A gel with acne-removing and repairing effects and preparation thereof

Abstract

An acne-removing and repairing gel suitable for daily skin care for people with mild to moderate acne and its preparation. The gel uses natural products such as Sophora flavescens, Panax notoginseng, American cockroach, and Portulaca oleracea, as well as colloidal sulfur, encapsulated salicylic acid, etc. as active raw materials; and uses aloe vera gel juice, carbomer, sodium hyaluronate, etc. as matrix materials, so that the gel has multiple functions: targeted mild antibacterial, anti-inflammatory and soothing, repairing skin barrier function, oil control, and improving excessive keratinization of hair follicles. At the same time, the acne-removing and repairing gel uses a formula of ingredients that are friendly and gentle to acne skin, avoiding the use of irritating ingredients.

Description

Technical Field

[0001] The invention relates to the field of cosmetics, and particularly to an acne-repairing gel which can be used for people with facial acne and has multiple functions of targeted mild antibacterial, anti-inflammatory and soothing, skin barrier repair, improvement of excessive keratinization of hair follicles and skin oil control, and a preparation thereof. Background Art

[0002] "Acne" is a chronic inflammatory skin disease that occurs frequently in the facial hair follicle sebaceous gland unit of adolescents. During adolescence, the hair follicle tissue, under the stimulation of androgens, induces the sebaceous glands to secrete a large amount of lipids on the one hand, and on the other hand, causes abnormal keratinization of the hair follicle sebaceous gland ducts, which hinders the discharge of sebum. The large accumulation of lipids causes the Propionibacterium acnes, which originally belonged to the normal flora of the skin, to proliferate abnormally in a lipophilic and anaerobic environment, triggering local inflammation and immune response, leading to redness and swelling of the skin, and then forming papules and pustules. The continuous aggravation of the inflammatory response can also induce the rupture of the hair follicle wall, and lipids, microorganisms and hair enter the dermis, producing a foreign body-like reaction, and even after the inflammation subsides, erythema, pigmentation or scars are often left behind. The incidence of acne in the Chinese population is 8.1%, and it is common among adolescents. As adolescents begin to pay attention to their appearance, the impact of acne on their appearance may directly affect their state, emotions, psychology and social life. Severe appearance anxiety may cause psychological diseases such as inferiority and depression.

[0003] Clinically, acne is divided into three degrees and four levels according to the degree of skin lesions: mild (level I, only acne), moderate (level II and III, with inflammatory papules and pustules), and severe (level IV with nodules and cysts). Among them, patients with moderate and severe conditions should be treated with medication, while patients with mild and moderate conditions should be treated with daily skin care. People with acne need to choose skin care products based on medical theory, including taking into account multiple functions: (1) Targeted mild antibacterial effect: It has a high antibacterial effect on skin pathogens such as Staphylococcus aureus to avoid infection, and has a mild antibacterial effect on normal skin flora such as Propionibacterium acnes and Staphylococcus epidermidis to prevent massive proliferation. (2) Anti-inflammatory and soothing: It inhibits the redness, swelling, and pain caused by the inflammatory response when acne occurs, soothes the skin's high response to various stimuli, and improves the skin's tolerance to stimuli. (3) Repair skin barrier function: It enhances the self-repair ability of the "brick + mortar" structure of the skin's stratum corneum and restores the skin barrier function. (4) Oil control: inhibit excessive oil secretion from the sebaceous glands in the hair follicles of the skin. (5) Improve excessive keratinization of hair follicles: soften and dissolve keratin, correct the keratinization of the hair follicle duct opening, and maintain the health of the hair follicles. At the same time, the skin care products used by people with acne should be mild and non-irritating, and avoid the use of irritating fragrances, preservatives and surfactants. Summary of the invention

[0004] The present invention provides an acne-removing and repairing gel suitable for daily skin care of people with mild and moderate acne and its preparation. The gel uses natural products such as Sophora flavescens, Panax notoginseng, American cockroach, Portulaca oleracea, and colloidal sulfur, encapsulated salicylic acid, etc. as active raw materials; and uses aloe vera gel juice, carbomer, sodium hyaluronate, etc. as matrix materials, so that the gel has multiple functions: targeted mild antibacterial, anti-inflammatory and soothing, repairing skin barrier function, oil control, and improving excessive keratinization of hair follicles. At the same time, the acne-removing and repairing gel uses a formula of ingredients that are friendly and mild to acne skin, avoiding the use of irritating ingredients.

[0005] To achieve the above objectives, the present invention provides an acne-repairing gel having multiple functions of targeted mild antibacterial, anti-inflammatory and soothing, skin barrier repair, improvement of excessive keratinization of hair follicles and skin oil control, and its preparation, and is achieved through the following technical scheme.

[0006] Specifically, an acne-repairing gel is provided, comprising the following active ingredients in percentage by weight: 1-5% of a preformulated solution of a Sophora flavescens extract, 2-8% of a preformulated solution of a Panax notoginseng extract, 0.1-0.8% of an American cockroach extract, 0.02-0.3% of colloidal sulfur, 0.1-0.4% of encapsulated salicylic acid, 0.05-2% of a Portulaca oleracea extract, 0.01-0.3% of asiatica glycoside, 0.01-0.08% of dipotassium glycyrrhizinate, 0.4-3% of ceramide NP, and 0.1-0.8% of oligopeptide-1.

[0007] Furthermore, an acne-repairing gel contains the following active ingredients in weight percentage: 1-3% of a preformulated solution of Sophora flavescens extract, 2-6% of a preformulated solution of Panax notoginseng extract, 0.15-0.4% of an American cockroach extract, 0.05-0.15% of colloidal sulfur, 0.15-0.4% of encapsulated salicylic acid, 0.12-0.8% of a Portulaca oleracea extract, 0.01-0.15% of asiatica glycoside, 0.02-0.06% of dipotassium glycyrrhizinate, 0.8-1.5% of ceramide NP, and 0.2-0.6% of oligopeptide-1.

[0008] Furthermore, an acne-repairing gel contains the following active ingredients in percentage by weight as shown in Table 1:

[0009] Table 1. Active ingredients of acne repair gel

[0010]

[0011] Furthermore, the preformulated solution of Sophora flavescens extract is a mixed solution (by weight percentage) containing 1% of Sophora flavescens extract, 10% of 1,3-butylene glycol, and 89% of propylene glycol.

[0012] Furthermore, the Panax notoginseng extract pre-formulated solution is a mixed solution (by weight percentage) containing 5% Panax notoginseng extract, 90% propylene glycol, and 5% PEG-40 hydrogenated castor oil.

[0013] Furthermore, the American cockroach extract is an extract powder of the American cockroach (PERIPLANETA AMERICANA) extract, which contains polyols and peptide active substances (including 18 amino acids) ≥ 82% and water ≤ 18% (weight percentage).

[0014] Furthermore, the colloidal sulfur is 75% elemental sulfur and 25% gum arabic (by weight); the elemental sulfur is an octagonal ring structure composed of 8 sulfur atoms.

[0015] Furthermore, the coated salicylic acid is a coated powder containing 40% salicylic acid (SALICYLIC ACID), 25% pullulan, 20% dextrin, and 15% xanthan gum (by weight percentage).

[0016] Furthermore, the purslane extract is a mixed solution containing 0.4% purslane (PORTULACA OLERACEA) extract, 69.6% water, and 30% butylene glycol (percentage by weight).

[0017] Furthermore, the ceramide NP is a mixed solution containing 58.8% water, 30% butanediol, 2.5% ceramide NP (CERAMIDENP), 7% phosphatidylcholine, 0.5% sodium stearoyl glutamate, 1% 1,2-hexanediol, and 0.2% p-hydroxyacetophenone (percentage by weight).

[0018] Furthermore, the oligopeptide-1 is a mixed solution containing 39.5% water, 60% glycerol and 0.5% oligopeptide-1 (OLIGOPEPTIDE-1) (weight percentage).

[0019] Furthermore, the asiatica glycoside is asiatica glycoside (ASIATICOSIDE).

[0020] Furthermore, the dipotassium glycyrrhizate is dipotassium glycyrrhizate (DIPOTASSIUM GLYCYRRHIZATE).

[0021] Furthermore, an acne-repairing gel is provided, which also contains the following matrix components in percentage by weight: 35-80% aloe vera gel juice, 0.02-0.08% disodium EDTA, 0.1-0.7% carbomer, 0.02-0.08% sodium hyaluronate, 1-4% glycerol, 0.4-1.5% PHL, 2-8% 1,3-butylene glycol, 0.1-0.7% L-arginine, and 3-7% water.

[0022] Furthermore, the matrix component formula information of the acne repair gel involved is shown in Table 2.

[0023] Table 2. Anti-acne repair gel base ingredients

[0024]

[0025] Furthermore, the aloe vera gel juice is Aloe barbadensis leaf juice.

