Acne removing and repairing composition and application thereof

Abstract

The invention relates to an acne removing and repairing composition and application thereof, and belongs to the technical field of cosmetics. The acne removing and repairing composition comprises a Chinese rose flower extract and an ormosia henryi extract, the concentration of the Chinese rose flower extract in the acne removing and repairing composition is 0.0001 mg / mL to 100 mg / mL, and the concentration of the ormosia henryi extract in the acne removing and repairing composition is 0.001 mu g / mL to 50000 mu g / mL. The acne removing and repairing composition has the advantages that the formation of biofilms of propionibacterium acnes, staphylococcus epidermidis and staphylococcus aureus is obviously inhibited through the multi-target synergistic effect, and the acne removing and repairing composition has the obvious activity of inhibiting gram-positive bacteria and shows excellent drug resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to an acne-repairing composition and an application thereof. Background Art

[0002] Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium tetani, Streptococcus, Pneumococcus, Proteus, and other pathogens. Common skin pathogens include Staphylococcus epidermidis, Propionibacterium acnes, and Staphylococcus aureus. Under certain conditions, an imbalance in the bacterial flora can easily cause acne. Currently known acne-causing bacteria include:

[0003] Propionibacterium acnes: As the most recognized pathogenic microorganism, its pathogenic mechanism is mainly to induce inflammatory response, promote keratinization of sebaceous gland epithelium of hair follicles, and promote sebaceous gland secretion.

[0004] Staphylococcus epidermidis (SEP) is a coagulase-negative, Gram-positive bacterium that is highly cryptic and able to evade the host's immune defenses. It typically causes low-toxic opportunistic infections. According to a study by M. Bek-Thomsen et al., the proportion of S. epidermidis colonizing the sebaceous glands of acne follicles is as high as 6.8%-47.3%, second only to Propionibacterium acnes. S. epidermidis is involved in superficial infections of the sebaceous glands of the skin, and infection with S. epidermidis can increase the incidence of acne vulgaris by 70% compared to controls. The main pathogenic factors include lipase and hemolysin. The role of S. epidermidis in the pathogenesis of acne is primarily related to the biofilm it forms.

[0005] Staphylococcus aureus, a Gram-positive coccus that produces multiple toxins, is a key pathogen in both hospital-acne and community-acquired infections. Studies have shown that S. aureus increases in abundance during acne outbreaks. In acne lesions, S. aureus is typically more abundant than in non-lesional areas. However, compared to Propionibacterium acnes, the specific role of S. aureus in acne pathogenesis may be more complex, as it can be both a pathogen and a component of the skin microbiome. In certain circumstances, excessive growth of S. aureus may exacerbate acne symptoms.

[0006] Inhibiting these Gram-positive bacteria is crucial to controlling the occurrence of acne. Common topical antibiotics currently on the market for controlling acne symptoms include erythromycin, lincomycin, its derivative clindamycin, chloramphenicol, clindamycin and fusidic acid. These drugs show low irritation when treating superficial inflammatory acne lesions such as papules and pustules, and are theoretically considered to be an effective choice for treating acne lesions. However, topical antibiotics may lead to increased bacterial resistance and are not recommended for single or long-term use. Guo Xianfei et al. isolated 12 strains of Propionibacterium acnes from clinical samples and conducted drug sensitivity tests for clindamycin, erythromycin, tetracycline and metronidazole. The results showed that all tested strains were resistant to clindamycin and metronidazole. Among them, 11 strains were resistant to erythromycin and only 1 strain showed moderate sensitivity. Zhang Liyuan et al. isolated 1,259 strains of P. acnes from 1,770 patients with acne, and found varying degrees of resistance to multiple antibiotics. Resistance rates to erythromycin, clindamycin, azithromycin, and metronidazole were 45%, 74%, 59%, and 96%, respectively. This widespread resistance has limited the use of antibiotics in the treatment of acne, and new antimicrobial strategies are urgently needed.

[0007] As traditional Chinese medicines, the active ingredients of roses and palm trees have been shown to possess antimicrobial activity. Gallic acid and quercetin in roses inhibit drug-resistant bacteria such as Staphylococcus aureus and Enterococcus faecalis; palm trees contain flavonoids, alkaloids, and volatile components with anti-inflammatory and antioxidant properties. However, roses and palm trees are primarily used in cosmetics for whitening and anti-wrinkle purposes, and their application in inhibiting acne-related Gram-positive bacteria has not been reported. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides an acne-repairing composition and its application. It is determined that the combination of rose extract and palmyra palm extract is superior to the use of single drugs, has a synergistic effect in inhibiting Gram-positive bacteria, and has an additive effect in inhibiting biofilms.

[0009] The first object of the present invention is to provide an acne-repairing composition, which comprises a rose extract and a palm tree extract.

[0010] In one embodiment of the present invention, the concentration of the rose extract in the acne-repairing composition is 0.0001 mg / mL-100 mg / mL, and the concentration of the palm tree extract is 0.001 μg / mL-50000 μg / mL.

[0011] In one embodiment of the present invention, the acne-removing and repairing composition further comprises a pharmaceutically acceptable carrier.

