Acne removal composition capable of acting on biological membrane as well as preparation method and application of acne removal composition

Abstract

The invention relates to the technical field of skin care products, in particular to an acne removal composition capable of acting on a biological membrane as well as a preparation method and application of the acne removal composition. The caesalpinia spinosa gum and the compound fermentation liquor are compounded according to a certain proportion, so that the synergistic interaction effect can be achieved, on one hand, the effect of inhibiting the formation of a propionibacterium acnes biological membrane can be better, and on the other hand, the generated propionibacterium acnes biological membrane can be disintegrated; on the other hand, the effects of removing acnes, relieving and controlling oil can be achieved; preferably, lactobacillus plantarum, lactobacillus acidophilus and lactobacillus reuteri are specifically selected for compound fermentation, so that the activity of the obtained composition is better, and the effect of inhibiting repeated acnes is more remarkable; according to the invention, reasonable active ingredient matching and a special preparation process are adopted, the effect of comprehensively inhibiting acne recurrence can be achieved through combined action of disintegration of the biological membrane dimension of generated propionibacterium acnes, relieving and oil control and adjustment of skin micro-ecological balance dimension, and the effect is more remarkable.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care products, and in particular to an anti-acne composition capable of acting on a biofilm, and a preparation method and application thereof. Background Art

[0002] Acne is one of the most common diseases in dermatology. It is a chronic inflammatory disease of the hair follicles and sebaceous glands. The main clinical manifestations include acne, papules, pustules, cysts, etc., and sebum secretion can usually be observed. It is most common on the faces of adolescent boys and girls, so it is also called "acne". In real life, many people's acne is often not completely cured. It will grow again and again, and even leave more acne marks and acne pits, which are difficult to eliminate, seriously affecting people's appearance, and personal self-confidence and social life will also be greatly affected.

[0003] Acne is a chronic inflammatory skin disease that occurs frequently in areas with high density of hair follicles and sebaceous glands. Its onset is related to a variety of factors, among which the massive reproduction of Propionibacterium acnes is closely related to the occurrence of acne. Systemic or topical application of antimicrobial drugs to inhibit Propionibacterium acnes is an important means of treating acne. In recent years, more and more studies have been conducted on the formation of bacterial biofilms leading to antimicrobial resistance. Bacterial biofilms are a kind of microbial aggregation community that forms and attaches to the surface of organisms or non-organisms and is surrounded by extracellular polymeric substances. The resistance of bacteria in biofilms to antimicrobial drugs is 1000 times that of planktonic bacteria. Bacteria can form mature biofilms 24 hours after attaching to the surface of organisms or the interface of non-organisms. During the formation of acne, microorganisms such as Propionibacterium acnes can form biofilms in the hair follicles and sebaceous glands. This biofilm can enhance the resistance of microorganisms to antibiotics and the host immune system. It provides a protective environment for microorganisms, making them more difficult to be removed, resulting in persistent or recurring inflammation, which makes acne prone to recurrence.

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

[0005] In view of the above problems, the object of the present invention is to provide an acne-removing composition that can act on biofilms, and a preparation method and application thereof.

[0006] The technical contents of the present invention are as follows:

[0007] On one hand, the present invention provides an anti-acne composition that can act on biofilm and a preparation method thereof. The composition comprises taro gum and composite fermentation liquid.

[0008] In a specific embodiment of the present invention, the composite fermentation broth is obtained by using red pine leaf extract and lotus seed extract as raw materials, through composite bacteria anaerobic fermentation for 2-5 days, sterilization, and centrifugal 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 leaves, mix with pure water in a ratio of 1:1, and grind to obtain slurry 1;

[0011] Step 2. Add 0.5-1% cellulase to the slurry 1 obtained in step 1, perform enzymolysis at 40-50° C. for 1-2 hours, inactivate in a water bath at 80-90° C. for 8-15 minutes, cool to room temperature, and centrifuge to obtain a residue and an enzymolysis solution;

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

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

[0014] Step 4. Evenly mix the enzymatic hydrolysate obtained in step 2 and the hydroalcoholic extract obtained in step 3 to obtain the red pine leaf extract.

