A traditional Chinese medicine composition with skin anti-inflammatory and repair effects and its application

Abstract

The present invention discloses a traditional Chinese medicine composition with skin anti-inflammatory and repair effects and its application. The traditional Chinese medicine composition includes extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens. Among them, the mass ratio of the extract of Hedyotis diffusa: the extract of Pyrola calliantha: the extract of Prunella vulgaris: the extract of Sophora flavescens = 1-100: 1-100: 1-50: 1-50, and its solid-liquid ratio is 0.001%-10%. The present invention prepares a traditional Chinese medicine composition with skin anti-inflammatory and repair effects through synergistic enhancement. Using the extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens as raw materials and with ingredient compatibility, it solves the problem of the anti-inflammatory efficacy limitation of a single raw material; preparing a traditional Chinese medicine composition of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens helps with the anti-inflammatory efficacy.

Description

Technical Field

[0001] The present invention belongs to the field of skin inflammation science, and particularly relates to a traditional Chinese medicine composition with skin anti-inflammatory and repair effects and its application. Background Art

[0002] Common skin phenomena such as damaged human skin barrier, allergy, and inflammation. When the skin is affected by stimulating factors such as ultraviolet rays, microorganisms, pollen, and improper use of cosmetics, symptoms such as skin redness, dry peeling, and burning pain occur. The main mechanism is the overexpression of inflammatory factors in skin cells (such as IL-6, IL-10, TNF-α, NO, etc.). Currently, anti-inflammatory active substances developed from natural and safe Chinese herbal medicine raw materials can be used to improve skin inflammation problems.

[0003] Skin inflammations such as rashes and atopic dermatitis are clinically characterized by skin erythema, redness, and dry peeling, often recurring, and having a serious impact on the quality of life and health status. In recent years, research has shown that microbial infectious factors, especially Staphylococcus aureus, can induce and exacerbate the inflammatory response. Currently, clinically, corticosteroid hormones and their compound preparations and other therapeutic drugs are mostly used for treatment. Due to their poor compliance and many side effects, the treatment effect is not good.

[0004] Hedyotis diffusa is a medicinal plant of the Rubiaceae family. It is cold in nature and bitter in taste, and has the functions of clearing heat and detoxifying. Hedyotis diffusa has good anti-inflammatory effects. Existing research has shown that the effective anti-inflammatory active ingredients of Hedyotis diffusa include flavonoids, terpenoids, phenylpropanoids, anthraquinones, sterols, etc. Clinically, it can be used to treat inflammation-related diseases such as appendicitis, hepatitis, and pelvic inflammation. Its active ingredients, pharmacological effects, and mechanism of action have become research hotspots. Pyrola calliantha is the dried whole herb of Pyrola calliantha of the Pyrolaceae family, with sweet and bitter tastes. Modern pharmacological research shows that Pyrola calliantha has multiple effects such as anti-infection and anti-inflammation, and has a wide range of pharmacological effects. Prunella vulgaris is the dried spike of Prunella vulgaris of the Lamiaceae family. It is cold in nature, bitter and pungent in taste, and has the effects of detumescence, anti-inflammation, and mass dissipation. It is often used in combination with other traditional Chinese medicines, and has no toxic or side effects. Sophora flavescens is the dried root of Sophora flavescens of the Fabaceae family, which is widely distributed and is a commonly used Chinese medicinal material in China, with a long history of medicinal use. A large number of modern pharmacological studies have found that Sophora flavescens has pharmacological effects such as anti-inflammation, anti-tumor, and antibacterial, and is widely used in the treatment of skin diseases, oncology, gynecology, and cardiovascular diseases, with significant effects and high safety.

[0005] Currently, there is no report on preparing a composition for treating skin inflammation by compounding Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. In this section, as well as in the abstract and title of the present application, some simplifications or omissions may be made to avoid obscuring the purpose of this section, the abstract, and the title, but such simplifications or omissions shall not be used to limit the scope of the present invention.

[0007] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0008] Therefore, an object of the present invention is to overcome the deficiencies in the prior art and provide a traditional Chinese medicine composition having a skin anti-inflammatory and repair effect.