[0012] In one embodiment of the present invention, the extraction method of the rose extract is: placing the rose in 15-25 times its weight of water, ultrasonically extracting at 150W-250W and 55°C-65°C for 15-25 minutes, and filtering and separating to obtain the rose extract.

[0013] In one embodiment of the present invention, the extraction method of the palm tree extract is: placing the palm tree in an alcohol solution 15-25 times its weight, ultrasonically extracting at 150W-250W and 75℃-85℃ for 20-30min, and filtering and separating to obtain the palm tree extract.

[0014] In one embodiment of the present invention, the acne-removing and repairing composition has at least one of the following effects:

[0015] (1) Destroy the biofilm structure: inhibit EPS synthesis, block bacterial adhesion and aggregation;

[0016] (2) Regulate the quorum sensing system: inhibit the expression of LuxS and SarA genes, weakening bacterial communication;

[0017] (3) Enhance antimicrobial permeability: destroy the biofilm barrier and promote the deep action of antimicrobial agents.

[0018] The second object of the present invention is to provide a use of the acne-repairing composition in inhibiting Gram-positive bacteria and the biofilm formed thereby.

[0019] In one embodiment of the present invention, the Gram-positive bacteria include one or more of Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus.

[0020] The third object of the present invention is to provide an acne-repairing cosmetic, wherein the cosmetic comprises the acne-repairing composition.

[0021] In one embodiment of the present invention, the cosmetics include creams, lotions, essences and facial masks.

[0022] The technical solution of the present invention has the following advantages over the prior art:

[0023] (1) The acne-removing and repairing composition of the present invention is compounded with rose extract and palmyra palm extract, which significantly inhibits the biofilm formation of Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus through multi-target synergistic action, has significant activity in inhibiting Gram-positive bacteria, and exhibits excellent anti-drug resistance.

[0024] (2) The cosmetics prepared from the acne-removing and repairing composition of the present invention are natural, harmless, and environmentally friendly. They can be applied directly to the skin, are highly safe, and have significant antibacterial effects. While beautifying the skin's appearance, they also enhance the resistance of the skin's flora to drug resistance, making the skin more resistant to irritation and healthier. This provides a new strategy for developing natural plant-derived anti-drug acne treatment products and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0026] Figure 1 The acne-removing and repairing results were observed with the naked eye 14 days after administration to the auricle skin of each group of rats in Test Example 5 of the present invention;

[0027] Figure 2 The scoring results of the apparent indexes of the auricle skin of rats in each group in Test Example 5 of the present invention (n=10); wherein K is the blank group, M is the model group, JZ is the matrix group, CH is the clindamycin hydrochloride gel group, QWJH is the seven-flavor turmeric liniment group, YJH is the rose extract gel group, HLM is the palm tree extract gel group, and ZHW is the combination extract gel group; * Compared with the model group, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001; # indicates that the combination group was compared with other groups. # P<0.05, ## P<0.01, ### P<0.001, #### P < 0.0001;

[0028] Figure 3 These are the H&E staining results of the auricle tissue of each group of rats after treatment in Test Example 5 of the present invention. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0030] In the present invention, unless otherwise specified, Propionibacterium acnes ATCC 11827 (C. acnes ATCC 11827), Propionibacterium acnes ATCC 6919 (C. acnes ATCC 6919), Staphylococcus epidermidis ATCC 35984 (SEP ATCC 35984), Bacteroides fragilis ATCC 25285, and Staphylococcus aureus ATCC 29213 used in the examples of the present invention were purchased from Guangdong Provincial Microbiological Culture Collection Center.

[0031] In the present invention, unless otherwise specified, the reagent information used in the examples of the present invention is shown in Table 1:

[0032] Table 1

[0033]

[0034]

[0035] In the present invention, unless otherwise specified, the culture medium involved in Examples 1-2 and Example 3 of the present invention is as follows:

[0036] Thioglycollate fluid medium: Weigh 2.8 g of dry powder into a beaker and add 100 mL of deionized water;

[0037] Nutrient broth: weigh 2.8 g of dry powder into a beaker and add 100 mL of deionized water;

[0038] Nutrient agar medium: Accurately weigh 3.2 g of dry powder into a beaker and add 100 mL of deionized water;

[0039] Brain heart infusion broth: Accurately weigh 5.2 g of dry powder into a beaker and add 100 mL of deionized water;

[0040] The above culture medium was stirred until completely dissolved, and the pH value was adjusted to 7.3±0.1 (Examples 1-2) and 7.4±0.2 (Example 3), respectively. The culture medium was sterilized by high pressure at 121°C for 30 min and stored in a refrigerator at 4°C.