[0026] Furthermore, the sodium hyaluronate is oligosaccharide HA (molecular weight 104Da).

[0027] Furthermore, the PHL is a mixed solution of 65% 1,3-propylene glycol, 30% 1,2-hexanediol, and 5% octanoylhydroxamic acid.

[0028] Furthermore, the water is deionized water.

[0029] Furthermore, an acne repair gel is provided, which contains 1kg of Sophora flavescens extract pre-formulated liquid, 2.5kg of Panax notoginseng extract pre-formulated liquid, 37.41kg of aloe vera gel juice, 0.025kg of disodium EDTA, 0.15kg of encapsulated salicylic acid, 0.245kg of carbomer, 0.025kg of sodium hyaluronate, 1.5kg of glycerin, 0.025kg of dipotassium glycyrrhizinate, 0.5kg of ceramide NP, 0.25kg of oligopeptide-1, 0.15kg of American cockroach extract, 0.5kg of Portulaca oleracea extract, 0.05kg of colloidal sulfur, 0.05kg of asiatica glycoside, 0.6kg of PHL, 2.5kg of 1,3-butanediol, 0.255kg of L-arginine, and 2.265kg of water.

[0030] The preparation method of the acne-removing and repairing gel comprises the following steps:

[0031] (1) After adding the amount of aloe vera gel juice in the formula to the emulsifier, add disodium EDTA and dipotassium glycyrrhizinate to the emulsifier and stir until they are completely dissolved. The temperature during this process is room temperature and the stirring speed is 20 rpm.

[0032] (2) Disperse the glycerol, carbomer and ceramide NP in the prescribed amount and add them to the emulsifying pot described in (1), and stir until the mixture is uniform. The temperature during the process is room temperature and the stirring speed is 20 rpm.

[0033] (3) Add the pre-prepared solution of Sophora flavescens extract, the pre-prepared solution of Panax notoginseng extract, the encapsulated salicylic acid, sodium hyaluronate, oligopeptide-1, American cockroach extract, Portulaca oleracea extract, colloidal sulfur, Centella asiatica glycoside, PHL, and 1,3-butylene glycol in the emulsifying pot described in (2) and stir until the mixture is uniform. The temperature during the process is room temperature and the stirring speed is 20 rpm.

[0034] (4) Dissolve the prescribed amount of L-arginine in water to prepare a solution.

[0035] (5) Add the solution obtained in (4) to the emulsifying pot described in (3), and stir and mix for 30 minutes to obtain a gel. The temperature during this process is room temperature, the stirring speed is 20 rpm, and the vacuum degree is -0.06 MPa.

[0036] (6) The gel obtained in step (5) is filtered through a 200-mesh filter cloth to obtain a transparent gel.

[0037] (7) The transparent gel prepared in (6) is tested for sensory, physical, chemical and microbiological indicators. The appearance should be transparent or translucent gel, free of foreign matter; conform to the specific aroma; pH value 5.5-6.5; total colony count ≤ 500 CFU / ; mold and yeast ≤ 50 CFU / g.

[0038] (8) Using a semi-automatic filling machine, fill the qualified transparent gel in (7) into a soft tube, and seal the soft tube with a sealing machine to obtain an acne-removing and repairing gel.

[0039] The characteristics of the acne-repairing gel involved in the present invention are:

[0040] 1. Targeted mild antibacterial

[0041] The bacteria that are easily infected when acne occurs on the face include Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes. Among them, Staphylococcus epidermidis and Propionibacterium acnes belong to the normal flora that parasitize on the skin. Propionibacterium acnes can decompose the oil produced by normal skin, release fatty acids, and maintain a weakly acidic environment on the skin surface, which plays a role in resisting pathogenic bacteria. Only when Propionibacterium acnes over-proliferates in blocked hair follicles will it cause local inflammation of the skin. Staphylococcus epidermidis is also a normal microorganism that parasitizes on human skin. When its number is within the normal range, it can stimulate and regulate the skin's immune response, but abnormal over-proliferation can also cause skin inflammation. Staphylococcus aureus is a highly contagious pathogen that mainly infects tiny wounds on the skin surface and forms small pustules. Areas with fragile skin barrier function (such as skin lesions caused by dryness, scratching, and squeezing acne) are more susceptible to infection by Staphylococcus aureus.

[0042] The extract of Sophora flavescens in this gel has antibacterial effect, and can significantly inhibit Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes. According to the test, this gel has a strong inhibitory effect on pathogenic Staphylococcus epidermidis, with an inhibition rate of >80%; while the inhibition of Staphylococcus epidermidis and Propionibacterium acnes, which belong to the normal flora of the skin, is relatively mild, with an inhibition rate between 25-50%.

[0043] 2. Anti-inflammatory and soothing

[0044] Inflammatory response runs through the entire process of acne. The massive reproduction of microorganisms such as Propionibacterium acnes destroys the microecological environment of the skin. Microorganisms and some abnormal lipids in the hair follicles activate Toll-like receptors (TLRs) to produce a variety of interleukins (such as IL-6, IL-1β, etc.). At the same time, activated macrophages mediate the release of tumor necrosis factor (TNF-α), inducing and strengthening local inflammatory response, thus causing a series of inflammatory reactions such as redness, swelling, and pain on the skin.

[0045] The notoginseng extract and sophora flavescens extract in the gel have anti-inflammatory and soothing effects, and can effectively inhibit the release of the above-mentioned inflammatory factors by cells. According to tests, the inhibition rates of the gel on inflammatory factors TNF-α, IL-6 and IL-1β were 38%, 83% and 39% respectively, which can effectively inhibit the redness, swelling and pain caused by local inflammatory reactions of the skin caused by acne, and can also play a soothing role and improve the tolerance to various stimuli.

[0046] 3. Skin barrier repair

[0047] The skin barrier is mainly realized by the "brick + mortar" structure of the stratum corneum. The "bricks" refer to the keratinocytes, and the extracellular components are tightly interlocked and cross-linked to form a non-water-soluble tough outer membrane, namely the keratin capsule; the "mortar" refers to the lipids in the intercellular space. The tight connection can limit the free flow of water in and out of cells or between cells. This barrier structure prevents the loss of water and electrolytes in the body, prevents the invasion of harmful substances from the outside world, and maintains the stability of the body's internal environment. Factors such as inflammatory reactions and microbial infections during the occurrence and development of acne can cause damage to the skin barrier function and reduce the skin's ability to resist the invasion of exogenous irritants and harmful substances.

[0048] The combination of American cockroach extract, hyaluronic acid, ceramide and other ingredients in this gel can effectively repair the barrier function of damaged skin and prevent the recurrence of acne. Transepidermal water loss (TEWL) and stratum corneum water content (SCH) are important indicators for evaluating skin barrier function. According to human tests, continuous use of this gel for 28 days can significantly improve the SCH of the subjects' skin and significantly reduce TEWL, and has the effect of repairing the skin barrier.

[0049] 4. Oil control

[0050] Excessive secretion of the skin's sebaceous glands is the cause of acne, and controlling the skin's oil secretion is another important factor in preventing the recurrence of acne / blackheads. In this process, 5α-reductase can directly catalyze the conversion of testosterone (T) into dihydrotestosterone (DHT), which promotes excessive proliferation of sebaceous gland cells and promotes the secretion of pro-inflammatory factors by sebaceous gland cells.

[0051] The flavonoids in the Sophora flavescens extract in this gel inhibit the activity of 5α-reductase, inhibit the sebaceous glands from secreting a large amount of oil, reduce the risk of hair follicle blockage, and prevent the abnormal proliferation of Propionibacterium acnes. After testing, the gel's inhibition rate on 5α-reductase was 53%, which has an oil control effect.

[0052] 5. Improve excessive keratinization of hair follicles

[0053] The sulfur in colloidal sulfur can interact with cysteine ​​in the stratum corneum to form hydrogen sulfide, which promotes the decomposition and shedding of keratin. Encapsulated salicylic acid can continuously release low-concentration salicylic acid, which can soften the keratin of hair follicles and promote keratin exfoliation.

[0054] 6. Mild and non-irritating

[0055] Factors such as inflammatory reactions and microbial infections during the occurrence and development of acne can damage the skin barrier function and reduce the skin's ability to resist invasion by exogenous irritants and harmful substances. Skin care products suitable for people with acne should be mild and non-irritating, and should avoid the use of flavors and fragrances. Preservatives and surfactants that are milder on the skin should be selected as much as possible.

[0056] This gel uses the natural surfactant molecules in the polyols (1,3-butylene glycol, glycerol, 1,3-propylene glycol, 1,2-hexanediol) and aloe vera gel juice to help dissolve the active ingredients, without adding any additional surfactants. In addition, carbomer is added to thicken and stabilize the dispersion. This gel also does not contain any flavors or fragrances. According to the safety evaluation test in the "Technical Specifications for Safety of Cosmetics" (2015 edition), this gel has no skin irritation.