[0015] In a specific embodiment of the present invention, the weight ratio of the Pinus densiflora leaf extract to the Lotus japonica seed extract is 3-5:1-2.

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

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

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

[0019] In a specific embodiment of the present invention, the acne-removing composition that can act on biofilms also includes solvent A and solvent B, wherein solvent A includes water and butanediol in a mass ratio of 5:1, and solvent B includes 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 anti-acne composition is as follows:

[0021] The thorny taro gum is dissolved in solvent A to obtain mixture 1, the composite fermentation liquid is dissolved in solvent B to obtain mixture 2, and the mixture 1 and the mixture 2 are evenly mixed to obtain the acne-removing composition.

[0022] In a specific embodiment of the present invention, the ratio of the taro gum to the 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 the solvent B is 45-70:2-5.

[0024] Another aspect of the present invention provides use of any one of the above acne-removing compositions in an essence.

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

[0026] In the present invention:

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

[0028] Lotus japonicus seed extract: Lotus japonicus is a plant of the genus Lotus japonicus in the leguminous family, also known as five-leaf clover, ox-horn flower, grass, bird's spur, etc. In my country, Lotus japonicus mainly grows in moist and weakly alkaline hillsides, fields, grasslands or riverbanks in the northwest, southwest, and the middle and upper reaches of the Yangtze River. Lotus japonicus is a

[0029] Traditional Chinese herbal medicine has the effects of clearing away heat and dampness, relieving cough and detoxifying, promoting blood circulation and removing blood stasis. Analysis of its ingredients revealed that it contains a variety of phytochemicals, including alkaloids, steroids, flavonoids, phenolic glycosides, etc., and the extract of Lotus japonicus was further studied and found to have an inhibitory effect on Gram-positive and Gram-negative bacteria such as Propionibacterium acnes, Staphylococcus aureus, Escherichia coli, and Salmonella typhi.

[0030] The gum of the spiny taro has a unique 3D colloidal structure. The polysaccharides in the 3D colloid will competitively attach to the microbial lectins, preventing the glycoproteins in the skin from combining with pathogenic bacteria, thereby avoiding the aggregation of colonies and skin inflammation.

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

[0032] (1) The acne-removing composition provided by the present invention can achieve a synergistic effect by compounding taro gum and composite fermentation liquid in a certain ratio. On the one hand, it can inhibit the formation of Propionibacterium acnes biofilm better, and at the same time, it can also disintegrate the biofilm of Propionibacterium acnes that has been formed; on the other hand, it can also achieve the effects of acne removal, soothing, and oil control.

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

[0034] (3) The present invention adopts a reasonable combination of active ingredients and a special preparation process to inhibit the formation of Propionibacterium acnes biofilm. It works together from the dimensions of disintegrating the biofilm of Propionibacterium acnes that has already been formed, soothing and controlling oil, and regulating the balance of skin microecology, which can achieve the effect of comprehensively inhibiting the recurrence of acne, and the effect is more significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a scanning electron microscope image of the essence of the present invention disintegrating biofilm;

[0036] Figure 2 Alpha diversity analysis chart (AAce index, B Chaos index, C Sobs index, D Coverage index);

[0037] Figure 3 is the species Venn diagram;

[0038] Figure 4 This is 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 DESCRIPTION

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

[0041] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0042] Example 1

[0043] The preparation of an anti-acne composition that can act on a biofilm comprises the following steps:

[0044] S1: Dissolve 0.2 parts of taro gum in 5 parts of deionized water and 1 part of butanediol to obtain a mixture of 1

[0045] S2: 60 parts of Pinus densiflora leaf extract and 20 parts of Lotus japonicus seed extract were mixed evenly to obtain a composite extract.