[0041] Example 1 Determination of in vitro antibacterial activity of single medication against acne pathogens

[0042] (1) Preparation of stock solution

[0043] Preparation of rose extract stock solution: Using ultrasonic microwave-assisted extraction-liquid chromatography (UAE-LC) combined preparation technology, rose (50 g) and water were placed in an ultrasonic extraction tank at a material-liquid ratio of 1:20, and ultrasonic extraction was performed at a power of 200 W and a temperature of 60°C for 20 minutes to release the active ingredients in the rose cells; then, the solution was coarsely filtered through a 200-mesh nylon filter cloth and then filtered through an organic membrane. The filtrate entered a combined preparative liquid phase system to separate, purify and enrich the flavonoid and phenolic acid compound samples, and dried to obtain a brown-red powder, namely, rose extract, recorded as sample A; an appropriate amount of rose extract was weighed, and a calculated amount of distilled water was added to dilute it in a clean bench to prepare a stock solution with a crude drug content of 100 mg / mL. The solution was sterilized by filtration using a 0.22 μm pore size filter membrane, sealed, and stored in a refrigerator at 4°C for later use.

[0044] Preparation of a stock solution of Pterocarpus chinensis extract: Using ultrasonic microwave-assisted extraction-liquid phase (UAE-LC) combined preparation technology, 50 g of Pterocarpus chinensis and an 80% alcohol solution were placed in an ultrasonic extraction tank at a material-liquid ratio of 1:20. Ultrasonic extraction was performed at a power of 200 W and a temperature of 80°C for 25 minutes to release the active ingredients in the Pterocarpus chinensis cells; the solution was then coarsely filtered through a 200-mesh nylon filter cloth and then filtered through an organic membrane. The filtrate entered a combined preparative liquid phase system to separate, purify and enrich the stilbene compound segment sample, and dried to obtain a yellow powder, i.e., the Pterocarpus chinensis extract, recorded as sample B; an appropriate amount of the Pterocarpus chinensis extract was weighed, and a calculated amount of DMSO was added to dilute the solution in a clean bench to prepare a stock solution with a crude drug content of 100 mg / mL. The solution was sterilized by filtration using a 0.22 μm pore size filter membrane, sealed, and stored in a refrigerator at 4°C for later use.

[0045] Stock solutions of Polygonum cuspidatum extract, Bletilla striata extract, Magnolia officinalis extract, Terminalia chebula extract, Clove extract, Rhubarb extract, and Alpinia officinalis extract were prepared as follows: 100 g of each medicinal material was added with 20 times the amount of water and decocted for 1.5 h. The extracts were filtered and freeze-dried to obtain the extracts. An appropriate amount of the extract was weighed and diluted with a calculated amount of distilled water in a clean bench to prepare a stock solution with a crude drug dosage of 100 mg / mL. The solution was sterilized by filtering with a 0.22 μm pore size filter membrane, and the solution was sealed and stored in a refrigerator at 4°C for later use.

[0046] Clindamycin hydrochloride stock solution: Weigh an appropriate amount of clindamycin hydrochloride and dilute it to a stock solution with a crude drug concentration of 256 mg / mL by adding the calculated amount of DMSO in a clean bench. Seal the solution and store it in a refrigerator at 4°C until use.

[0047] Seven-ingredient turmeric liniment stock solution: Weigh an appropriate amount of Seven-ingredient turmeric liniment, add the calculated amount of distilled water in a clean bench to dilute it to a stock solution with a crude drug dosage of 100 mg / mL, seal it and store it in a 4°C refrigerator until use.

[0048] (2) Strain activation and preparation of bacterial suspension

[0049] Activation of strains: In a sterile workbench, take appropriate amounts of bacterial suspension from a glycerol tube of Propionibacterium acnes and a glycerol tube of Staphylococcus epidermidis and inoculate them onto brain heart infusion broth and nutrient agar, respectively. C. acnes is cultured anaerobically in a 37°C incubator for 48-72 hours, and SEP is cultured in a 37°C incubator for 18-24 hours to obtain activated strains.

[0050] Preparation of bacterial suspension: Wash the activated strain on the culture medium with physiological saline to obtain a high concentration of bacterial suspension. Take a small amount of bacterial suspension in a test tube filled with physiological saline and compare the turbidity with the 0.5 McFarland turbidimetric tube. If the turbidity is consistent, the concentration is 1.0×10 8 CFU / mL bacterial suspension; 200 μL of the above bacterial suspension was diluted with thioglycollate fluid medium and nutrient broth medium respectively to obtain a concentration of 1.0×10 6 CFU / mL of experimental bacterial suspension;

[0051] Quality control bacteria: For Propionibacterium acnes, Bacteroides fragilis is used as the quality control strain and metronidazole is used as the quality control drug. Under parallel operation, if the MIC value of the quality control strain is within the range of 0.25μg / mL-2μg / mL, the test results are valid and reliable. For Staphylococcus epidermidis, Staphylococcus aureus is used as the quality control strain and oxacillin is used as the quality control drug. If the MIC value of the quality control strain is within the range of 0.12μg / mL-0.5μg / mL, the experimental results are true and reliable.