[0057] 7. High stability

[0058] This acne repair gel contains a large amount of water and a certain concentration of peptides and amino acids. In order to avoid the growth of microorganisms and deterioration, this acne repair gel uses a trace amount of octanoyl hydroxamic acid as a preservative. It is produced in a conventional 100,000-level purification workshop and sealed with conventional PET hoses to ensure that the microbial indicators meet the limit requirements of the "Cosmetic Safety Technical Specifications" (2015 edition) when leaving the factory. After accelerated stability experiments, the physical and chemical properties and microbial indicators of the gel meet the requirements of a shelf life of 36 months. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 .Compare the OD450 of each test sample

[0060] Figure 2 .Inhibition zones of different concentrations of test samples against three test bacteria

[0061] A: Propionibacterium acnes B: Staphylococcus epidermidis C: Staphylococcus aureus

[0062] Figure 3 .VISIA imaging images of some subjects DETAILED DESCRIPTION

[0063] Example 1 Preparation of acne-repairing gel (500 g)

[0064] The reagents and instruments used in the following examples are as follows:

[0065] Reagents:

[0066] Sophora flavescens extract pre-prepared solution and Panax notoginseng extract pre-prepared solution were prepared by themselves. Aloe vera gel juice, EDTA-2Na, encapsulated salicylic acid, carbomer, sodium hyaluronate, glycerin, dipotassium glycyrrhizinate, ceramide NP, oligopeptide-1, American cockroach extract, purslane extract, colloidal sulfur, asiaticoside, PHL, 1,3-butylene glycol, and L-arginine were purchased from Yunnan Bainilan Resources Development Co., Ltd.

[0067] instrument:

[0068] Laboratory vacuum homogenizer, purchased from Shanghai Huashuo Intelligent Equipment Co., Ltd., model 1

[0069] Electronic balance, purchased from Mettler Toledo, model PL1002E / 02

[0070] Sealing machine, Guangzhou Changxin Packaging Materials Co., Ltd.

[0071] Contains 10g of Sophora flavescens extract pre-formulated liquid, 25g of Panax notoginseng extract pre-formulated liquid, 375g of aloe vera gel juice, 0.25g of disodium EDTA, 1.5g of encapsulated salicylic acid, 2.5g of carbomer, 0.25g of sodium hyaluronate, 15g of glycerin, 0.25g of dipotassium glycyrrhizinate, 5g of ceramide NP, 12.5g of oligopeptide-1, 1.5g of American cockroach extract, 5g of Portulaca oleracea extract, 0.5g of colloidal sulfur, 0.5g of asiatica glycoside, 6g of PHL, 25g of 1,3-butylene glycol, 2.5g of L-arginine, and 2.3g of water.

[0072] Preparation method:

[0073] (1) After adding the amount of aloe vera gel juice in the formula to the emulsifier, add disodium EDTA and dipotassium glycyrrhizinate to the emulsifier and stir until they are completely dissolved. The temperature during this process is room temperature and the stirring speed is 20 rpm.

[0074] (2) Disperse the glycerol, carbomer and ceramide NP in the prescribed amount and add them to the emulsifying pot described in (1), and stir until the mixture is uniform. The temperature during the process is room temperature and the stirring speed is 20 rpm.

[0075] (3) Add the pre-prepared solution of Sophora flavescens extract, the pre-prepared solution of Panax notoginseng extract, the encapsulated salicylic acid, sodium hyaluronate, oligopeptide-1, American cockroach extract, Portulaca oleracea extract, colloidal sulfur, Centella asiatica glycoside, PHL, and 1,3-butylene glycol in the emulsifying pot described in (2) and stir until the mixture is uniform. The temperature during the process is room temperature and the stirring speed is 20 rpm.

[0076] (4) Dissolve the prescribed amount of L-arginine in water to prepare a solution.

[0077] (5) Add the solution obtained in (4) to the emulsifying pot described in (3), and stir and mix for 30 minutes to obtain a gel. The temperature during this process is room temperature, the stirring speed is 20 rpm, and the vacuum degree is -0.06 MPa.

[0078] (6) The gel obtained in step (5) is filtered through a 200-mesh filter cloth to obtain a transparent gel.

[0079] (7) Conduct sensory, physical, chemical and microbiological tests on the transparent gel prepared in (6). The appearance should be transparent or translucent gel, free of foreign matter, conform to the specified fragrance, and have a pH value of 5.5.

[0080] Example 2 Preparation of Anti-Acne Repair Gel (50kg)

[0081] The reagents and instruments used in the following examples are as follows:

[0082] Reagents:

[0083] Sophora flavescens extract pre-prepared solution and Panax notoginseng extract pre-prepared solution were prepared by themselves. Aloe vera gel juice, EDTA-2Na, encapsulated salicylic acid, carbomer, sodium hyaluronate, glycerin, dipotassium glycyrrhizinate, ceramide NP, oligopeptide-1, American cockroach extract, purslane extract, colloidal sulfur, asiaticoside, PHL, 1,3-butylene glycol, and L-arginine were purchased from Yunnan Bainilan Resources Development Co., Ltd.

[0084] instrument:

[0085] 100L vacuum homogenizing emulsifying pot, purchased from Gaoyou Instrument Factory, Jiangsu Province, model XY-A

[0086] Electronic platform scale, purchased from Xiamen Bailunsi Technology, model TCS-T01R-150

[0087] Electronic balance, purchased from Mettler Toledo, model PL1002E / 02

[0088] Semi-automatic filling machine, purchased from Shanghai Jiajue Machinery Co., Ltd., model GFC-25

[0089] Sealing machine, Guangzhou Changxin Packaging Materials Co., Ltd.

[0090] It contains 1kg of Sophora flavescens extract pre-formulated liquid, 2.5kg of Panax notoginseng extract pre-formulated liquid, 37.41kg of aloe vera gel juice, 0.025kg of disodium EDTA, 0.15kg of encapsulated salicylic acid, 0.245kg of carbomer, 0.025kg of sodium hyaluronate, 1.5kg of glycerin, 0.025kg of dipotassium glycyrrhizinate, 0.5kg of ceramide NP, 0.25kg of oligopeptide-1, 0.15kg of American cockroach extract, 0.5kg of Portulaca oleracea extract, 0.05kg of colloidal sulfur, 0.05kg of asiatica glycoside, 0.6kg of PHL, 2.5kg of 1,3-butylene glycol, 0.255kg of L-arginine, and 2.265kg of water.

[0091] Preparation method:

[0092] (1) This preparation was produced in a 100,000-class cleanroom. Before preparation, it was ensured that the on-site functional room had been cleared, the equipment could operate normally, and the equipment had been cleaned and disinfected.

[0093] (2) After adding the amount of aloe vera gel juice to the emulsifier, add disodium EDTA and dipotassium glycyrrhizinate to the emulsifier and stir until they are completely dissolved. The temperature during this process is room temperature and the stirring speed is 20 rpm.

[0094] (3) Disperse the glycerol, carbomer and ceramide NP in the prescribed amount and add them to the emulsifying pot described in (2), and stir until the mixture is uniform. The temperature during this process is room temperature and the stirring speed is 20 rpm.

[0095] (4) Add the pre-prepared solution of Sophora flavescens extract, the pre-prepared solution of Panax notoginseng extract, the encapsulated salicylic acid, sodium hyaluronate, oligopeptide-1, American cockroach extract, Portulaca oleracea extract, colloidal sulfur, Centella asiatica glycoside, PHL, and 1,3-butylene glycol in the emulsifying pot described in (3) and stir until the mixture is uniform. The temperature during the process is room temperature and the stirring speed is 20 rpm.

[0096] (5) Dissolve the prescribed amount of L-arginine in water to prepare a solution.

[0097] (6) Add the solution obtained in (5) to the emulsifying pot described in (4), and stir and mix for 30 minutes to obtain a gel. The temperature during this process is room temperature, the stirring speed is 20 rpm, and the vacuum degree is -0.06 MPa.

[0098] (7) The gel obtained in step (6) is filtered through a 200-mesh filter cloth to obtain a transparent gel.

[0099] (8) The transparent gel prepared in (7) is tested for sensory, physical, chemical and microbiological indicators. The appearance should be transparent or translucent gel, free of foreign matter; conform to the specific aroma; pH value is 5.5; total colony count ≤ 500 CFU / ; mold and yeast ≤ 50 CFU / g.

[0100] (9) Use a semi-automatic filling machine to fill the qualified transparent gel in (8) into a soft tube, 15 g / tube, and seal the soft tube with a sealing machine to obtain an acne-removing and repairing gel.

[0101] The following test examples are used to illustrate the effect of the acne-removing and repairing gel in this application.