[0046] S3: Add 0.8×10 5 cfu / mL, fermented at a constant temperature of 30°C for 3 days, sterilized, and centrifuged to obtain a composite fermentation liquid. The composite bacteria are Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri in a live cell count ratio of 1:1:1.

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

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

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

[0050] 1) Wash and dry red pine leaves, mix with pure water in a ratio of 1:1 and grind to obtain slurry 1;

[0051] 2) adding 0.6% cellulase to the slurry 1 obtained in step 1, performing enzymolysis at 45° C. for 1.5 h, inactivating in a water bath at 80° C. for 10 min, cooling to room temperature, and centrifuging to obtain a residue and an enzymolysis solution;

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

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

[0054] 4) The enzymatic hydrolyzate obtained in step 2 and the hydroalcoholic extract obtained in step 3 are mixed evenly to obtain the red pine leaf extract.

[0055] The Lotus japonicus seed extract was purchased from Shanghai Jiakai Biotechnology Co., Ltd.

[0056] The Lactobacillus plantarum was purchased from Beijing Biobo Biotechnology Co., Ltd.

[0057] The Lactobacillus acidophilus was purchased from Beijing Biobo Biotechnology Co., Ltd.

[0058] The Lactobacillus reuteri was purchased from Beijing Biobo Biotechnology Co., Ltd.

[0059] This embodiment provides an essence, comprising the following components in percentage by mass: 0.1% allantoin, 0.02% EDTA-2 sodium, 2% glycerol, 3% 1,3-propylene glycol, 0.15% carbomer, 0.15% arginine, 0.5% 1,2-hexanediol, 0.5% p-hydroxyphenylethanol, 2% anti-acne composition, and the balance water;

[0060] The preparation method of the essence is as follows: allantoin, EDTA-2 sodium, glycerol, 1,3-propylene glycol, carbomer and water are mixed evenly according to proportion, the obtained mixture is kept at 85° C. for 20 minutes, when the temperature is lowered to 70° C., arginine, 1,2-ethylene glycol and p-hydroxyphenylethanol are added, and the mixture is stirred evenly, when the temperature is lowered to 45° C., the acne-removing composition is added, and the mixture is stirred evenly to obtain the essence.

[0061] Example 2

[0062] The preparation of an anti-acne composition that can act on a biofilm comprises the following steps:

[0063] S1: Dissolve 0.3 parts of taro gum in 6.25 parts of deionized water and 1.25 parts of butanediol to obtain a mixture of 1

[0064] S2: Evenly mix 50 parts of Pinus densiflora leaf extract and 10 parts of Lotus japonicus seed extract to obtain a composite extract.

[0065] S3: Add 1.2×10 5 cfu / mL, fermented at a constant temperature of 33°C for 2 days, sterilized, and centrifuged to obtain a composite fermentation liquid. The composite bacteria are Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri in a live cell count ratio of 1:1:1.

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

[0067] S5: Mixing 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 red pine leaves, mix with pure water in a ratio of 1:1 and grind to obtain slurry 1;

[0070] 2) adding 0.7% cellulase to the slurry 1 obtained in step 1, performing enzymolysis at 40° C. for 2 h, inactivating in a water bath at 85° C. for 8 min, cooling to room temperature, and centrifuging to obtain a residue and an enzymolysis solution;

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

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

[0073] 4) The enzymatic hydrolysate obtained in step 2 and the hydroalcoholic extract obtained in step 3 are mixed evenly to obtain the red pine leaf extract.

[0074] The Lotus japonicus seed extract was purchased from Shanghai Jiakai Biotechnology Co., Ltd.

[0075] The Lactobacillus plantarum was purchased from Beijing Biobo Biotechnology Co., Ltd.

[0076] The Lactobacillus acidophilus was purchased from Beijing Biobo Biotechnology Co., Ltd.

[0077] The Lactobacillus reuteri was purchased from Beijing Biobo Biotechnology Co., Ltd.