[0009] To solve the above technical problems, the present invention provides the following technical solution: a traditional Chinese medicine composition having a skin anti-inflammatory and repair effect, the traditional Chinese medicine composition comprising extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens; wherein,

[0010] The mass ratio of the extract of Hedyotis diffusa: the extract of Pyrola calliantha: the extract of Prunella vulgaris: the extract of Sophora flavescens = 1 to 100: 1 to 100: 1 to 50: 1 to 50, and the solid-liquid ratio is 0.001% to 10%.

[0011] As a preferred embodiment of the traditional Chinese medicine composition of the present invention, wherein: the mass ratio of the extract of Hedyotis diffusa: the extract of Pyrola calliantha: the extract of Prunella vulgaris: the extract of Sophora flavescens = 1 to 50: 1 to 50: 1 to 10: 1 to 20; and the final solid-liquid ratio is 0.001% to 1%.

[0012] As a preferred embodiment of the traditional Chinese medicine composition of the present invention, wherein: the mass ratio of the extract of Hedyotis diffusa: the extract of Pyrola calliantha: the extract of Prunella vulgaris: the extract of Sophora flavescens = 5: 2: 1: 15.

[0013] As a preferred embodiment of the traditional Chinese medicine composition of the present invention, wherein: the mass ratio of the extract of Hedyotis diffusa: the extract of Pyrola calliantha: the extract of Prunella vulgaris: the extract of Sophora flavescens = 20: 5: 5: 6.

[0014] As a preferred embodiment of the traditional Chinese medicine composition of the present invention, wherein: the mass ratio of the extract of Hedyotis diffusa: the extract of Pyrola calliantha: the extract of Prunella vulgaris: the extract of Sophora flavescens = 12: 12: 4: 3.

[0015] As a preferred embodiment of the traditional Chinese medicine composition of the present invention, wherein the preparation methods of the extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens include,

[0016] The raw materials are separately pulverized, screened through a 10-120 mesh sieve, and extracted with a 70-95% ethanol-aqueous solution for 1-2 h, with a solid-liquid ratio of 1:5-20. Ultrasonic extraction is carried out at 20-25 °C for 1-3 times, with each extraction lasting 30-40 min;

[0017] The filtrate is obtained by filtration, rotary evaporation is performed, and it is dried at 50-60 °C for 1-2 days to obtain the extract.

[0018] As a preferred embodiment of the traditional Chinese medicine composition of the present invention, the solvent of the traditional Chinese medicine composition includes pure water and absolute ethanol.

[0019] Another object of the present invention is to overcome the deficiencies in the prior art and provide a preparation method of a traditional Chinese medicine composition with skin anti-inflammatory and repair effects, including,

[0020] The extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens are added to a solvent and mixed evenly to prepare the composition; wherein, the solvent includes pure water and absolute ethanol.

[0021] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of a traditional Chinese medicine composition in the preparation of skin care products with anti-inflammatory and skin damaged barrier repair effects. The addition amount of the traditional Chinese medicine composition in the skin care products is 0.1%-10% by mass fraction, and the skin care products include essence water, lotion, and cream.

[0022] Beneficial effects of the present invention:

[0023] (1) The present invention prepares a traditional Chinese medicine composition with skin anti-inflammatory and repair effects through synergistic enhancement. Using the extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens as raw materials and formulating the ingredients, it solves the problem of the anti-inflammatory effect limitation of a single raw material; preparing a traditional Chinese medicine composition of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens helps to exert the anti-inflammatory effect.

[0024] (2) The present invention is not simply a combination of the extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens, but through relevant inflammation experiments, it is confirmed that the combined score of the extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris, and Sophora flavescens is greater than 10, showing a significant synergistic enhancement of the anti-inflammatory effect; by further selecting excipients allowed to be used in cosmetics and drugs, it can be made into a dosage form acceptable for cosmetics and drugs, and applied to the fields of cosmetics for improving skin inflammation and skin barrier repair and skin external medicines.