[0102] Test Example 1 Evaluation of the Skin Irritation of Acne Repair Gel

[0103] Purpose of the test

[0104] According to the standard method of "Technical Specification for Safety of Cosmetics" (2015 edition), the multiple skin irritation of the acne repair gel (prepared in Example 2) was evaluated.

[0105] Test Method Animals: Rabbits (Japanese large white rabbits), ordinary grade, 4 (2 males, 2 females), weight 2-2.3 kg. Feeding: temperature 17-25°C, humidity 40-60%, 12 / 12h day and night light and dark alternation. Feed with standard experimental animal feed, free drinking water.

[0106] Test material: Anti-acne repair gel (prepared in Example 2).

[0107] Negative control: purified water.

[0108] Skin preparation: 24 hours before the test, the hair on both sides of the dorsal spine of the experimental animals was trimmed, the hair removal area was 3×3cm each, and the application area was 2.5×2.5cm.

[0109] Administration: Select 4 rabbits with no wounds, plaques or papules on the prepared skin as test animals, apply about 0.5g of anti-acne repair gel on the left side of the animal skin, and apply pure water on the right side as a negative control, apply once a day for 14 consecutive days. From the second day, the hair should be trimmed before each application, and the residual test substance should be removed with warm water. Observe the results after one hour and score according to Table 3 and Table 4.

[0110] Table 3. Skin irritation response scoring table

[0111]

[0112] Table 4. Correspondence between mean integral value and stimulus intensity

[0113]

[0114]

[0115] Test Results

[0116] Apply once a day for 14 consecutive days. During the test, no abnormal results were found on the animal's treated side and control skin. See Table 5.

[0117] Table 5. Test samples for multiple skin irritation and reaction scores on rabbits

[0118]

[0119] in conclusion

[0120] According to the requirements of the "Technical Specifications for Safety of Cosmetics" (2015 edition), the acne repair gel can be judged to be non-irritating to the skin of rabbits after multiple exposure.

[0121] Test Example 2 Evaluation of the Oil Control Effect of Acne Repair Gel

[0122] Purpose of the test

[0123] The oil control efficacy of the acne repair gel (prepared in Example 2) was evaluated using a laboratory method based on Elisa oil control efficacy test evaluation based on inhibition of 5α-reductase activity.

[0124] principle

[0125] Oily skin is caused by excessive secretion of human sebaceous glands and abnormal sebaceous gland function. Abnormal secretion of sebaceous glands is usually caused by excessive male hormones. Testosterone (T) and dihydrotestosterone (DHT) are both male hormones. The ability of DHT to bind to androgen receptors is more than 5 times higher than that of T. In the process of T converting to DHT, 5α-reductase is the direct catalytic enzyme. Both can promote excessive proliferation of sebaceous gland cells. At the same time, dihydrotestosterone will also promote the secretion of pro-inflammatory factors by sebaceous gland cells, further leading to skin diseases such as acne and seborrheic dermatitis.

[0126] This experiment uses the "one-step sandwich method" to detect the inhibition rate of the test substance on 5α-reductase. The inhibitory factor (a substance that may exist in the sample) acts on the 5α-reductase adsorbed on the antibody (the antibody has been fixed to the concave well of the ELISA plate), inhibiting its activity. The 5α-reductase still remaining on the microwell after treatment is connected to the HRP-labeled immunoenzyme, catalyzing the color development of the substrate, and the absorbance value is tested after the reaction is terminated. Make a negative control group (no sample or inhibitor is added) and a positive control group (this experiment uses EGCG with a certain amount of concentration of substance), and evaluate the inhibitory effect of the test substance on 5α-reductase by comparing and analyzing the data. And use this data to scientifically and reasonably evaluate the oil control effect of cosmetics.

[0127] Test Method

[0128] 1. Experimental reagents:

[0129] Catechin gallate (CAS: 981-51-5), concentration 3 μg / μL, stored at 18-26°C;

[0130] 5α-reductase, concentration>80U / ml, the titer of the enzyme in this experiment was about 100U / ml, stored at -20℃;

[0131] Tris-HCl buffer (CAS: 1185-53-1) concentration 0.1 M, pH = 7.0-7.2.

[0132] 2. Test materials and instruments:

[0133] ELISA analyzer; 5α-reductase antigen Elisa reagent test kit.

[0134] 3. Test environment: temperature 20-25℃, relative humidity 50-70%

[0135] 4. Test group: see Table 6

[0136] Table 6. Experimental group design

[0137]

[0138] Note: The molar concentration ratio of the positive control and sample should be appropriate, and its molar concentration is calculated and set with reference to the "molar mass" of the sample (considering the molar mass of several raw materials with the highest mass concentration in the formula, and the molar mass of the remaining raw materials / plant raw materials is replaced by the molar mass of glucose). The experimenter believes that the ideal situation for this experiment is: the molar concentration of the sample substance: the molar concentration of the positive control substance is approximately 1:0.372, and it is adjusted with reference to other factors.

[0139] 5. Test steps

[0140] (1) Dilute the sample N times (20<N<50) with 0.1M tris-HCl buffer and use it as the sample for the next test; take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips with a ziplock bag and return them to 4°C.

[0141] (2) Set up blank wells, positive control wells, sample wells, and negative control wells; except for the blank group, no enzyme was added. Add 50 μL of 5α-reductase to each well of the other groups and incubate in a 37°C constant temperature incubator for 10 min. Add samples to the sample wells and positive controls to the positive control wells. Note that the molar concentration ratio between the sample and the positive control should be appropriate (at least less than 10 times). Incubate the microplate after adding the sample for 10 min and wash the plate 5 times (the washing solution is provided by the kit, and the original washing solution is diluted 20 times for later use).

[0142] (3) Add 100uL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the negative control group, positive control group, and sample wells, seal the wells with a sealing film, and incubate at 37°C in a water bath or incubator for 60min.

[0143] (4) Discard the liquid, pat dry on absorbent paper, and wash the plate three times in a plate washer.

[0144] (5) Add 50uL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0145] (6) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0146] 6.5 Calculation of α-reductase inhibition rate

[0147] (1) The percentage calculation formula of sample inhibition rate is as follows:

[0148] Inhibition rate (%) = 100 × (OD 空白 +OD 阴性对照 -OD 样品 ) / (OD 空白 +OD 阴性对照 )

[0149] (2) The percentage calculation formula of the positive control inhibition rate is as follows:

[0150] Inhibition rate (%) = 100 × (OD 空白 +OD 阴性对照 -OD 阳性对照 ) / (OD 空白 +OD 阴性对照 )

[0151] Test results:

[0152] The inhibition of 5α-reductase by the test sample is as follows: Figure 1 And as shown in Table 7.

[0153] Table 7. Inhibition of 5α-reductase by each test sample

[0154]

[0155] The average OD value of the acne repair gel on 5α-reductase inhibition was 0.481, which was significantly lower than the positive control group (0.851) and the negative control group (0.701) using a certain molar concentration of EGCG. After calculation, the test sample's inhibition rate on 5α-reductase = 52.95%, the positive control's inhibition rate on 5α-reductase = 16.73%, and the sample's inhibition rate > 30%, indicating that the sample has inhibitory activity on 5α-reductase.

[0156] in conclusion

[0157] Under the conditions of this test, the anti-acne repair gel (prepared in Example 2) has an inhibitory effect on 5α-reductase, and the inhibition rate is 54.43%, indicating that the test sample has an oil control effect.

[0158] Test Example 3 Evaluation of the antibacterial properties of acne repair gel

[0159] Purpose of the test

[0160] The antibacterial activity of the acne-repairing gel prepared in Example 2 against Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus was evaluated.

[0161] principle

[0162] 1. Paper method

[0163] The method is to stick a filter paper containing a quantitative antimicrobial drug on the surface of an agar inoculated with test bacteria. The drug in the paper diffuses in the agar, and the concentration of the antimicrobial drug decreases logarithmically with the increase of the diffusion distance, thereby forming a concentration gradient around the paper. At the same time, the test bacteria within the antibacterial concentration range around the paper cannot grow, while the strains outside the antibacterial concentration range continue to grow. The size of the antibacterial circle formed around the paper can reflect the sensitivity of the test bacteria to the measured drug.

[0164] 2. Colorimetry

[0165] When the light beam passes through the suspension, its transmission is reduced due to scattering or absorption. The concentration of the bacterial suspension is proportional to the optical density within a certain range and inversely proportional to the transmittance. According to the method in the literature, the optical density value OD600 of the 6×108cfu bacterial suspension at a wavelength of 600nm is measured by an enzyme marker to indicate the concentration of the bacterial suspension to verify the inhibitory sensitivity of the test sample to the selected test bacteria. The colorimetric method uses the absorbance of bacteria to measure the concentration of bacterial culture fluid and estimate the growth of bacteria. Therefore, it is usually used to refer to the bacterial cell density. It is a standard indicator for tracking the density of microorganisms in liquid culture and indicates the bacterial cell density.