[0052] (3) Determination of the minimum inhibitory concentration (MIC) of drugs against acne pathogens

[0053] The minimum inhibitory concentration (MIC) of the drug against the three test bacteria was determined by the microdilution method of the M11-A7 and M07-A9 protocols developed by the Clinical and Laboratory Standards Institute (CLSI), as briefly described below:

[0054] Propionibacterium acnes drug susceptibility testing: Clindamycin hydrochloride stock solution was diluted with thioglycollate fluid medium to obtain a 1 μg / mL clindamycin hydrochloride test solution. A 6.25 mg / mL test solution of tamarisk extract was prepared by referring to the preparation of its stock solution. The remaining test solutions, including rose extract, were prepared by referring to their stock solution preparations to obtain a 50 mg / mL test solution. 100 μL of thioglycollate fluid medium was added to columns 2 through 11 of a 96-well plate, and 200 μL of the same medium was added to column 12. 200 μL of each test solution was added to column 1 and pipetted horizontally, diluting the solution twofold to column 10. 100 μL of the P. acnes test bacterial suspension was added to columns 1 through 11, bringing the final volume of each well to 200 μL. Column 11 served as a growth control well, and column 12 served as a blank control well. The concentrations of extracts from rose, knotweed, white peony root, magnolia bark, terminalia chebula, clove, red peony root, rhubarb, galangal, and seven-flavor turmeric liniment ranged from 50 mg / mL to 0.0976 mg / mL; the concentration of palmatum extract ranged from 6.25 mg / mL to 0.0122 mg / mL; and the concentration of clindamycin hydrochloride ranged from 0.5 mg / mL to 0.0010 μg / mL. The cells were incubated anaerobically at 37°C for 48 to 72 hours in a constant-temperature incubator and the results were observed. The MIC (MIC) of the wells where no bacterial growth was observed was determined by visual inspection, compared with the growth control wells (x ± SD, n = 3).

[0055] Staphylococcus epidermidis drug susceptibility test: Clindamycin hydrochloride stock solution was diluted with nutrient broth to obtain a clindamycin hydrochloride test solution with a concentration of 8 mg / mL. The remaining test solutions were prepared using the same experimental method as above, except that the SEP culture medium was nutrient broth and incubated at 37°C for 18-24 hours. The concentration of clindamycin hydrochloride ranged from 4 mg / mL to 0.0078 mg / mL, and the remaining drug concentrations were the same as above.

[0056] The specific test results are shown in Table 2:

[0057] Table 2

[0058] Drugs / strains C. acnes ATCC 11827 C. acnes ATCC 6919 SEP ATCC 35984 Rose extract (mg / mL) 1.56±0.36 1.56±0.36 3.12±0.00 Palmatum truncatum extract (mg / mL) 0.049±0.00 0.049±0.00 0.391±0.00 Polygonum cuspidatum extract (mg / mL) 3.125±0.00 4.167±1.80 12.5±0.00 Bletilla striata extract (mg / mL) 6.25±0.00 5.21±1.80 10.42±3.61 Magnolia bark extract (mg / mL) 25±0.00 33.33±14.43 41.67±14.43 Terminalia chebula extract (mg / mL) 3.125±0.00 3.125±0.00 10.42±3.61 Clove extract (mg / mL) 6.25±0.00 6.25±0.00 8.33±3.61 Red Peony Root Extract (mg / mL) 6.25±0.00 8.33±3.61 20.83±7.22 Rhubarb extract (mg / mL) 1.5625±0.00 2.125±0.97 4.17±1.80 Galangal extract (mg / mL) 12.5±0.00 12.5±0.00 25±0.00 Clindamycin hydrochloride (μg / mL) 0.0078±0.00 0.0078±0.00 250.00±0.00 Seven-ingredient turmeric liniment (mg / mL) 12.50±0.00 12.50±0.00 25±0.00

[0059] As shown in Table 2, the MICs for inhibition of Propionibacterium acnes were as follows: P. acnes extract < Chinese rose extract / rhubarb extract < Terminalia chebula extract / Polygonum cuspidatum extract < Ampelopsis radiata extract / Clove extract / Red peony root extract < Alpinia officinalis extract < Magnolia officinalis extract. Chinese rose extract exhibited significant antibacterial activity against both C. acnes and SEP. Compared to the positive drug, Seven-ingredient Curcuma Liniment, the MICs for P. acnes extract, Chinese rose extract, rhubarb extract, Terminalia chebula extract, Polygonum cuspidatum extract, Ampelopsis radiata extract, Clove extract, and Red peony root extract against C. acnes and SEP were significantly lower than those of Seven-ingredient Curcuma Liniment (P < 0.0001). Due to the stability and safety of Rhubarb extract, it is not currently considered as a target plant for later product development. In clinical practice, combining multiple drugs is recommended, and drugs should be selected to exhibit potentiative or additive effects without antagonism.

[0060] Therefore, rose extract, palmyra palm extract, terminalia chebula extract, and knotweed extract were selected as the objects of further study and combined use was conducted to see if there would be synergistic effects.

[0061] Example 2 Effect of combined drug use on drug sensitivity of Propionibacterium acnes and Staphylococcus epidermidis

[0062] The MIC of the test bacteria was determined using the microdilution method of the M11-A7 and M07-A9 protocols developed by the Clinical and Laboratory Standards Institute (CLSI). The antibacterial effects of the extracts were investigated and their fractional inhibitory concentration index (FICI) values were calculated. The following is a brief description:

[0063] According to the MIC results of Example 1, the stock solution in Example 1 was diluted with culture medium to obtain extract solutions with concentrations of 4×MIC-1 / 8×MIC.