[0025] (3) The traditional Chinese medicine composition with skin anti-inflammatory and repair effects provided by the present invention is simple to prepare, selects natural plant ingredients, is safe and has no toxic side effects, and is worthy of popularization and application. Description of the Drawings

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings. Among them:

[0027] Figure 1 It is the effect curve diagram of the DPPH free radical scavenging rate of a single herb in the traditional Chinese medicine composition of Embodiment 1 of the present invention;

[0028] Figure 2 It is the effect curve diagram of the ABTS free radical scavenging rate of a single herb in the traditional Chinese medicine composition of Embodiment 1 of the present invention;

[0029] Figure 3 It is the effect curve diagram of the NO free radical scavenging rate of a single herb in the traditional Chinese medicine composition of Embodiment 1 of the present invention;

[0030] Figure 4 It is the result diagram of the DPPH free radical scavenging rate after the compounding of the traditional Chinese medicine composition in Embodiment 1 of the present invention;

[0031] Figure 5 It is the synergy effect diagram obtained by calculating the DPPH free radical scavenging rate using the ZIP model in statistics;

[0032] Figure 6 It is the result diagram of the ABTS free radical scavenging rate after the compounding of the traditional Chinese medicine composition in Embodiment 1 of the present invention;

[0033] Figure 7 It is the synergy effect diagram obtained by calculating the ABTS free radical scavenging rate using the ZIP model in statistics;

[0034] Figure 8 It is the result diagram of the NO free radical scavenging rate after the compounding of the traditional Chinese medicine composition in the embodiments of the present invention;

[0035] Figure 9 It is the synergy effect diagram obtained by calculating the NO free radical scavenging rate using the ZIP model in statistics;

[0036] Figure 10 It is the cytotoxicity diagram of different ratios of the traditional Chinese medicine composition in the embodiments of the present invention;

[0037] Figure 11 It is the schematic diagram of the inhibition rate of the traditional Chinese medicine composition in the embodiments of the present invention on the inflammatory factor NO;

[0038] Figure 12 It is the schematic diagram of the inhibition rate of the traditional Chinese medicine composition in the embodiments of the present invention on the inflammatory factor NO;

[0039] Figure 13This is the result diagram of the chicken embryo chorioallantoic membrane vascular test in the embodiments of the present invention. Detailed implementation manners

[0040] To make the above objects, features and advantages of the present invention more obvious and understandable, the following describes the detailed implementation manners of the present invention in combination with the embodiments of the specification.

[0041] In the following description, many specific details are set forth to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0042] Secondly, the so-called "one embodiment" or "embodiment" herein refers to specific features, structures or characteristics that can be included in at least one implementation manner of the present invention. The "in one embodiment" appearing in different places in this specification does not all refer to the same embodiment, nor is it a separate or alternative embodiment that mutually excludes other embodiments.

[0043] The raw materials in the present invention are all ordinary commercially available products.

[0044] Preparation methods of extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris and Sophora flavescens in the present invention:

[0045] The raw materials are respectively crushed, passed through a 100-mesh sieve, extracted with 70% ethanol-aqueous solution for 2 h, the solid-liquid ratio is 1:20, ultrasonically extracted 3 times at 25 °C, and each extraction is 40 min;

[0046] Filter to obtain the filtrate, perform rotary evaporation, and dry at 60 °C for 2 days to obtain the extract.

[0047] Example 1

[0048] This example provides a traditional Chinese medicine composition with skin anti-inflammatory and repair effects. The efficacy extract is dissolved in 70% ethanol. The concentrations of extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris and Sophora flavescens after dissolution are all 10 mg / mL, which are experimental samples.

[0049] (1) Experimental materials and instruments:

[0050] DPPH, ABTS, sodium nitroferricyanide dihydrate (SNP) are all provided by Shanghai Aladdin Biochemical Technology Co., Ltd., RAW264.7 cell line is provided by Beijing NaChuangLian Biotechnology Co., Ltd., DMEM high glucose (10% FBS, 1% double antibody), 96- and 24-well plates, lipopolysaccharide LPS, etc.