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

[0079] Example 3

[0080] The preparation of an anti-acne composition that can act on a biofilm comprises the following steps:

[0081] S1: Dissolve 0.5 parts of taro gum in 10 parts of deionized water and 2 parts of butanediol to obtain a mixture of 1

[0082] S2: 65 parts of Pinus densiflora leaf extract and 18 parts of Lotus japonicus seed extract were mixed evenly to obtain a composite extract.

[0083] S3: Add 1.1×10 5 cfu / mL, fermented at a constant temperature of 35°C for 4 days, sterilized, and centrifuged to obtain a composite fermentation liquid. The composite bacteria are Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri in a live cell count ratio of 1:1:1.

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

[0085] S5: Mixing 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 red pine leaves, mix with pure water in a ratio of 1:1 and grind to obtain slurry 1;

[0088] 2) adding 0.8% cellulase to the slurry 1 obtained in step 1, performing enzymolysis at 40° C. for 1 h, inactivating in a water bath at 88° C. for 5 min, cooling to room temperature, and centrifuging to obtain a residue and an enzymolysis solution;

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

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

[0091] 4) The enzymatic hydrolyzate obtained in step 2 and the hydroalcoholic extract obtained in step 3 are mixed evenly to obtain the red pine leaf extract.

[0092] The Lotus japonicus seed extract was purchased from Shanghai Jiakai Biotechnology Co., Ltd.

[0093] The Lactobacillus plantarum was purchased from Beijing Biobo Biotechnology Co., Ltd.

[0094] The Lactobacillus acidophilus was purchased from Beijing Biobo Biotechnology Co., Ltd.

[0095] The Lactobacillus reuteri was purchased from Beijing Biobo Biotechnology Co., Ltd.

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

[0097] Comparative Example 1

[0098] An anti-acne composition capable of acting on a biofilm and a preparation method thereof, the difference from Example 3 being that the composition does not contain taro gum

[0099] The anti-acne composition of this comparative example was used to prepare the essence of this comparative example according to the composition and preparation method of the essence of Example 1.

[0100] Comparative Example 2

[0101] An anti-acne composition capable of acting on biofilm and a preparation method thereof, the difference from Example 3 being that the red pine leaf extract in the composite extract is only a hydroalcoholic extract

[0102] The anti-acne composition of this comparative example was used to prepare the essence of this comparative example according to the composition and preparation method of the essence of Example 1.

[0103] Comparative Example 3

[0104] An anti-acne composition capable of acting on biofilm and a preparation method thereof, the difference from Example 3 is that the composite extract is a single red pine leaf extract

[0105] The anti-acne composition of this comparative example was used to prepare the essence of this comparative example according to the composition and preparation method of the essence of Example 1.

[0106] Comparative Example 4

[0107] An anti-acne composition capable of acting on biofilm and a preparation method thereof, the difference from Example 3 being that the composite extract is a single Lotus japonicus seed extract

[0108] The anti-acne composition of this comparative example was used to prepare the essence of this comparative example according to the composition and preparation method of the essence of Example 1.

[0109] Comparative Example 5

[0110] An anti-acne composition capable of acting on biofilm and a preparation method thereof, compared with Example 3, the only difference is that the composite extract is red pine leaf extract and fucus vesiculosus extract

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

[0112] The anti-acne composition of this comparative example was used to prepare the essence of this comparative example according to the composition and preparation method of the essence of Example 1.

[0113] Comparative Example 6

[0114] An anti-acne composition that can act on biofilm and a preparation method thereof, compared with Example 3, the only difference is that only Lactobacillus plantarum is inoculated during the fermentation process

[0115] The anti-acne composition of this comparative example was used to prepare the essence of this comparative example according to the composition and preparation method of the essence of Example 1.

[0116] Comparative Example 7

[0117] An anti-acne composition capable of acting on biofilm and a preparation method thereof, compared with Example 3, the only difference is that only Lactobacillus acidophilus is inoculated during the fermentation process

[0118] The anti-acne composition of this comparative example was used to prepare the essence of this comparative example according to the composition and preparation method of the essence of Example 1.