[0064] Propionibacterium acnes drug sensitivity test: Add 50 μL of the corresponding bacterial culture medium to rows AG and columns 2-10 of a 96-well plate. Take the above 4×MIC rose extract solution and add 100 μL to columns A1-G1 respectively, and dilute it horizontally by two times to the 6th column. At this time, the volume of each well is 50 μL; add 50 μL of a series of concentrations of palm tree extract solution to rows AG (the final concentration range of rose extract, palm tree extract, terminalia chebula extract and knotweed extract is 1×MIC-1 / 16×MIC). Set up growth control wells and blank control wells. Except for the blank control wells with culture medium, 100 μL of experimental bacterial suspension is added to the rest, and the final volume of all wells is 200 μL. Place in a 37°C incubator for anaerobic culture for 48h-72h; compared with the growth control wells, the microwells with no bacterial growth observed with the naked eye are their MICs. 联合 (x±SD, n=3), calculate FICI: FICI=MIC A药联合 / MIC A药单用 +MIC B药联合 / MIC B药单用 . The judgment basis for the combined drug sensitivity test is FICI: FICI ≤ 0.5 indicates synergistic effect; 0.5 < FICI ≤ 1 indicates additive effect; 1 < FICI ≤ 2 indicates no effect; FICI > 2 indicates antagonistic effect.

[0065] Drug sensitivity test of Staphylococcus epidermidis: Dilute the stock solution of clindamycin hydrochloride with nutrient broth medium to obtain the test solution of clindamycin hydrochloride with a concentration of 8 mg / mL, and prepare the other test solutions; the experimental method is the same as above. Among them, the culture medium for SEP is nutrient broth medium, and it is incubated at 37°C for 18 h - 24 h; the drug concentration is the same as above.

[0066] The specific test results are shown in Table 3:

[0067] Table 3

[0068]

[0069]

[0070] As can be seen from Table 3, after the combination of each pair of drugs, they all have a certain effect on the 3 strains of bacteria. After the combined use of the extract of Rosa chinensis and the extract of Ormosia henryi, the FICI values for C. acnes ATCC 11827, C. acnes ATCC 6919, and SEP ATCC 35984 are all less than 0.5, indicating a synergistic antibacterial effect, which can enhance the inhibitory effect of the drugs on Propionibacterium acnes and Staphylococcus epidermidis. For C. acnes ATCC 11827, C. acnes ATCC 6919, and SEP ATCC 35984, the combined MIC concentrations of the two extracts can reach 1 / 4 of the single MIC concentration respectively, achieving the effect of reducing the dosage and increasing the efficacy. Other combinations mainly show additive or no effects.

[0071] Therefore, study the effect of the combined use of the extract of Rosa chinensis and the extract of Ormosia henryi on biofilms, and preliminarily explore the anti-drug resistance mechanism after their combination.

[0072] Example 3 Determination of the minimum biofilm inhibitory concentration (MBIC) of Propionibacterium acnes and Staphylococcus epidermidis by single drug use

[0073] Drug preparation: The preparation method is the same as that of Example 1, except that the concentration of the rose extract is 50 mg / mL-0.0976 mg / mL, the concentration of the palmyra palm extract is 6.25 mg / mL-0.0244 mg / mL, the concentration of clindamycin hydrochloride is 0.5 mg / mL-0.0010 μg / mL, and the concentration of the seven-flavor turmeric liniment is 50 mg / mL-0.0976 mg / mL.

[0074] Activation of strains: Take an appropriate amount of bacterial liquid from the glycerol tube of Propionibacterium acnes and the glycerol tube of Staphylococcus epidermidis and inoculate it into brain heart infusion broth culture medium and nutrient agar culture medium respectively. The inoculated culture medium is placed in an anaerobic culture device. C. acnes is anaerobically cultured in a 37°C constant temperature incubator for 48h-72h, and SEP is cultured in a 37°C constant temperature incubator for 18h-24h. Then, a single colony is selected and transferred to 50mL of thioglycollate fluid culture medium. It is shaken and cultured at 37°C for 48h-72h to obtain the activated strain.

[0075] Preparation of bacterial suspension: Wash the activated strain on the culture medium with physiological saline to obtain a high concentration of bacterial suspension. Take a small amount of bacterial suspension in a test tube filled with physiological saline and compare the turbidity with the 0.5 McFarland turbidimetric tube. If the turbidity is consistent, the concentration of 1.0×10 8 CFU / mL bacterial suspension. Take 200 μL of the above bacterial suspension and dilute it with thioglycollate fluid medium and nutrient broth medium respectively to obtain a concentration of 1.0×10 6 CFU / mL of experimental bacterial suspension.

[0076] Biofilm formation: 200 μL of the above-prepared Propionibacterium acnes suspension and Staphylococcus epidermidis suspension were inoculated into a 96-well plate, respectively. P. acnes was cultured anaerobically at 37°C for 144-168 h, and S. epidermidis for 48-72 h until biofilms formed. The wells were rinsed three times with sterile PBS to remove the culture medium and floating bacteria, and the wells were air-dried for later use. 200 μL of each diluted solution was added to a 96-well plate, with the 11th column as a growth control well and the 12th column as a blank control well. P. acnes was cultured anaerobically in a 37°C incubator for 72 h, and S. epidermidis was cultured in a 37°C incubator for 24 h. The results were observed, and the wells in which no bacterial growth was observed by the naked eye were designated as the MBIC (x±SD, n=3).