[0051] STERI-CYCLE i160 cell culture incubator, Synergy-H4 multi-functional microplate reader, inverted microscope, 1300SERIES A2 laminar flow hood, etc.

[0052] (2) Detection of free radical scavenging ability of plant formula:

[0053] (I) Determination of DPPH free radical scavenging rate:

[0054] DPPH is a stable free radical that can exist stably and can damage cells and tissues, causing diseases. In the presence of free radical scavengers, its single electron is scavenged and the color becomes lighter. The absorbance value at the maximum light absorption wavelength of 517 nm decreases. The lower the absorbance level and the greater the inhibition rate, the stronger the antioxidant property. Based on this, the antioxidant ability of the test sample is judged.

[0055] The test is carried out according to the following method:

[0056] Solution preparation: Weigh 1.2 mg of DPPH solid and dissolve it in 10 mL of alcohol. The absorbance at 517 nm is 1.6 - 1.65. Prepare it freshly for use and take the test sample solution in Example 1 above.

[0057] For the positive control group, dissolve 25 mg of VC in 1 mL of water, dilute it to a concentration of 25 μg / mL, and then serially dilute it to aqueous solutions with concentrations of 12.5, 6.26, 3.13, and 1.5625 μg / mL. Add each reagent according to Table 1 below. React in the dark for 30 min and measure the absorbance values of A, B, C, and D at 517 nm.

[0058] Table 1 DPPH experimental operation steps

[0059] DPPH solution Solvent (methanol) Sample solution Sample solvent (ethanol) A 100 μL 0 μL 100 μL 0 μL B 0 μL 100 μL 100 μL 0 μL C 100 μL 0 μL 0 μL 100 μL D 0 μL 100 μL 0 μL 100 μL

[0060] Calculation formula:

[0061] DPPH free radical inhibition rate (%) = [1 - (A - B / C - D)] × 100%

[0062] (II) Determination of ABTS free radical scavenging rate

[0063] ABTS is oxidized to green ABTS cationic free radical under the action of potassium persulfate. In the presence of antioxidants, this free radical is inhibited. The antioxidant ability of the sample can be calculated and evaluated by measuring the absorbance at 734 nm.

[0064] The test is carried out according to the following method:

[0065] Solution preparation: Weigh 6.6 mg of potassium persulfate solid and dissolve it in 10 mL of deionized water to obtain a 2.45 mM potassium persulfate solution; weigh 7.7 mg of ABTS and dissolve it in 2 mL of the potassium persulfate solution, and use it after reacting for 16 h.

[0066] Dilute the concentrated ABTS solution with deionized water until the absorbance at 734 nm is 0.9. Prepare it immediately before use, and take the test sample solution in the above Experimental Example 1.

[0067] Add each reagent according to Table 2 below. React in the dark for 10 min, and measure the absorbance value of AB at 734 nm.

[0068] Table 2 ABTS experimental operation steps

[0069] ABTS solution Sample solution Sample solvent (ethanol) A 150 μL 50 μL 0 μL B 150 μL 0 μL 50 μL

[0070] Calculation formula:

[0071] DPPH free radical inhibition rate (%) = (1 - A / B) × 100%

[0072] (III) Determination of NO free radical scavenging rate:

[0073] Use the Griess colorimetric method to determine the in vitro NO scavenging activity: Sodium nitroprusside (SNP) is dissolved in PBS solution and decomposes to produce nitric oxide after light irradiation. NO and oxygen react with water through a series of reactions to generate nitrite ions and nitrate ions in the solution. Add Griess reagent A and Griess reagent B to the solution successively, and finally a diazo compound is formed, and the solution turns pink. The absorbance value can be measured at 550 nm, which can reflect the amount of NO produced by the decomposition of SNP.

[0074] Perform the test according to the method in Table 3 below:

[0075] 50 μL of 0.5 mM SNP solution + 50 μL of the test sample solution in the above Experimental Example 1, irradiate under UVB of a UV crosslinker for 60 min, first add Griess reagent A, and detect the absorbance value at 550 nm with an enzyme-linked immunosorbent assay reader as the blank group. Then add Griess reagent B, react in the dark for 10 minutes, and detect the absorbance value at 550 nm with an enzyme-linked immunosorbent assay reader.