[0119] Comparative Example 8

[0120] An anti-acne composition that can act on biofilm and a preparation method thereof, compared with Example 3, the only difference is that only Lactobacillus reuteri is inoculated during the fermentation process

[0121] The anti-acne composition of this comparative example was used to prepare the essence of this comparative example according to the composition and preparation method of the essence of Example 1.

[0122] Effect verification

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

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

[0125] Experimental group: Weigh 2.5 g of each of the anti-acne composition prepared in Examples 1-3 and the anti-acne composition prepared in Comparative Examples 1-8, add them into 97.5 g of TSB culture medium, respectively, and mix them evenly.

[0126] Step 2: Take a fresh culture of Propionibacterium acnes within 5 generations, use a pipette to absorb an appropriate amount of 0.03 mol / L phosphate buffer solution, and repeatedly blow and wash the bacterial moss. Use an electric mixer to mix for 20 seconds to make the bacteria evenly suspended. Inoculate the bacterial suspension into the culture medium prepared in step 1. The initial concentration of Propionibacterium acnes in the culture medium is required to be about 10 8 CFU / ml.

[0127] Step 3: Inoculate the culture medium supplemented with Propionibacterium acnes suspension into a 6-well plate, 5 ml per well, and inoculate 3 wells in parallel. Incubate at 37°C for 48 hours. When the culture reaches 48 hours, discard the bacterial solution, add PBS to wash away the non-adherent bacteria, and repeat the washing 3 times.

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

[0129] 10% acetic acid was dissolved in the biofilm, and after the crystals combined with the biofilm were fully dissolved, the absorbance (A) value at 570 nm was measured using an ultraviolet 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 can be seen from Table 1, compared with the blank control group, Examples 1-3 have a significant inhibitory effect on the formation of Propionibacterium acnes biofilm, while Comparative Examples 1-8 have a weaker inhibitory effect on the formation of Propionibacterium acnes biofilm, indicating that tsugae gum and the composite fermentation liquid have a synergistic effect on inhibiting the formation of Propionibacterium acnes biofilm. At the same time, the compounding of different extracts, different extraction processes, and different fermentation strain ratios during composite fermentation all have an effect on the acne-removing composition inhibiting the formation of Propionibacterium acnes biofilm.

[0133] 2. Human efficacy experiment

[0134] The test method is as follows: 176 volunteers with grade 1-2 acne on their faces were recruited, 16 people in each group, the essence of the blank group did not contain the acne-removing composition, and the composition and content of the other components were the same as the essence of Example 1. The test group used the essences obtained in Examples 1-3 and Comparative Examples 1-8, respectively, for a total of 28 days, and the number of acne was clinically evaluated on day 0, day 14, and day 28, and the a value (a value, i.e., the redness content of the skin, indicating the erythema of the dermis of the skin, representing the inflammatory state of the skin) and the amount of skin oil secretion were tested using a VISA instrument in the acne area, and the improvement rate was calculated with the data on day 0; the volunteers were asked whether they had acne again after a return visit 56 days after using the product. The test results are shown in Table 2.

[0135] Table 2 Skin effects

[0136]

[0137] From the above results, it can be seen that the acne removal essences of Examples 1 to 3 have significant acne removal effects, and the combination of thorny taro gum and composite fermentation liquid has a good effect of inhibiting the recurrence of acne, and has a good soothing and oil control effect. From the comparative example, it can be seen that the process of enzymatic hydrolysis of red pine leaves before fermentation can make its soothing and oil control effect stronger and reduce the possibility of acne recurrence; the combination of lotus seed extract during the fermentation process has a good promoting effect on acne removal; the reasonable combination of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri can fundamentally improve the skin condition and make acne less likely to recur.