[0077] The specific test results are shown in Table 4:

[0078] Table 4

[0079]

[0080] Example 4 Determination of the Minimum Inhibitory Concentration (MBIC) of Combination Drugs for Propionibacterium acnes and Staphylococcus epidermidis

[0081] 200 μL of the Propionibacterium acnes suspension and Staphylococcus epidermidis suspension prepared above were respectively inoculated into a 96-well plate, and anaerobically cultured at a constant temperature of 37°C for 120 h and 72 h to form a biofilm. The culture medium and planktonic bacteria in each well were removed by rinsing three times with sterile PBS; the culture medium and planktonic bacteria in each well were removed by rinsing three times with sterile PBS, and the wells were dried and set aside.

[0082] According to the MBIC values determined in Example 3, the prepared stock solutions of the rose extract and the palmyra palm extract were diluted with brain heart infusion broth to a concentration of 4×MBIC-1 / 8×MBIC.

[0083] 100 μL of each concentration of rose extract was added horizontally from the 1st to the 6th column of the 96-well plate of the biofilm; 100 μL of the series of concentrations of palm tree extract were added vertically from rows A to G (the final concentration range of rose extract and palm tree extract was 1×MIC-1 / 16×MIC); the plate was placed in a 37°C incubator and continued to be anaerobic incubated for 72 h and 24 h; the results were observed, and the microwells with no bacterial growth observed by the naked eye were considered MBICs. 联合 (x±SD, n=3).

[0084] The specific test results are shown in Table 5:

[0085] Table 5

[0086]

[0087] As shown in Tables 4 and 5, the combined use of the two agents demonstrated FICI values of 0.5 < 0.916 < 1 for Propionibacterium acnes and 0.5 < 0.839 < 1 for Staphylococcus aureus, respectively, demonstrating a relatively good additive inhibitory effect on the biofilm growth of acne-related bacteria. This suggests that combined use can effectively inhibit the metabolic activity of bacteria within biofilms, inhibit or eliminate bacterial extracellular matrix secretion, reduce the adhesion of test strains to each other and to the surface of the medium, and disrupt the three-dimensional structure of the biofilm. It can also delay the growth of acne-causing bacteria C. acnes and SEP. By inhibiting their extracellular matrix secretion, reducing interbacterial adhesion, and disrupting the biofilm structure, the two agents can inhibit the biofilm growth of acne-causing bacteria, thereby combating drug resistance, reducing drug dosage, and improving drug efficiency.

[0088] Example 5 Effect of the composition on rat acne model

[0089] Experimental animals: 80 SPF-grade Sprague-Dawley rats, half male and half female, aged 4-6 weeks, weighing (200 ± 20) g, were purchased from the Guangdong Medical Laboratory Animal Center. They were housed under a 12-h light / 12-h dark cycle, room temperature of 22°C-24°C, and relative humidity of 50%-60%. The rats were fed normal food and water for one week before the experiment.

[0090] Preparation of gel: Phase A (2g Aristoflex AVC, 5g glycerin, and an appropriate amount of pure water) is mixed and heated to 80°C to fully dissolve and stir; Phase B (1g SIL 034, 0.75g SIMULGEL EG) and Phase C (2g penetration enhancer DMI, 3g extract powder (rose extract, palm tree extract, and combination extract, respectively), 0.6g K350 preservative) are heated to 60°C and 45°C, respectively, and stirred to dissolve completely; Phase B is added to Phase A for emulsification, cooled to 45°C, and then Phase C is added and stirred evenly. Finally, pure water is added to make the total amount of gel 100g, and stirred to room temperature to form the extract gel; the preparation of the base cream is the same as the extract gel, except that the extract powder is not added.

[0091] Preparation of Propionibacterium acnes suspension: Take an appropriate amount of bacterial suspension from the C. acnes ATCC 6919 glycerol tube and inoculate it onto the surface of brain heart infusion broth. Place the inoculated culture medium in an anaerobic incubator at 37°C for 48-72 hours. After anaerobically incubating at 37°C for 48-72 hours, select a single colony and transfer it to 50 mL of thioglycollate fluid medium. Incubate at 37°C with shaking for 48-72 hours. After shaking, adjust the bacterial suspension concentration to 1.5×10 8 CFU / mL, and obtain Propionibacterium acnes suspension.

[0092] Animal Grouping and Model Establishment: 80 rats were randomly divided into 8 groups (10 rats each): blank group (A), model group (B), matrix group (C), clindamycin hydrochloride gel group (D), seven-flavor turmeric liniment group (E), rose flower extract gel group (F), palm tree extract gel group (G), and combination extract gel group (H). Except for the blank group, the remaining 7 groups were used to establish a rat auricular acne model: For the model group, 100% oleic acid was applied to the opening of the auricular duct of the right ear of the rat using a cotton swab, 0.5 mL / time, once daily. Every other day, 50 μL of Propionibacterium acnes bacterial solution was injected into the modeling site for 2 weeks.