[0076] Table 3 NO experimental operation steps

[0077]

[0078] Calculation formula:

[0079] NO inhibition rate (%) = [1 - (As - Ab) / (Ac - Acb)] × 100

[0080] A is the absorbance value of the experimental group; Ac is the absorbance value of the control group; Ab is the absorbance value of the blank group.

[0081] For the graph showing the DPPH radical scavenging rate effect of each single herb in the traditional Chinese medicine composition of this embodiment, see Figure 1 ; For the graph showing the ABTS radical scavenging rate effect of each single herb in the traditional Chinese medicine composition of this embodiment of the present invention, see Figure 2 ; For the graph showing the NO radical scavenging rate effect of each single herb in the traditional Chinese medicine composition of this embodiment of the present invention, see Figure 3 .

[0082] Compound of the traditional Chinese medicine composition with skin anti-inflammatory and repair effects:

[0083] (3) Explore the synergistic antioxidant capacity of extracts of Hedyotis diffusa (BHS), Pyrola calliantha (LXC), Prunella vulgaris (XKC) and Sophora flavescens (KS). The four samples in Experimental Example 1 were compounded in pairs according to a ratio of 1:1 (v / v): Hedyotis diffusa: Pyrola calliantha = 1:1, Hedyotis diffusa: Prunella vulgaris = 1:1, Hedyotis diffusa: Sophora flavescens = 1:1, Pyrola calliantha: Prunella vulgaris = 1:1, Pyrola calliantha: Sophora flavescens = 1:1, Prunella vulgaris: Sophora flavescens = 1:1. After being diluted to the corresponding concentrations respectively, their in vitro antioxidant activities were determined by DPPH, ABTS and NO methods, and the Synergy Finder software was further used to evaluate the synergistic effect by the ZIP model.

[0084] This software is an interactive analysis and visualization web application that can be used for multi-drug (two or more drugs) multi-dose combination response data. When evaluating the potential synergistic effect of combined drugs, usually the drug combination response is further compared with the expected combination response calculated by the synergistic scoring model. Thus, according to the deviation between the observed response and the expected response, the drug combination can be classified as synergistic or antagonistic. Among them, the synergistic effect score is shown as a surface plot of the delta score (δ-score) of an algorithm obtained by selecting one of the four reference models (HSA, Loewe, Bliss and ZIP models), and shows the average over-response caused by drug interaction. δ-score > 10, < -10 and between -10 and 10 indicate that the drug combination has synergistic, antagonistic and additive effects respectively

[0085] The results show that extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris and Sophora flavescens all have certain antioxidant capacities, such as Figure 4 , 6 , as shown in Figure 8, the graphs of the scavenging rates of DPPH radicals, ABTS cation radicals and NO radicals after compounding the extracts at different concentrations in a ratio of 1:1;

[0086] Figure 5 ,7 Figures 9 are the collaborative effect diagrams of their corresponding ZIP models. Among them, red indicates synergistic effect, green indicates antagonistic effect, and the darker the color, the stronger the synergistic and antagonistic effects. The average synergistic value greater than 10 in the figure indicates a synergistic effect. The concentration corresponding to the area with the darkest color (marked with a black arrow) is selected as the subsequent compounding concentration.

[0087] Figure 5 Among them, the mass ratio is BHS:LXC:XKC:KS = 125:50:25:375 = 5:2:1:15; Figure 7 Among them, the mass ratio is BHS:LXC:XKC:KS = 62.5:15.625:18.75:18.75 ≈ 20:5:5:6; Figure 9 Among them, the mass ratio is BHS:LXC:XKC:KS = 150:150:50:37.5 = 12:12:4:3. Therefore, the following mass ratio is selected:

[0088] Hedyotis diffusa extract: Pyrola calliantha extract: Prunella vulgaris extract: Sophora flavescens extract mass ratio = 5:2:1:15;

[0089] Hedyotis diffusa extract: Pyrola calliantha extract: Prunella vulgaris extract: Sophora flavescens extract mass ratio = 20:5:5:6;

[0090] Hedyotis diffusa extract: Pyrola calliantha extract: Prunella vulgaris extract: Sophora flavescens extract are compounded and used in a mass ratio of 12:12:4:3, and have strong synergistic antioxidant ability.