[0166] Test Method

[0167] 1. Bacteria

[0168] Propionibacterium acnes (ATCC11827), Staphylococcus epidermidis (ATCC 12228), and Staphylococcus aureus (ATCC6538) were obtained from the Microbial Culture Collection Center of Guangdong Institute of Microbiology.

[0169] 2. Instruments

[0170] Bacterial incubator (Jintan City Jerel Electric Company, MJ-150B), multifunctional microplate reader (Beckman DTX800, USA), anaerobic culture bag (Mitsubishi Lijiu Chemical Co., Ltd., Japan)

[0171] 3. Test samples

[0172] Acne-removing and repairing gel prepared in Example 2

[0173] 4. Test steps

[0174] (1) Paper disc method: The test samples of different dilution concentrations were dissolved in anhydrous ethanol as solvent and diluted in half. The concentration gradient was: original solution, 50%, 25%, 12.5%, 6.25%. 6 mm paper discs were soaked in the test samples of different concentrations and plated at 6.0 × 108 CFU / mL. The paper discs were pressed Figure 1 The plates were placed on agar plates in sequence. Propionibacterium acnes was placed in an anaerobic culture box at 37°C for 48 hours, and Staphylococcus epidermidis and Staphylococcus aureus were placed in a 37°C constant temperature incubator for 24 hours. The size of the inhibition zone was observed and measured. The experiment was repeated 3 times and the results were judged.

[0175] (2) Colorimetric method: In the experimental group, 20 μl of the test sample was added to a 96-well plate, and then 180 μl of the bacterial suspension was added; in the positive group, 20 μl of mupirocin ointment was added to a 96-well plate, and then 180 μl of the bacterial suspension was added; in the blank group, 200 μl of blank liquid culture medium was added; in the control group, 200 μl of bacterial liquid was added. Propionibacterium acnes was placed in an anaerobic culture box in a 37°C constant temperature incubator for 48 h, and Staphylococcus epidermidis and Staphylococcus aureus were placed in a 37°C constant temperature incubator for 24 h. The OD value at 600 nm was measured by an enzyme marker, and the growth inhibition rate was calculated as follows = (control group OD - experimental group OD) / (control group OD - blank group OD). The experiment was repeated 3 times and the results were judged.

[0176] Test Results

[0177] 1. Paper disc method to test the inhibition zone

[0178] See Figure 2 .

[0179] Figure 2 The inhibition zones of different concentrations of the test samples against Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus are shown in Table 8. The 100% and 50% concentration test samples showed significant inhibition against the three test bacteria.

[0180] Table 8. Inhibition zones of different concentrations of acne repair gel on each test bacteria

[0181]

[0182] Note: “*” indicates P<0.05 compared with the anhydrous ethanol group, and “**” indicates P<0.01.

[0183] According to the t-test analysis, the antibacterial effect of the test sample at 100% concentration on the three test bacteria was higher than that of anhydrous ethanol, and the difference was statistically significant (P<0.01 for Propionibacterium acnes, P<0.05 for Staphylococcus aureus, and P<0.01 for Staphylococcus epidermidis); the antibacterial effect of the test sample at 50% concentration on Staphylococcus epidermidis was also significantly higher than that of anhydrous ethanol (P<0.05). In other low-concentration groups, there was no significant difference in the antibacterial effect of the test sample on the three test bacteria compared with anhydrous ethanol.

[0184] 2. Colorimetric test results

[0185] Table 9 shows the comparison of the inhibitory effects of the test sample and the positive group (mupirocin ointment) on the three test bacteria calculated by the colorimetric test. The results show that the test sample has an inhibitory effect on Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus, with inhibition rates of 25.7%, 41.4% and 84.1% respectively.

[0186] Table 9. Comparison of the inhibition rate of the test samples on the test bacteria

[0187]

[0188] Note: Inhibition rate = (control group OD - experimental group OD) / (control group OD - blank group OD)

[0189] in conclusion

[0190] Through the paper disc method and colorimetric method, under the experimental conditions, the acne repair gel has a significant antibacterial effect on Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus. Among them, the inhibitory effect on Staphylococcus aureus is stronger, and the antibacterial effect on Propionibacterium acnes and Staphylococcus epidermidis is relatively mild. Experimental Example 4 Evaluation of the soothing and anti-inflammatory activity of the prepared gel

[0191] Purpose of the test

[0192] The anti-inflammatory efficacy of the acne repair gel (prepared in Example 2) was evaluated by using an anti-inflammatory efficacy model in which lipopolysaccharide (LPS)-induced macrophage inflammatory cells (RAW264.7) secreted inflammatory factors TNF-α, IL-1β and IL-6.

[0193] principle

[0194] LPS-induced RAW264.7 is a classic cell model for studying inflammatory factors. LPS binds to the antigen recognition receptors on the surface of macrophages and can induce macrophages to secrete a variety of inflammatory factors such as TNF-α, IL-1β and IL-6.

[0195] 1. TNF-α can activate three signaling pathways: Caspase, JNK and transcription factor NF-κB, to achieve its biological functions such as cytotoxicity, antiviral, immunomodulation and cell apoptosis.

[0196] 2. IL-1β is an important mediator of inflammatory response. When the body is in an inflammatory state or an immune response occurs, it can participate in a variety of cell activities, including cell proliferation, differentiation and apoptosis. IL-1β can also activate endothelial cells, cause bone marrow neutrophil mobilization (leukocytosis) and activate various types of white blood cells, causing an increase in acute phase response proteins such as C-reactive protein, thereby leading to systemic inflammation.

[0197] 3. The pattern of IL-6 production in different inflammations is different. In the early stages of infectious inflammation, different pathogens stimulate monocytes and macrophages to produce IL-6 through Toll-like receptors (TLRs) of associated molecular patterns (PAMPs). In non-infectious inflammation (such as burns or traumatic injuries), damaged cells are stimulated to produce IL-6 through TLRs of damage-associated molecular patterns (DAMPs). This acute IL-6 expression plays a central role in host defense by stimulating various cell populations.

[0198] This method evaluates the effect of acne repair gel in inhibiting the secretion of TNF-α, IL-1β and IL-6 by comparing the difference in the secretion of TNF-α, IL-1β and IL-6 by RAW264.7 cells after administration of negative control and acne repair gel. The content was determined by enzyme-linked immunosorbent assay (ELISA), and the specific principle is (taking TNF-α as an example): after the inflammatory factor TNF-α specifically binds to the TNF-α antibody coated on the ELISA plate, the anti-TNF-α antibody labeled with the substrate binds, and the substrate is catalyzed to generate a colored product. The TNF-α content and the colored product are measured at a wavelength of 450nm. The optical density (OD value) is used to calculate the TNF-α content.

[0199] Test Method

[0200] 1. Cells: Mouse mononuclear macrophage leukemia cell line RAW264.7, the cell line was obtained from the Kunming Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences, and the selected model was KCB 200603YJ.

[0201] 2. Culture conditions: DMEM medium, fetal bovine serum, trypsin / EDTA solution (0.25% trypsin solution and 0.02 mol / L EDTA solution mixed in a ratio of 1:1), phosphate buffered saline (PBS), and cell culture flask.

[0202] 3. Stimulant: Bacterial lipopolysaccharide (LPS from E. coli)

[0203] 4. Positive control: dexamethasone (Solebo D8040)

[0204] 5. Materials and instruments: 96-well plate; TNF-α ELISA detection kit, IL-1β ELISA detection kit, IL-6 ELISA detection kit ( ELISA kit); CCK-8 cytotoxicity detection kit (Solabo CA1210); microplate reader; clean bench; dimethyl sulfoxide (DMSO).

[0205] 6. Test sample: acne-repairing gel, prepared according to Example 2.

[0206] 7. Test Grouping

[0207] Table 10. Experimental group design

[0208]

[0209] 8. Test steps (1) Cell preparation:

[0210] a. Cell recovery:

[0211] 1) Preheat a water bath to 37°C, prepare clean disposable PE gloves, and add 9 ml of sterile DMEM medium into a sterile centrifuge tube;

[0212] 2) Take the cells out of the liquid nitrogen tank and put them in PE gloves, quickly immerse them in a water bath, and shake the cryotubes to accelerate the lysis. It is best to completely dissolve them within 1 minute.

[0213] 3) Add the revived cell solution into a centrifuge tube containing PBS culture medium in a clean bench.

[0214] Centrifuge at 1000 rpm / min for 3 minutes and remove the supernatant;

[0215] 4) Resuspend the cells with 6 ml of DMEM medium containing 10% serum, inoculate into a T25 culture flask, add medium to 5 ml, and place in an incubator for culture.