[0093] Grouped medication: After the rat auricle acne model was successfully established, topical medication was started. The remaining 70 rats in the modeling group were weighed again and numbered and grouped. Each group was given medication on the rat auricle modeling site for 15 consecutive days, with 0.5g of medication applied to the entire ear each time, twice a day.

[0094] Experimental animal sampling: After 15 days of drug intervention, all rats were fasted but not watered for 24 h and anesthetized with 10% chloral hydrate injected intraperitoneally. After the rats entered the anesthetized state, about 5 mL of abdominal aortic blood was quickly collected, centrifuged, and frozen in a -20°C refrigerator. The hair around the back of the rat's ear was shaved with an electric shaver, and the right auricle tissue was cut off and repeatedly rinsed with PBS. Part of the auricle tissue was fixed with 4% paraformaldehyde for histopathological examination. The remaining auricle tissue was placed in a cryovial and quickly frozen in liquid nitrogen. After sampling, it was transferred to a -80°C refrigerator for storage.

[0095] Morphological observation: On the 3rd, 7th and 14th days of treatment, the appearance and morphology of the right auricle of each group of rats were systematically observed and evaluated at the same time period before medication. The main observation indicators included the swelling degree of auricle tissue, skin color change, epidermal keratinization and the formation of papules and pustules. After 14 days of topical treatment, the morphology of the auricle skin lesions of the rats was observed with the naked eye. The results are as follows Figure 1 As shown. Figure 1 The auricles of the rats in the blank group appeared pale pink, smooth, thin, and slightly transparent, with clear capillaries, no swelling, and a soft touch. In the model group, the auricles appeared red, rough, and swollen. The capillaries in the upper dermis at the injection site were significantly dilated, and the auricles felt noticeably harder and thicker. Compared with the model group, the auricles of the rats in the clindamycin hydrochloride gel, seven-flavor turmeric liniment, rose flower extract gel, palm tree extract gel, and combined extract gel groups showed varying degrees of relief from redness, swelling, roughness, thickening, and nodules and pustules. The auricles also felt softer and thinner. Compared with the clindamycin hydrochloride gel group, the ear tubes in each extract gel group were lighter in color, the auricle surface roughness and thickness were reduced, no obvious keratinization was observed, and the nodules and pustules were significantly smaller and fewer. In contrast, some rats in the clindamycin hydrochloride gel group experienced redness and swelling within the auricles, with a few scattered pustules. Compared with the seven-ingredient turmeric liniment, there was no significant difference in the swelling degree of the right auricle, skin color, epidermal keratinization and papule-pustule appearance of rats in the combination extract gel group.

[0096] The following changes were observed in the auricles of the rats in each treatment group after receiving the different drugs: On the third day of treatment, the auricles were generally red, with reduced hyperplasia and hypertrophy, accompanied by mild to moderate swelling. Compared to pre-treatment, the number of crusts and scales on the auricle surface decreased significantly, but the morphology of the papules and pustules remained unchanged. By the seventh day, the auricle margins had softened. While some redness and swelling remained within the auricle, the keratinization at the follicle openings had decreased. Furthermore, minor desquamation occurred at the ear canal, while the papules and pustules decreased in diameter. After 14 days of treatment, the auricles of the rats in the Seven Turmeric Liniment and various extract gel groups became softer. The color of the external auricles was not significantly different from that of the control ears. While the ear canal was slightly reddish, no obvious keratinization was observed. Furthermore, the number of nodules and pustules in these groups also decreased significantly. In contrast, the auricles of some rats in the clindamycin hydrochloride gel group were still red and swollen, with a few pustules scattered around; while the auricles of rats in the rose extract gel group were still filled with a small amount of keratinized material, and the pustules of some rats were still protruding.

[0097] Observation of auricle appearance indicators: The appearance and morphology of the right auricle of each group of rats were evaluated. The evaluation indicators and scoring system of this experiment were formulated with reference to the "Guidelines for the Treatment of Acne in China (2019 Revised Edition)" and the "Acne Animal Model Preparation Standards (Draft)", as shown in Table 6:

[0098] Table 6

[0099]

[0100] The changes in swelling, color, keratinization, and papules and pustules of the rat auricle at different treatment time points were observed and recorded, and the scores were compared according to Table 6. The specific scoring results are as follows: Figure 2 As shown. Figure 2 It can be seen that the auricle scores of the model group rats did not show significant changes during the treatment period (P>0.05). In each treatment group, the auricle skin lesions were observed to improve to varying degrees at different time points. On the third day of treatment, the skin lesions in the seven-flavor turmeric liniment group and the composite extract gel group were improved compared with the model group (P<0.05); on the seventh day of treatment, the skin lesions in each treatment group improved significantly, with a significant difference compared with the model group (P<0.001); on the 14th day of treatment, the skin lesion scores of the rats in each treatment group were significantly reduced compared with the model group (P<0.0001). The composite extract gel group had significantly better improvement in skin lesions than the clindamycin hydrochloride gel group, the palm tree extract gel group, and the rose extract gel group (P<0.05, P<0.0001). Compared with the seven-flavor turmeric liniment group, the composite extract gel group showed comparable therapeutic effects, with no statistically significant difference (P>0.05).