[0138] 3. Security Testing

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

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

[0141] According to the general method, 34 qualified volunteers were selected to participate in the test, and the samples prepared from the anti-acne compositions of the embodiments were used as the test objects, and the control samples were sterile deionized water. The patch tester with the test objects and the control samples was attached to the inner forearm of the test subject, and then removed after 24 hours. The skin reaction was observed 30 minutes after the patch tester was removed, 24 hours and 48 hours later according to the skin reaction grading standard of the skin occlusion patch test in Table 3, and the observation results were recorded, as shown in Table 4.

[0142] Table 3 Skin reaction grading standards for skin occlusive patch test

[0143]

[0144] Table 4 Patch test results

[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 anti-acne composition prepared by using tsara gum and the composite fermentation liquid in the present invention is mild and non-irritating.

[0147] 4. Biofilm Disintegration Experiment

[0148] Control group: Essence without adding acne-removing composition

[0149] Experimental group: Essence prepared in Example 3

[0150] Take the activated Propionibacterium acnes and add 20 ml of BHI medium to make a bacterial suspension. The initial bacterial concentration is adjusted to 1.0 (OD 600) for later use. Inoculate the bacterial solution on a 6-well plate cell slide and culture anaerobically at 37°C for 5 days, replacing the culture medium with fresh culture medium once a day. After 5 days, remove the supernatant and add PBS (2ml) to wash once. Add the sample dissolved in (2mL) as the sample group and PBS (2ml) as the control group, and incubate anaerobically at 37°C for 12h. After incubation, wash 3 times with PBS (2ml). Fix with 2.5% glutaraldehyde fixative for 4h, then wash 3 times with PBS, and let stand for 20min each time. Spray gold on the sample and observe the morphological changes of bacteria using a field emission scanning electron microscope.

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

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

[0153] Test sample: The test sample is the essence prepared in Example 3

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

[0155] All volunteers signed the informed consent form during the first visit and were screened according to the inclusion and exclusion criteria. The day of the formal trial was counted as 0 day. The qualified subjects arrived at the visit center and skin swab sampling and microecological related testing were performed on the acne area of ​​4*4cm2 of the volunteers. The test samples were then used. The follow-up time after 4 weeks was counted as 28 days, and the same sampling and testing as 0 day were performed. Before each skin sampling, do not wash your face in the morning to ensure sufficient biological samples.

[0156] All tests were conducted in a laboratory with a controlled temperature of 20-22°C and a relative humidity of 40-60%. 64 valid samples were collected and recorded as D0 (before the test substance was used) and D28 (after the test substance was used for 28 consecutive days) for 32 volunteers as the D0 and D28 groups.

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

[0158] The indices reflecting community richness are: sobs, chao, and ace.

[0159] Indices reflecting community coverage include: coverage; the higher its value, the higher the probability that a sequence in the sample is detected and the lower the probability that it is not detected. This index reflects whether the sequencing results of this time represent the true situation of 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 (Marked as * when 0.01 < p ≤ 0.05 and marked as *** when 0.0001 < p ≤ 0.001) 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 balance of skin flora and consolidating the immune homeostasis of the skin.

[0162] 6. Community composition analysis

[0163] Venn analysis focuses on showing the number of unique and common species in different groups, facilitating the understanding of the 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 microorganisms increased by 56 species. 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. Significance analysis of species differences

[0166] The differential test at the phylum level was performed on the top 5 bacterial species with changing abundances (the genera with no significant changes were not summarized and shown). The results are as Figure 4. It was found that the abundance of Bacteroidota in the skin resident bacteria increased significantly (P = 0.00652), while the abundance of Cyanobacteria, which was less abundant, decreased significantly (p = 0.000296). Studies have shown that the abundance of Bacteroidetes in acne patients decreased significantly compared with healthy controls (P < 0.05). In the above results, after 28 days of skin intervention with the test substance, the abundance of Bacteroidota increased significantly, indicating that it adjusted towards the trend of healthy skin.