[0216] b. Cell digestion and passaging:

[0217] Use trypsin / EDTA (preferably 0.25%), 1 ml of trypsin for T25 culture flasks and 3 mL of trypsin for T75 culture flasks to digest the cells. Observe under a microscope until most cells become round and suspended, add about 2-3 times the volume of trypsin in DMEM medium containing 10% serum to terminate digestion, collect the cells in a centrifuge tube, centrifuge at 1000r / min for 5 minutes, discard the supernatant after centrifugation, add DMEM medium containing 10% serum to the centrifuge tube, pipette to mix the cells, and transfer them to two T25 culture flasks.

[0218] (2) Preparation of acne repair gel solution

[0219] Before testing, the emulsion sample was dissolved in DMSO and ultrasonicated in a water bath for 5 minutes to prepare an acne-repairing gel solution with a concentration of 0.8 mg / ml. The solution was uniform without precipitation or flocs. The aqueous phase sample was directly used as the test solution.

[0220] (3) CCK-8 cell viability assay

[0221] Inoculate cells into 96-well plates, 200 μl of cell suspension per well (80,000 cells per well). Culture in an incubator for 24 hours, add different concentrations of the test substance to the culture plate, incubate for 24 hours, remove the supernatant and discard, add 100 μl of culture medium and 10 μl of CCK-8 solution mixture to each well, incubate for 2 hours, and measure the absorbance at 450 nm with an enzyme marker.

[0222] (4) Preparation of stimulant solution

[0223] Preparation of LPS stock solution (5 mg / ml): Dissolve 10 mg LPS in 2000 μl PBS, vortex mix for 5 min, filter with a 0.22 μm filter membrane for sterilization, and store in a -80°C ultra-low temperature refrigerator.

[0224] Preparation of LPS working solution (1 μg / ml): Prepare it at a dilution ratio of 1:5000 using high-glucose DMEM culture medium.

[0225] (5) Preparation of positive control solution:

[0226] Preparation of dexamethasone stock solution (100 mg / ml): Dissolve 100 mg of dexamethasone in 1000 μl of DMSO, shake for 2 hours, mix thoroughly, and package in 20 μl / tube volumes. Store at -80°C. After preparation, filter with a 0.22 μm filter membrane for sterilization and set aside.

[0227] (6) Cell inoculation

[0228] Dilute the cells with cell culture medium to the inoculation density (80,000 cells / well, 45%-60% confluence 24 hours after inoculation), and inoculate them into 96-well plates, with 200 μl per well. After inoculation, place in a CO2 incubator for 24 hours. Ensure that the confluence of the cells is in the range of 45-60% 24 hours after inoculation.

[0229] (7) Induction and administration

[0230] Discard the culture medium in the 96-well plate, add culture medium containing a certain concentration of test substance and LPS (1 μg / ml) to the test wells, add cell culture medium containing LPS to the negative control wells, add culture medium containing positive control and LPS to the positive control wells, and add cell culture medium to the blank / solvent control wells, 200 μl per well, and the concentration of acne repair gel is 3.2 mg / ml. After administration, place the 96-well plate in a CO2 incubator and culture for 24 hours.

[0231] (8) Collection of cell supernatant

[0232] After the incubation, collect 200 μl of cell culture supernatant into a 1.5 ml sterile centrifuge tube and place it in a -80°C ultra-low temperature refrigerator for cryopreservation.

[0233] (9) ELISA test:

[0234] a. Preparation before testing:

[0235] 1) Please take the kit out of the refrigerator 20 minutes in advance to equilibrate to room temperature.

[0236] 2) Dilute the 20× concentrated washing solution to 1× working solution with deionized water. Return the unused solution to 4°C.

[0237] 3) Standard: Centrifuge at 1000 rpm for 1 min before opening the lid. Add 0.5 ml of the universal diluent for standard and specimen to the freeze-dried standard, let it stand for 15 minutes, and mix gently after it is fully dissolved (concentration is 2000

[0238] pg / ml). Then dilute as needed (the following concentrations are used in the standard curve of this assay: 500, 250, 125, 62.5, 31.25, 15.625, 7.813 pg / ml).

[0239] 4) Biotinylated antibody working solution: Dilute the 30× concentrated biotinylated antibody into 1× working solution with biotinylated antibody diluent 20 minutes before use according to the amount required for the test. Use on the same day.

[0240] 5) Enzyme conjugate working solution: Dilute the 30× concentrated enzyme conjugate into 1× working solution with enzyme conjugate diluent 20 minutes before use according to the amount required for the test. Use on the same day.

[0241] b. Operation steps:

[0242] 1) Take out the required panels from the sealed bag that has been equilibrated to room temperature. Put the unused panels and desiccant back into the aluminum foil bag, press the self-sealing strip, seal the bag, and return it to 4°C.

[0243] 2) Add universal diluent for standards and specimens to the blank wells, add specimens or standards of different concentrations (100 μl / well) to the other corresponding wells, seal the reaction wells with sealing tape, and incubate in a 37°C incubator in the dark for 90 minutes.

[0244] 3) Prepare the biotinylated antibody working solution 20 minutes in advance.

[0245] 4) Wash the plate 5 times.

[0246] 5) Add biotinylated antibody diluent to the blank wells, and add biotinylated antibody working solution (100 μl / well) to the remaining wells. Seal the reaction wells with new sealing tape and incubate in a 37°C incubator in the dark for 60 minutes.

[0247] 6) Prepare the enzyme conjugate working solution 20 minutes in advance and place it at room temperature (22-25°C) away from light.

[0248] 7) Wash the plate 5 times.

[0249] 8) Add enzyme conjugate diluent to the blank wells and add enzyme conjugate working solution (100 μl / well) to the remaining wells.

[0250] Seal the reaction wells with new sealing tape and incubate in a 37°C incubator in the dark for 30 minutes.

[0251] 9) Turn on the power of the microplate reader, preheat the instrument, and set the detection program.

[0252] 10) Wash the plate 5 times.

[0253] 11) Add 100 μl / well of chromogenic substrate (TMB) and incubate in a 37°C incubator for 15 minutes in the dark.

[0254] 12) Add the reaction stop solution (100 μl / well), mix well and measure the OD immediately. 450nm Value (within 3 minutes). c. Result judgment:

[0255] The OD value of the blank well should be subtracted from the OD value of each standard and sample. With the concentration of the standard as the horizontal axis and the OD value as the vertical axis, the software draws and selects the best fitting curve. It is recommended to use a quadratic polynomial equation to fit. The concentration of the sample can be found on the standard curve through its OD value.

[0256] 9. Calculation of TNF-α, IL-1β and IL-6 content and inhibition rate

[0257] (1) Content

[0258] The standard solutions of TNF-α, IL-1β and IL-6 were prepared into standard solutions, and then diluted step by step into a series of solutions with known concentrations. The OD values ​​(OD) at 450 nm were measured by the above ELISA method. 450 ), the regression equation of the standard curve was fitted and the OD of each acne repair gel was calculated. 450 The values ​​were put into the equation to calculate the content of TNF-α, IL-1β and IL-6 in the acne repair gel. Three replicate wells were tested in each group in parallel, and the mean value and standard deviation (SD) were calculated. SPSS statistical software was used to perform t-test analysis on the TNF-α, IL-1β and IL-6 content of each sample group measured in the above test and the negative control group, and P < 0.05 was considered statistically significant.

[0259] (2) TNF-α, IL-1β and IL-6 inhibition rates

[0260] Inhibition rate (%) = (1-T / C) × 100%, wherein T is the average TNF-α, IL-1β or IL-6 content of each sample group, and C is the average TNF-α, IL-1β or IL-6 content of the negative control group.

[0261] Study test results

[0262] 1.CCK-8 cell viability assay

[0263] The effects of different doses of acne repair gel on RAW264.7 cell viability were tested using the CCK-8 kit as shown in Table 11:

[0264] Table 11. Comparison of the effects of different concentrations of acne repair gel on RAW264.7 cell viability

[0265]

[0266] According to the requirements of standard T / SHRH033-2020, the dosage concentration of acne repair gel should be controlled within the range that can ensure cell viability ≥ 90%. Therefore, 3.2 mg / ml was selected as the dosage concentration for subsequent inflammatory factor content testing.

[0267] 2. Detection of the content and inhibition rate of inflammatory factor TNF-α

[0268] Table 12. Inhibition of acne repair gel on LPS-induced secretion of inflammatory factor TNF-α by RAW264.7 cells

[0269]

[0270]

[0271] Note: Inhibition rate (%) = (1-T / C) × 100%

[0272] T——average value of TNF-α content in sample group

[0273] C——average value of TNF-α content in negative control group

[0274] Table 12 compares the content and inhibition rate of TNF-α secretion by LPS-induced RAW264.7 cells in each test group. The TNF-α secretion by LPS-induced RAW264.7 cells using 3.2 mg / ml acne repair gel was 74.10 pg / ml, significantly lower than the negative control group (119.76 pg / ml, p<0.05), and the TNF-α inhibition rate was 38.12%; the positive control group using 100 mg / ml dexamethasone was 37.25 g / ml, also significantly lower than the negative control group (p<0.05), and the TNF-α inhibition rate was 68.90%.