[0101] Pathological observation of auricle tissue: After the auricle tissue was fixed in 4% paraformaldehyde universal tissue fixative for 24 hours, it was taken out and placed in a dehydration box, and then went through a series of processing steps: tissue dehydration, paraffin embedding, sectioning, dewaxing and hematoxylin and eosin staining. Finally, the auricle tissue was dehydrated and mounted. The H&E staining results of the auricle tissue were observed under a ×100 microscope. The results are as follows Figure 3 As shown. Figure 3 As can be seen, the auricle structure of the rats in the blank group was clear, the stratum corneum was thin, the hair follicles were normal in size, and the contents were small. No significant inflammatory cell infiltration or capillary dilation was observed in the subcutaneous region. In addition, there was no proliferation of the hair follicle walls, and the sebaceous gland volume remained normal. The collagen fibers in the dermis were stretched, spindle-shaped, and arranged in an orderly manner, indicating good tissue structural integrity (A). Compared with the blank group, the auricle structure of the rats in the model and matrix groups was significantly damaged. The epidermis showed hyperkeratosis, with significantly dilated hair follicle openings and a large accumulation of keratin. The spinous layer was significantly thickened, with a large number of inflammatory cells and capillary dilation in the dermis. In addition, the collagen fibers in the dermis were significantly thickened and arranged in a disordered and interwoven manner (B). Compared with the model group: the auricle structure and tissue of the clindamycin hydrochloride gel group, seven-flavor turmeric liniment group, rose flower extract gel group, palmyra palm extract gel group, and combination extract gel group (D, E, G, H) basically returned to normal, epidermal keratinization was alleviated to varying degrees, the spinous layer tissue became thinner, and the inflammatory cell infiltration and capillary dilation in the dermis were significantly improved, but some hair follicle openings still had slight dilation; in the matrix group and rose flower extract group (C, F), there was still some keratin accumulation in the infundibulum of the hair follicles, the spinous layer tissue thickness became thinner, the inflammatory cell infiltration in the dermis was reduced compared with the model group, and the capillary dilation was alleviated.

[0102] Compared with the clindamycin hydrochloride gel group, the Rosa Chinensis extract, Palmaria officinalis extract, and combined extract gel groups showed reduced epidermal spinous layer thickness and decreased inflammatory cell infiltration in the dermis. Compared with the Seven-flavor Curcuma Liniment group, the combined extract gel group showed reduced epidermal spinous layer thickness and decreased inflammatory cell infiltration in the dermis. This suggests that the combined extract gel group is effective in repairing auricular tissue structure and alleviating inflammatory responses. This is because the mechanism of P. acnes in acne inflammation involves: (1) the formation of biofilms can enhance intercellular adhesion and thus promote the formation of comedones; (2) the production of lipase, protease, hyaluronidase and neutrophil chemotactic factor, leading to skin tissue damage; (3) P. acnes promotes the release of inflammatory factors, including TNF-α, IL-8 and IL-1β, by activating the TLR2 pathway of tissue cells; (4) after P. acnes infection, host cells produce antimicrobial peptides and antimicrobial lipopeptides, which synergistically release proinflammatory cytokines through TLR4 and CD14-dependent pathways; (5) P. acnes induces keratinocytes to express matrix metalloproteinase-9, which causes the rupture of hair follicles and sebaceous glands, thereby exacerbating inflammation.

[0103] In summary, the experimental results show that the acne-repairing composition of the present invention can effectively enhance antibacterial permeability, destroy the biofilm barrier, promote the deep action of antibacterial agents, destroy the formation of biofilm, reduce the colonization of Propionibacterium acnes in the skin, and further reduce the production of pro-inflammatory factors, thereby repairing acne.

[0104] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An acne-removing and repairing composition, characterized in that: The acne-repairing composition comprises rose extract and palm tree extract.

2. The acne-removing and repairing composition according to claim 1, characterized in that: The concentration of the rose extract in the acne-repairing composition is 0.0001 mg / mL-100 mg / mL, and the concentration of the palmyra palm extract is 0.001 μg / mL-50000 μg / mL.

3. The acne-removing and repairing composition according to claim 1, characterized in that: The acne-removing and repairing composition further includes a pharmaceutically acceptable carrier.

4. The acne-removing and repairing composition according to claim 1, characterized in that: The functions of the acne-repairing composition include: destroying the biofilm barrier and promoting the penetration of antibacterial agents.

5. Use of the acne-repairing composition according to any one of claims 1 to 4 in inhibiting Gram-positive bacteria and biofilms formed thereby.

6. The use according to claim 5, characterized in that The Gram-positive bacteria include one or more of Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus.

7. An acne-repairing cosmetic, characterized in that: The cosmetic comprises the acne-repairing composition according to any one of claims 1 to 4.

8. The acne-repairing cosmetic according to claim 7, characterized in that: The cosmetics include creams, lotions, essences and facial masks.

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