[0091] Example 2

[0092] A traditional Chinese medicine composition with skin anti-inflammatory and repair effects, the efficacy extract is dissolved in 0.3% DMSO, and is mixed in the following ratio: Hedyotis diffusa extract: Pyrola calliantha extract: Prunella vulgaris extract: Sophora flavescens extract mass ratio = 5:2:1:15

[0093] Example 3

[0094] A traditional Chinese medicine composition with skin anti-inflammatory and repair effects, the efficacy extract is dissolved in 0.3% DMSO, and is mixed in the following ratio: Hedyotis diffusa extract: Pyrola calliantha extract: Prunella vulgaris extract: Sophora flavescens extract mass ratio = 20:5:5:6

[0095] Example 4

[0096] A traditional Chinese medicine composition with skin anti-inflammatory and repair effects, the efficacy extract is dissolved in 0.3% DMSO, and is mixed in the following ratio: Hedyotis diffusa extract: Pyrola calliantha extract: Prunella vulgaris extract: Sophora flavescens extract mass ratio = 12:12:4:3

[0097] Example 5

[0098] Cytotoxicity detection of a traditional Chinese medicine composition with skin anti - inflammatory and repair effects:

[0099] Experimental steps:

[0100] Set up a blank group, a model group, and samples of Examples 2 - 4.

[0101] (1) Cell culture: In a 5% CO2, 37°C incubator, use DMEM high - glucose medium to culture RAW264.7 cells until the cell density reaches 80% - 90%.

[0102] (2) Cell seeding: Wash down the adherent cells according to the above steps, centrifuge and resuspend for counting, dilute to a cell density of 105 cells / mL, and add 100 μL of cell suspension to each well of a 96 - well plate.

[0103] (3) Cell drug administration: After culturing for 24 h, discard the medium in each well, add 100 μL of drug - containing medium respectively, continue to culture for 24 h, add 0.5 mg / mL MTT solution, discard the supernatant after culturing for 4 h, add DMSO, and measure the absorbance value (OD) of the sample at a wavelength of 490 nm.

[0104] (4) Data processing: Calculate the cell survival rate in the sample according to the absorbance.

[0105] (5) Experimental results: As Figure 10 shown, at the same experimental concentration, the cell activity of Example 2 is lower than 80%, and Examples 3 - 4 do not show cytotoxicity.

[0106] Example 6

[0107] NO inhibition rate detection of a traditional Chinese medicine composition with skin anti - inflammatory and repair effects:

[0108] Experimental steps:

[0109] Set up a blank group, a model group, and samples of Examples 3 - 4.

[0110] (1) Cell culture: In a 5% CO2, 37°C incubator, use DMEM high - glucose medium to culture RAW264.7 cells until the cell density reaches 80% - 90%.

[0111] (2) Cell seeding: Wash down the adherent cells according to the above steps, centrifuge and resuspend for counting, dilute to a cell density of 10 5 cells / mL, and add 500 μL of cell suspension to each well of a 24 - well plate.

[0112] (3) Cell administration: After culturing for 24 h, discard the culture medium in each well, add 500 μL of drug-containing culture medium respectively. After culturing for 1 h, discard the culture medium. Finally, add 500 μL of drug-containing + 1 μg / mL LPS culture medium respectively and continue culturing for 24 h.

[0113] (4) Sample determination: Take 50 μL of the centrifuged cell supernatant, add it to a 96-well plate, and successively add 50 μL of Griess A and Griess B. Measure the absorbance value (OD) of the sample at a wavelength of 540 nm.

[0114] (5) Data processing: Calculate the concentration of NO in the sample based on the absorbance.

[0115] Experimental results: As Figure 11 、 12 shown, the NO clearance rate of Example 3 is 43.4% - 61.8%, and the NO clearance rate of Example 4 is 45.7 - 65.8%. Both have a significant effect on inhibiting NO production and have a good anti-inflammatory effect.