[0275] 3. Detection of the content and inhibition rate of inflammatory factor IL-1β

[0276] Table 13. Inhibition of acne repair gel on LPS-induced secretion of inflammatory factor IL-1β by RAW264.7 cells

[0277]

[0278] Note: Inhibition rate (%) = (1-T / C) × 100%

[0279] T——average value of IL-1β content in sample group

[0280] C——average IL-1β content in negative control group

[0281] Table 13 compares the content and inhibition rate of IL-1β secretion by RAW264.7 cells induced by LPS in each test group. The 3.2mg / ml acne repair gel was used to secrete 2.73pg / ml IL-1β from RAW264.7 cells induced by LPS, which was significantly lower than the negative control group (16.36pg / ml, p<0.05), and the IL-1β inhibition rate was 83.29%; the positive control group using 100mg / ml dexamethasone was 3.18pg / ml IL-1β secretion from RAW264.7 cells induced by LPS, which was also significantly lower than the negative control group (p<0.05), and the IL-1β inhibition rate was 80.56%.

[0282] 4. Detection of the content and inhibition rate of inflammatory factor IL-6

[0283] Table 14 Inhibition of acne repair gel on LPS-induced secretion of inflammatory factor IL-6 by RAW264.7 cells

[0284]

[0285] Note: Inhibition rate (%) = (1-T / C) × 100%

[0286] T——average IL-6 content in sample group

[0287] C——average IL-6 content in negative control group

[0288] Table 14 compares the content and inhibition rate of IL-6 secretion by LPS-induced RAW264.7 cells in each test group. The IL-6 secretion by LPS-induced RAW264.7 cells using 3.2 mg / ml acne repair gel was 9.87 pg / ml, significantly lower than the negative control group (16.17 pg / ml, p<0.05), and the IL-6 inhibition rate was 38.96%; the positive control group using 100 mg / ml dexamethasone was 0.11 pg / ml for LPS-induced RAW264.7 cells, which was also significantly lower than the negative control group (p<0.05), and the IL-1β inhibition rate was 99.31%. Summary:

[0289] Under the conditions of this experiment, 3.2 mg / ml of acne repair gel had an effect on the CCK-8 cell viability of RAW264.7 cells by 95.98%, and had an inhibitory effect on the secretion of TNF-α, IL-1β and IL-6 by RAW264.7 cells induced by LPS, with inhibition rates of 38.12%, 83.29% and 38.96%, respectively. The acne repair gel has soothing and anti-inflammatory effects.

[0290] Test Example 5: Safety Evaluation of Acne Repair Gel and Evaluation of Its Acne Removal Efficacy in Human Body

[0291] Purpose of the test

[0292] Asian adult subjects normally used the acne repair gel prepared in Example 2 for 14 days and 28 days, and dermatologists conducted clinical evaluations on the acne, papules, pustules, and nodules on the subjects' faces. The safety and acne removal efficacy of the acne repair gel were evaluated by high-definition facial photography and self-evaluation by the subjects.

[0293] Test Method

[0294] 1. Test sample: acne-repairing gel, prepared according to Example 2.

[0295] 2. Subjects: Asian adults, aged 20-35, including 22 males and 11 females, all of whom are prone to acne and have visible pimples on their faces (including whiteheads and blackheads). One week before the test, the subjects stopped using cosmetics or topical medications. The night before the test, the subjects did not apply any skin care products on their faces after washing their faces at home. On the morning of the test, the subjects did not apply any skin care products on their faces after washing their faces with clean water at home.

[0296] 3. Application area: acne and pimples.

[0297] 4. Directions: Squeeze out an appropriate amount of Anti-Acne Repair Gel and apply it to the acne area. Use it twice a day. Ask the subjects to return for a self-assessment on the 14th and 28th days of continuous use, and ask a dermatologist to conduct a clinical evaluation.

[0298] 5. Clinical evaluation of efficacy

[0299] On the 14th and 28th days of continuous use of the acne repair gel, dermatologists recorded the number of skin lesions (acne, papules, pustules, nodules) on the subjects' faces, and comprehensively evaluated the degree of inflammation and the number of skin lesions in the form of a total score.

[0300] Before the evaluation, the subjects rested for 30 minutes in an environment with a temperature of 21±1°C and a relative humidity of 50±10%RH. The dermatologist then used the facial high-definition imager VISIA-CR to collect facial images of the subjects.

[0301] The change rates of various facial lesions (acne, papules, pustules, nodules) at each time point were calculated. Each subject used the data collected before and after to conduct the Shapiro-Wilk normal distribution test and variance homogeneity test. After statistical significance, a paired sample t-test was performed, and the test level was a=0.05, and p<0.05 indicated that the difference was significant.

[0302] 6. Safety Assessment

[0303] Through the use of the subjects, the safety of the acne repair gel on human skin is tested (such as causing dry skin, scaling, redness, papules, etc.). The subjects are revisited, and any adverse events that occur during the use of the acne repair gel are carefully questioned, tested and recorded, including the discovery, occurrence time, treatment measures and outcome of the adverse events, and the relationship between the adverse events and the use of the acne repair gel is judged.

[0304] Table 15 Classification standards for skin reactions in human trial tests

[0305]

[0306] Note: The classification of adverse skin reactions shall be determined according to the skin reaction classification standards for human trial tests specified in the "Technical Specifications for Safety of Cosmetics" (2015 edition).

[0307] Table 16. Judgment criteria for the relationship between adverse events and test samples

[0308]

[0309] 7. Subject Self-Assessment

[0310] The subjects filled in a usage log based on their use of the acne repair gel for 14 and 28 consecutive days to record the adverse reactions caused to the skin.

[0311] Table 17. Subject self-assessment criteria

[0312]

[0313]

[0314] Test Results

[0315] 1. Skin can be clinically evaluated by a doctor

[0316] Table 18. Clinical evaluation of acne treatment efficacy

[0317]

[0318] Note: Change rate = [(after use - before use) / before use] × 100%

[0319] 2. Clinical safety evaluation

[0320] Table 19. Summary of adverse reactions

[0321]

[0322] During the entire test, 33 subjects did not feel any discomfort when using the acne repair gel.

[0323] 3. Subject Self-Assessment

[0324] Table 20. Subjects’ self-assessment satisfaction before using the sample

[0325]

[0326] Table 21. Subjects' self-assessment satisfaction on the 14th day of using the sample

[0327]

[0328]

[0329] Table 22. Subjects' self-assessment satisfaction on the 28th day of using the sample

[0330]

[0331] Table 23. Self-assessment of satisfaction of subjects on the 14th and 28th days of using the sample

[0332]

[0333] After using the acne repair gel for 28 days, the overall satisfaction of the 33 subjects with the acne repair gel was 56.6%.

[0334] in conclusion

[0335] Under the control of dermatologists, 33 Asian adult subjects aged 20-35 were observed to use the acne repair gel continuously for 28 days under normal conditions to evaluate the potential possibility of adverse reactions to human skin caused by the acne repair gel. The results showed that none of the 33 subjects experienced adverse reactions after using the acne repair gel continuously for 28 days, indicating that the acne repair gel has good safety.

[0336] 33 Asian adult subjects aged 20-35 years old used the acne repair gel continuously for 14 days and 28 days under normal circumstances. Dermatologists conducted clinical evaluations on the subjects' facial acne, papules, pustules, and nodules. The results showed that the acne repair gel had an acne removal effect after 14 days and 28 days.

[0337] Test Example 6: Human Evaluation of the Barrier Repairing Effect of Anti-Acne Repair Gel

[0338] Purpose of the test

[0339] The acne-repairing gel (prepared in Example 2) was used continuously by Asian adult subjects under normal conditions for 28 days, and the moisture content of the stratum corneum and the transepidermal water loss (TEWL) of the skin were measured to evaluate the moisturizing and repairing effects of the test sample.

[0340] Test Method

[0341] 1. Test sample: acne-repairing gel, prepared according to Example 2.

[0342] 2. Subjects: Healthy subjects, Asian healthy adults, aged 22-50 years old. Subjects self-assessed that their facial skin was sensitive skin, with the stratum corneum water content of one cheek area <60 C.U., and the transepidermal water loss rate of one cheek area >12 g / m 2 ·h, and the skin in the test area has no obvious skin lesions, scars, hair, etc. The subjects are required to cooperate with the test according to the requirements of the test plan and maintain a regular life during the test period.

[0343] 3. Application area: face.

[0344] 4. Usage: Apply all over the face, use twice without dosage, and use continuously for 28 days.

