the richness, diversity, and coverage of species in the community can be obtained. In this study, the between-group difference test method was used to detect whether there were significant differences in the Alpha diversity index values between 0 day and 28 days. The community richness was reflected by sobs, chao, and ace indices, and the coverage rate of the sample library was reflected by coverage.

[0161] The results are as Figure 2 (0.01 < p ≤ 0.05 is marked as *, 0.0001 < p ≤ 0.001 is marked as ***). As shown, from the start of the intervention of the test samples (D0) to 28 days later (D28), there were obvious differences in the Alpha diversity of skin bacterial microorganisms (p < 0.05). At D28, the Ace, Sobs, Chao, and Coverage indices all increased significantly, indicating that the test samples had a significant regulatory effect on the skin bacterial diversity of the subjects (p < 0.05). The significant increase in skin species diversity is beneficial to adjusting the skin flora balance and consolidating the skin immune homeostasis.

[0162] 6. Analysis of community composition

[0163] Venn analysis focuses on showing the number of unique and common species in different groups, facilitating the understanding of changes in the presence or absence of species in different research situations. This application can select biomarkers for research based on unique or common species.

[0164] As Figure 3 , different colors represent the 0-day and 28-day groups respectively. The overlapping part represents the species common to multiple groups (or samples), and the non-overlapping part represents the species unique to that group (or sample). The numbers represent the corresponding number of species. The figure shows that from D0 when the test samples were used until D28, the number of skin bacterial microbial species increased by 56. The trend of rich skin species is beneficial to the balance of healthy skin flora and consolidates the skin barrier and its homeostasis.

[0165] 7. Analysis of species difference significance

[0166] Differential tests at the phylum level were performed on the top 5 bacterial species with changing abundances (genera with no significant changes were not summarized and displayed). The results are shown as Figure 4The study found a significant increase in the abundance of Bacteroidetes among the resident flora of the skin (P = 0.00652), while among species with lower abundance, the abundance of Cyanobacteria significantly decreased (p = 0.000296). Previous studies have shown a significant decrease in Bacteroidetes in acne-prone individuals compared to healthy controls (P < 0.05). The significant increase in Bacteroidetes abundance after 28 days of skin intervention with the test substance indicates a trend towards healthier skin.

[0167] A differential test was performed on the top 10 bacterial species with the highest abundance that showed changes (genera without significant changes were not included in the summary). The results showed that... Figure 5 This study investigated the changes in the levels of skin bacteria before and after intervention with the test substance on acne-prone (or pimple) skin. The results showed a significant increase in the abundance of Bacillus (p = 1.633e-7). Bacillus has antibacterial effects on the skin, and can also moisturize, promote cell growth, and exfoliate. This trend suggests that it can better regulate and strengthen the skin barrier in acne-prone skin.

[0168] In summary, under the experimental conditions described above, the serum prepared in Example 3 improved the skin's bacterial microecological balance and was able to regulate the distribution of bacteria in acne-prone skin towards that of healthy individuals.

[0169] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An acne-removing composition that can act on biological membranes, characterized in that, The composition comprises 0.1-1 parts of prickly ash gum and 45-70 parts of compound fermentation broth; The compound fermentation broth is obtained by anaerobic fermentation of red pine leaf extract and coix seed extract by compound bacteria for 2-5 days, followed by sterilization and centrifugal filtration. The compound bacteria consist of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri in a live bacteria ratio of 1:1:

1. The preparation process of the red pine leaf extract is as follows: Step 1. Wash and dry the red pine needles, then mix them with pure water in a 1:1 ratio and grind them into a slurry to obtain slurry one; Step 2. Add 0.5-1% cellulase to the slurry obtained in Step 1, and perform enzymatic hydrolysis at 40-50℃ for 1-2 hours. Inactivate the enzyme in a water bath at 80-90℃ for 8-15 minutes. After cooling to room temperature, centrifuge and filter to obtain filter residue and enzymatic hydrolysate. Step 3. Add 10-15 times the volume of ethanol aqueous solution to the filter residue obtained in step 2, perform ultrasonic extraction, centrifuge, filter and dry to obtain water-ethanol extract; The volume fraction of ethanol is 50-60%, the temperature of ultrasonic extraction is 50-60℃, the working frequency of ultrasonic extraction is 30-40KHz, the ultrasonic power of ultrasonic extraction is 400-600W, and the ultrasonic extraction time is 20-40min. Step 4. Mix the enzymatic hydrolysate obtained in Step 2 and the hydroalcohol extract obtained in Step 3 evenly to obtain the red pine leaf extract.

2. The acne-removing composition that can act on biological membranes according to claim 1, characterized in that, The weight ratio of the red pine leaf extract to the spider vine seed extract is 3-5:1-2.

3. The acne-removing composition that can act on biological membranes according to claim 1, characterized in that, The inoculation ratio of the compound bacteria is 0.5 × 10⁻⁶. 5 -1.2×10 5 cfu / mL; The fermentation temperature is 28-37℃.

4. The acne-removing composition that can act on biological membranes according to claim 1, 2, or 3, characterized in that, It also includes solvent A and solvent B, wherein solvent A comprises water and butanediol in a mass ratio of 5:1, and solvent B comprises 1,2-pentanediol and 1,2-hexanediol in a mass ratio of 2:

1.

5. A method for preparing the acne-removing composition that can act on biological membranes as described in claim 4, characterized in that, Includes the following steps: Dissolve the prickly pear gum in solvent A to obtain mixture 1, dissolve the compound fermentation liquid in solvent B to obtain mixture 2, and mix mixture 1 and mixture 2 evenly to obtain the acne-removing composition.

6. The method for preparing the acne-removing composition applicable to biological membranes according to claim 5, characterized in that, The ratio of the prickly ash jelly to solvent A is 0.1-1:20-30.

7. The method for preparing the acne-removing composition applicable to biological membranes according to claim 5, characterized in that, The ratio of the compound fermentation broth to solvent B is 45-70:2-5.

8. An acne-removing essence, characterized in that, The acne-removing essence includes the acne-removing composition according to any one of claims 1-4; The acne-removing composition is 2-10% by mass in the acne-removing essence.

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