[0116] Example 7

[0117] Safety evaluation of a traditional Chinese medicine composition with skin anti-inflammatory and repair effects:

[0118] Experimental steps:

[0119] (1) Human skin patch test

[0120] Conducted according to the method specified in the 2015 Cosmetics Technical Specification.

[0121] Volunteer requirements: 30 normal subjects (aged 20 - 40), without clinically unresolved inflammatory skin diseases, skin test sites without scars, pigmentation, atrophy, nevus flammeus or other defects, not participating in other clinical trials, not highly sensitive in constitution, and not having had a patch test in the past month.

[0122] Experimental method: Dilute the traditional Chinese medicine prescriptions prepared in Examples 3 and 4 successively with deionized water to obtain 10%, 5.0%, 1.0%, 0.5%, and 0.1% traditional Chinese medicine prescriptions. Add the above solutions to the filter paper attached to the patch tester and place them in the patch tester, with a dosage of about 0.020 - 0.025 mL. The control well is a blank control (deionized water). Apply the patch tester with the test substance to the flexor side of the forearm with non-irritating tape, and gently press with the palm to make it evenly adhere to the skin for 24 h. Observe the skin reaction 30 min after removing the test substance patch tester, and wait for the indentation to disappear. If the result is negative, observe again at 24 h and 48 h after the patch test and record the reaction results. The results are shown in Table 4.

[0123] Table 4 Results of human skin patch test

[0124]

[0125] Note: -(0): Negative reaction; ±(1): Suspected reaction (weak erythema); +(2): Weak positive reaction (erythema reaction); erythema, infiltration, edema

[0126] Experimental results: All 30 subjects in the human patch test showed grade 0 reaction. According to the "Hygiene Standard for Cosmetics 2015", the test samples did not cause adverse reactions to human skin. Thus, it can be seen that the safety of this traditional Chinese medicine formula is very high and there is no risk of irritation.

[0127] (2) Chicken embryo chorioallantoic membrane vascular assay

[0128] The chicken embryo chorioallantoic membrane vascular assay evaluates irritation by detecting the damage of chemicals to the chorioallantoic membrane.

[0129] Experimental design: 1. Positive control: 0.1N sodium hydroxide (NaOH) solution, 1% SDS (Dodecyl Sulfate Sodium Salt) solution;

[0130] 2. Negative control: 0.9% sterile normal saline (NaCl) solution;

[0131] 3. Test sample: The traditional Chinese medicine formula prepared in Examples 3 and 4 was formulated into a 5% aqueous solution;

[0132] Experimental results: As Figure 13 shown, in the HET-CAM assay, there was no obvious change in the 0.9% sterile normal saline; capillary bleeding gradually occurred in the 1% SDS solution; in the 0.1N sodium hydroxide (NaOH) solution, the edges of some thinner blood vessels gradually became blurred at 30 s, the thin blood vessels began to disappear at 3 min, and severe bleeding and gradual blurring of the main blood vessels occurred at 5 min; there was no obvious change in the 5% traditional Chinese medicine composition.

[0133] Therefore, the traditional Chinese medicine composition at a high concentration (5%) showed no obvious irritation.

[0134] Example 8

[0135] Application of a traditional Chinese medicine composition with skin anti-inflammatory and repair effects:

[0136] The traditional Chinese medicine formula has good safety and stability, and has good effects in anti-inflammatory, anti-inflammatory and repair aspects. It can be applied to emulsion skin care products with anti-inflammatory and repair effects, as shown in Table 5.