[0345] 5. Efficacy test

[0346] Before using the acne treatment and on the 28th day of continuous use of the acne repair gel, the dermatologist measured the water content of the stratum corneum on the same side of the cheek of the subjects using a skin moisture tester (Corneometer CM825, Courage+Khazaka, Germany). At the same time, the transepidermal water loss of the cheek skin of the subjects was tested using a skin moisture loss test probe (Tewameter TM300, Courage+Khazaka, Germany). Before the evaluation, the subjects rested for 30 minutes in an environment with a temperature of 21±1℃ and a relative humidity of 50±10%RH.

[0347] The water content of the stratum corneum of the facial skin and the rate of change of transepidermal water loss at each time point were calculated. The Shapiro-Wilk normal distribution test and variance homogeneity test were performed on the data collected before and after each subject. After statistical significance, paired sample t-test was performed, and the test level was a=0.05, and p<0.05 indicated that the difference was significant.

[0348] Test Results

[0349] Table 24. Comparison of skin physiology of subjects using acne repair gel for 28 days

[0350]

[0351] in conclusion

[0352] 30 Asian adult subjects aged 22-50 years old used the acne repair gel continuously for 28 days under normal conditions. The moisture content of the skin's stratum corneum and the transepidermal water loss (TEWL) value of the skin were tested. The results showed that the acne repair gel had moisturizing and repairing effects after 28 days.

Claims

1. A moisturizing, repairing, mild, non-irritating, and highly stable acne-repairing gel, wherein the active ingredients are composed of the following active ingredients in percentage by weight: Sophora flavescens extract preformulated solution 1.5-2.5%, Panax notoginseng extract preformed solution 4-6%, American cockroach extract 0.2-0.4%, colloidal sulfur 0.05-0.15%, encapsulated salicylic acid 0.2-0.4%, Portulaca oleracea extract mixture 0.12-0.8%, Centella asiatica glycoside 0.1-0.15%, dipotassium glycyrrhizinate 0.04-0.06%, ceramide NP mixture 0.8-1.5%, oligopeptide-1 mixture 0.4-0.6%; The pre-formulated solution of the Sophora flavescens extract is a mixed solution containing 1% of Sophora flavescens extract, 10% of 1,3-butylene glycol and 89% of propylene glycol; and The Panax notoginseng extract pre-formulated solution is a mixed solution containing 5% Panax notoginseng extract, 90% propylene glycol, and 5% PEG-40 hydrogenated castor oil; and The American cockroach extract is an extract powder of an American cockroach (PERIPLANETA AMERICANA) extract, which contains polyols and peptide active substances ≥ 82% and water ≤ 18%; and The colloidal sulfur is 75% elemental sulfur and 25% gum arabic; and The elemental sulfur is an octagonal ring structure consisting of 8 sulfur atoms; and The coated salicylic acid is a coated powder containing 40% salicylic acid (SALICYLIC ACID), 25% pullulan, 20% dextrin, and 15% xanthan gum; and The purslane extract mixture is a mixed solution containing 0.4% purslane (PORTULACAOLERACEA) extract, 69.6% water, and 30% butylene glycol; and The ceramide NP mixture is a mixed solution containing 58.8% water, 30% butanediol, 2.5% ceramide NP (CERAMIDE NP), 7% phosphatidylcholine, 0.5% sodium stearoyl glutamate, 1% 1,2-hexanediol, and 0.2% p-hydroxyacetophenone; and The oligopeptide-1 mixture is a mixed solution containing 39.5% water, 60% glycerol and 0.5% oligopeptide-1.

2. The moisturizing, repairing, mild, non-irritating, and highly stable acne-repairing gel according to claim 1 further comprises a matrix component, which is composed of the following matrix components in the following weight percentages: Aloe vera gel juice 35-80%, disodium EDTA 0.02-0.08%, carbomer 0.1-0.7%, sodium hyaluronate 0.02-0.08%, glycerol 1-4%, PHL 0.4-1.5%, 1,3-butylene glycol 2-8%, L-arginine 0.1-0.7%, water 3-7%.

3. The moisturizing, repairing, mild, non-irritating, and highly stable acne-repairing gel according to claim 2, wherein: The aloe vera gel juice is Aloe barbadensis leaf juice; and The sodium hyaluronate is an oligosaccharide with a molecular weight of 104 Da; and The PHL is a mixed solution of 65% 1,3-propylene glycol, 30% 1,2-hexanediol, and 5% octanoylhydroxamic acid; and The water is deionized water.

4. The moisturizing, repairing, mild, non-irritating, and highly stable acne-repairing gel according to claim 3, wherein the preparation method comprises the following steps: (1) After adding the amount of aloe vera gel juice to the emulsifier, add disodium EDTA and dipotassium glycyrrhizinate to the emulsifier and stir until they are completely dissolved; the temperature during this process is room temperature and the stirring speed is 20 rpm; (2) Dispersing the mixture of glycerol, carbomer and ceramide NP in the formula amount and then adding it to the emulsifying pot described in (1), stirring until the mixture is uniform; the temperature during the process is room temperature and the stirring speed is 20 rpm; (3) Add the pre-prepared solution of Sophora flavescens extract, the pre-prepared solution of Panax notoginseng extract, the encapsulated salicylic acid, sodium hyaluronate, the oligopeptide-1 mixture, the American cockroach extract, the Portulaca oleracea extract mixture, colloidal sulfur, Centella asiatica glycoside, PHL, and 1,3-butylene glycol in the formula amount into the emulsifying pot described in (2), and stir until the mixture is uniform; the temperature during the process is room temperature and the stirring speed is 20 rpm; (4) dissolving the L-arginine in a prescribed amount in water to prepare a solution; (5) adding the solution obtained in (4) to the emulsifying pot described in (3), stirring and mixing for 30 minutes to obtain a gel; during this process, the temperature was room temperature, the stirring speed was 20 rpm, and the vacuum degree was -0.06 MPa; (6) The gel obtained in step (5) is filtered through a 200-mesh filter cloth to obtain a transparent gel.

5. The moisturizing, repairing, mild, non-irritating, and highly stable acne-repairing gel according to claim 4, wherein the preparation method thereof further comprises the following steps: (7) The transparent gel prepared in (6) is tested for sensory, physical, chemical and microbiological indicators; the appearance should be transparent or translucent gel, free of foreign matter; conform to the specific aroma; pH value is 5.5-6.5; total colony count ≤ 500 CFU / g; mold and yeast ≤ 50 CFU / g; (8) Using a semi-automatic filling machine, fill the qualified transparent gel in (7) into a soft tube, and seal the soft tube with a sealing machine to obtain an acne-removing and repairing gel.

6. A method for preparing a moisturizing, repairing, mild, non-irritating, and highly stable acne-repairing gel according to any one of claims 2 to 3, comprising the following steps: (1) After adding the amount of aloe vera gel juice to the emulsifier, add disodium EDTA and dipotassium glycyrrhizinate to the emulsifier and stir until they are completely dissolved; the temperature during this process is room temperature and the stirring speed is 20 rpm; (2) Dispersing the mixture of glycerol, carbomer and ceramide NP in the formula amount and then adding it to the emulsifying pot described in (1), stirring until the mixture is uniform; the temperature during the process is room temperature and the stirring speed is 20 rpm; (3) Add the pre-prepared solution of Sophora flavescens extract, the pre-prepared solution of Panax notoginseng extract, the encapsulated salicylic acid, sodium hyaluronate, the oligopeptide-1 mixture, the American cockroach extract, the Portulaca oleracea extract mixture, colloidal sulfur, Centella asiatica glycoside, PHL, and 1,3-butylene glycol in the formula amount into the emulsifying pot described in (2), and stir until the mixture is uniform; the temperature during the process is room temperature and the stirring speed is 20 rpm; (4) dissolving the L-arginine in a prescribed amount in water to prepare a solution; (5) adding the solution obtained in (4) to the emulsifying pot described in (3), stirring and mixing for 30 minutes to obtain a gel; during this process, the temperature was room temperature, the stirring speed was 20 rpm, and the vacuum degree was -0.06 MPa; (6) filtering the gel obtained in step (5) through a 200-mesh filter cloth to obtain a transparent gel; (7) The transparent gel prepared in (6) is tested for sensory, physical, chemical and microbiological indicators; the appearance should be transparent or translucent gel, free of foreign matter; conform to the specific aroma; pH value is 5.5-6.5; total colony count ≤ 500 CFU / g; mold and yeast ≤ 50 CFU / g; (8) Using a semi-automatic filling machine, fill the qualified transparent gel in (7) into a soft tube, and seal the soft tube with a sealing machine to obtain an acne-removing and repairing gel.

7. Use of the moisturizing, repairing, mild, non-irritating and highly stable acne-repairing gel according to any one of claims 1 to 5 for preparing products for antibacterial, anti-inflammatory and soothing, skin barrier repair, improvement of excessive keratinization of hair follicles and skin oil control.

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