[0167] The top 10 bacterial species with changed abundance were tested for differences (the genera without significant changes were not summarized and displayed). The results showed that Figure 5 . This study investigated the changes in the levels of skin bacteria before and after the test substance intervened in acne (or blackheads) skin. The results showed that the abundance of Bacillus increased significantly (p = 1.633e-7). Bacillus has antibacterial effects on the skin, and can also moisturize, promote cell growth and exfoliate. This change trend can better regulate and consolidate the barrier of acne skin.

[0168] In summary, under the experimental conditions, the essence prepared in Example 3 improved the microecological balance of skin bacterial flora and was able to adjust the distribution of flora in acne skin toward the trend of healthy people.

[0169] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. An anti-acne composition that can act on biofilm, characterized in that: The composition comprises 0.1-1 parts of taro gum and 45-70 parts of composite fermentation liquid.

2. The anti-acne composition capable of acting on biofilm according to claim 1, characterized in that: The composite fermentation liquid is obtained by using red pine leaf extract and lotus seed extract as raw materials, through composite bacteria anaerobic fermentation for 2-5 days, sterilization and centrifugal filtration.

3. The anti-acne composition capable of acting on biofilm according to claim 2, characterized in that: The preparation process of the red pine leaf extract is as follows: Step 1. Wash and dry the red pine leaves, mix with pure water in a ratio of 1:1, and grind to obtain slurry 1; Step 2. Add 0.5-1% cellulase to the slurry 1 obtained in step 1, perform enzymolysis at 40-50° C. for 1-2 hours, inactivate in a water bath at 80-90° C. for 8-15 minutes, cool to room temperature, and centrifuge to obtain a residue and an enzymolysis solution; Step 3. Add 10-15 times of ethanol aqueous solution to the filter residue obtained in step 2, perform ultrasonic extraction, centrifuge filtration and dry to obtain a hydroalcoholic extract; The volume fraction of the ethanol is 50-60%, the temperature of the ultrasonic extraction is 50-60° C., the working frequency of the ultrasonic extraction is 30-40 KHz, the ultrasonic power of the ultrasonic extraction is 400-600 W, and the time of the ultrasonic extraction is 20-40 min. Step 4. Evenly mix the enzymatic hydrolysate obtained in step 2 and the hydroalcoholic extract obtained in step 3 to obtain the red pine leaf extract.

4. The anti-acne composition that can act on biofilm according to claim 2, characterized in that: The weight ratio of the Pinus densiflora leaf extract to the Lotus japonica seed extract is 3-5:1-2.

5. The anti-acne composition that can act on biofilm according to claim 2, characterized in that: The composite bacteria are Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri in a live bacteria ratio of 1:1:1; The inoculation ratio of the composite bacteria is 0.5×10 5 -1.2×10 5 cfu / mL; The fermentation temperature is 28-37°C.

6. The anti-acne composition capable of acting on biofilm according to claim 1, characterized in that: The invention also comprises solvent A and solvent B, wherein the solvent A comprises water and butanediol in a mass ratio of 5:1, and the solvent B comprises 1,2-pentanediol and 1,2-hexanediol in a mass ratio of 2:

1.

7. A method for preparing the anti-acne composition capable of acting on biofilm according to any one of claims 1 to 6, characterized in that: The steps include: The thorny taro gum is dissolved in solvent A to obtain mixture 1, the composite fermentation liquid is dissolved in solvent B to obtain mixture 2, and the mixture 1 and the mixture 2 are evenly mixed to obtain the acne-removing composition.

8. The method for preparing the anti-acne composition capable of acting on biofilm according to claim 7, characterized in that: The ratio of the taro gum to the solvent A is 0.1-1:20-30.

9. The method for preparing the anti-acne composition capable of acting on biofilm according to claim 7, characterized in that: The ratio of the composite fermentation liquid to the solvent B is 45-70:2-5.

10. An anti-acne essence, characterized in that: The anti-acne essence comprises the anti-acne composition according to any one of claims 1 to 6; The mass percentage of the anti-acne composition in the anti-acne essence is 2-10%.

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