[0137] Table 5 Anti-inflammatory and repair emulsion

[0138]

[0139] Sample A (the emulsion prepared above with the traditional Chinese medicine formula ratio of Example 3), Sample B (the emulsion prepared above with the traditional Chinese medicine formula ratio of Example 4) were used as the experimental groups, and Sample C (the basic emulsion formula) was used as the control group. After the skin of both arms of the subjects was stimulated with SDS for 15 minutes, Sample A and C were used on one arm, and Sample B and C were used as a control on the other arm to compare the changes in skin physiological indexes before and after the subjects used the products and relative to the control group. Mark the measurement area on the inner side of the forearm of the subjects. At least 30 minutes of sitting quietly should be done in a standard room before the formal test. The forearm should be exposed and placed in a test state, keeping relaxed. In the experiment, mark a 3×3 cm 2 experimental area on the inner side of the forearm. Multiple areas can be marked on the same arm at the same time, with an interval of 1 cm between areas. The test products and the blank control are randomly distributed on the forearm. First, measure the blank value 30 minutes after cleaning each test area. Add 0.2 g of the test sample amount, and evenly coat the test sample on the experimental area. After smearing, measure the skin heme content of the test area and the control area at 0.5, 1, 1.5, and 2 hours respectively.

[0140] Table 6 Analysis and test of skin heme content

[0141]

[0142]

[0143] Note: In Table 6, the blank spaces are replaced by " / ", "-" represents P>0.05, "*" represents P<0.05, and "**" represents P<0.01.

[0144] The skin heme content was measured by Mexameter MX 18. The larger the value, the higher the skin heme content, and vice versa, the lower the heme content. The less the skin heme content, the better the soothing effect of the sample on skin irritation.

[0145] It can be seen from Table 6 that after the subjects were smeared with SDS to stimulate the skin, the heme content increased. After using the samples in the test areas of Sample A, Sample B, and Sample C, the skin heme content values were all lower than before. Among them, the skin heme content of using Sample A and B was significantly lower than that of Sample C, indicating that the soothing effects of Sample A and B were significantly higher than that of Sample C, and had a good effect on skin repair, thus improving the anti-inflammatory repair ability of the skin.

[0146] The present invention uses Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris and Sophora flavescens as raw materials. Based on the skin inflammation effects of the four, they are rationally combined to produce a synergistic effect. The combination of the four raw material components solves the problem of the anti-inflammatory effect limitation of a single raw material. A traditional Chinese medicine composition of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris and Sophora flavescens is prepared. Through experimental verification, the combined score of the four is >10, showing a significant synergistic anti-inflammatory effect. The purpose of the present invention is to overcome the deficiencies in the prior art and provide a preparation method of a traditional Chinese medicine formula for anti-inflammatory repair. By further selecting excipients permitted by cosmetics and drug regulations, it can be made into a dosage form acceptable for cosmetics and drugs, and applied to the fields of cosmetics for improving skin inflammation and skin barrier repair, as well as topical skin medications.

[0147] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the present invention.

Claims

1. A traditional Chinese medicine composition with skin anti-inflammatory and repair effects, characterized in that: [[ID=|1]] The traditional Chinese medicine composition is composed of extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris and Sophora flavescens; wherein, The mass ratio of the extract of Hedyotis diffusa: the extract of Pyrola calliantha: the extract of Prunella vulgaris: the extract of Sophora flavescens = 20:5:5:6 or 12:12:4:3; Wherein, the preparation methods of the extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris and Sophora flavescens are as follows: The raw materials are respectively crushed, passed through a 10-120 mesh sieve, leached with a 70-95% ethanol-aqueous solution for 1-2 h, the solid-liquid ratio is 1:5-20, ultrasonically extracted at 20-25 °C for 1-3 times, and each extraction is 30-40 min; Filter to obtain the filtrate, perform rotary evaporation, and dry at 50-60 °C for 1-2 days to obtain the extract.

2. The preparation method of the traditional Chinese medicine composition according to claim 1, characterized in that: Including, Add the extracts of Hedyotis diffusa, Pyrola calliantha, Prunella vulgaris and Sophora flavescens into a solvent and mix evenly to obtain the composition; wherein, the solvent is an ethanol solution with a concentration of 70%.

3. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of skin care products with anti-inflammatory and skin damaged barrier repair effects.

4. The application according to claim 3, characterized in that: The addition amount of the traditional Chinese medicine composition in the skin care products is 0.1%-10% by mass fraction, and the skin care products include essence water, emulsion and cream.

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