Capsaicin stimulation inhibitor for cosmetics as well as preparation method and application of capsaicin stimulation inhibitor

Abstract

The invention discloses a capsaicin stimulation inhibitor for cosmetics and a preparation method and application thereof.The preparation method of the capsaicin stimulation inhibitor for cosmetics comprises the following steps that A, medicinal materials are soaked in an extraction solvent with the weight being 15-20 times that of the medicinal materials for 2-16 h, and a soaking solution is obtained; the extraction solvent is an aqueous alkali solution with the concentration of 0.2 mol / L-0. 8mol / L, and the medicinal materials are at least one of calendula officinalis, purslane and radix scrophulariae; b, extracting the soak solution for 1-3 hours under the condition of 95-105 DEG C; the capsaicin stimulation inhibitor for cosmetics prepared by the preparation method disclosed by the invention can obviously inhibit secretion of keratinocyte IL-1alpha and expression of TRPV-1 protein caused by capsaicin stimulation, and the combination of every two inhibitors has a certain synergistic effect; therefore, the compound can be applied to inhibition of TRPV1 expression induced by capsaicin, inhibition of IL-1alpha secretion induced by capsaicin and preparation of skin external preparations.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a capsaicin irritation inhibitor for cosmetics and a preparation method and application thereof. Background Art

[0002] TRPV1, the full name of Transient Receptor Potential Vanilloid 1, is a non-selective cation channel belonging to the vanilloid subfamily of the transient receptor potential (TRP) ion channel family. It was originally discovered as a capsaicin receptor, so it is also called capsaicin receptor or vanilloid receptor 1. TRPV1 can be activated by a variety of physical and chemical stimuli, such as high temperature (>43°C), endogenous lipid molecules and exogenous ligands, and is involved in a variety of physiological and pathological processes such as pain perception, temperature regulation, and inflammatory response. TRPV1 is one of the targets in the cosmetics field that is most closely related to allergic reactions such as skin burning and stinging. Screening plant extracts and combinations that inhibit TRPV1 expression is an important means to develop anti-irritation and anti-sensitivity agents for cosmetics. The present invention is based on this technical means, and extracts from different plants and processes have been screened to find plant extracts and combinations with specific anti-allergic effects.

[0003] The expression of TRPV1 in sensitive skin increases significantly, making the skin more sensitive to various stimuli, causing discomfort such as tingling and itching. Therefore, the use of skin care products that can inhibit the expression of TRPV1 receptors is very important for alleviating the symptoms of sensitive skin. Secondly, TRPV1 is involved in the occurrence and development of a variety of inflammatory skin diseases. The activation of TRPV1 can lead to the release of neuropeptides or proinflammatory transmitters, such as prostaglandin E2 (PGE2), interleukins and other inflammatory factors related to skin inflammatory reactions, inducing the occurrence and development of skin inflammation. In summary, TRPV1 is of great significance in the research of skin care products. It is not only related to the symptom relief of sensitive skin, but also involves the treatment and links of inflammatory skin diseases. Therefore, targeting TRPV1 to develop skin care ingredients is expected to be a new solution for the care and treatment of sensitive and inflammatory skin.

[0004] IL-1α is a pro-inflammatory cytokine secreted by a variety of cells (such as macrophages, monocytes, endothelial cells, keratinocytes, etc.) when stimulated (such as infection, tissue damage, etc.). It mainly exerts its biological effects by activating the NF-κB and MAPK signaling pathways, thereby inducing the production of other cytokines, promoting the activation and migration of immune cells, and participating in the regulation of inflammatory and immune responses. For example, when the skin is stimulated or damaged, IL-1α can stimulate monocytes, neutrophils, and fibroblasts to secrete pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α, which further promote the occurrence and development of inflammatory responses. In addition, IL-1α can also attract neutrophils, causing the release of inflammatory mediators, thereby exacerbating the inflammatory response.

[0005] In summary, IL-1α is an important proinflammatory cytokine that plays an important role in inflammatory response, immune response and tissue repair. Studying the biological role and clinical application of IL-1α is of great significance for understanding the pathogenesis of diseases and developing new treatments. Summary of the invention

[0006] The present invention has found through research that the capsaicin stimulation inhibitor for cosmetics obtained by selecting specific ingredients to be prepared alone, in combination of two or three ingredients can improve the inhibition rate of capsaicin-induced TRPV1 expression and the inhibition rate of capsaicin-induced IL-1α secretion. Therefore, the first purpose of the present invention is to provide a method for preparing a capsaicin stimulation inhibitor for cosmetics; the second purpose of the present invention is to provide a capsaicin stimulation inhibitor for cosmetics prepared by the method for preparing the capsaicin stimulation inhibitor for cosmetics described above; the third purpose of the present invention is to provide the use of the capsaicin stimulation inhibitor for cosmetics described above in inhibiting capsaicin-induced TRPV1 expression. The fourth purpose of the present invention is to provide the use of the capsaicin stimulation inhibitor for cosmetics described above in inhibiting capsaicin-induced IL-1α secretion.

[0007] To achieve the above object, the present invention adopts the following technical solution: As a first aspect of the present invention, a method for preparing a capsaicin irritation inhibitor for cosmetics comprises the following steps: A. Soaking the medicinal material in 15-20 times the weight of an extraction solvent for 2-16 hours to obtain an immersion solution; the extraction solvent is a 0.2 mol / L-0.8 mol / L alkaline aqueous solution, and the medicinal material is at least one of calendula, purslane, and Scrophularia; B. Extract the soaked solution at 95-105℃ for 1-3h.

[0008] According to the present invention, the following steps are also included after step B: C. Remove the filter residue, decolorize, filter until clear, desalt, and concentrate to obtain an extract to prepare a capsaicin irritation inhibitor for cosmetics.

[0009] Preferably, the concentration of the aqueous alkaline solution is 0.2 mol / L-0.5 mol / L.

[0010] According to the present invention, the medicinal materials are two of marigold, purslane and Scrophularia ningpoensis, and the weight ratio of marigold to purslane is 30-50:50-70, or, The weight ratio of the marigold to the Scrophularia ningpoensis is 40:60, or, The weight ratio of the purslane to the Scrophularia ningpoensis is 50-60:40-50.

[0011] According to the present invention, the medicinal materials are marigold, purslane and Scrophularia ningpoensis, and the weight ratio of marigold, purslane and Scrophularia ningpoensis is 30:50:20.

[0012] According to the present invention, the alkaline aqueous solution in step A is one or more combinations of an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, an aqueous sodium carbonate solution, and an arginine solution. Preferably, the alkaline aqueous solution in step A is one or more combinations of an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, and an arginine solution; more preferably, the alkaline aqueous solution in step A is an arginine solution.

[0013] According to the present invention, the decolorization in step C is performed by activated carbon adsorption decolorization; and / or, The desalination in step C is carried out by ion exchange resin desalination.

[0014] As a second aspect of the present invention, a capsaicin irritation inhibitor for cosmetics is prepared by the above-mentioned preparation method of the capsaicin irritation inhibitor for cosmetics.

[0015] As a third aspect of the present invention, the cosmetic capsaicin stimulation inhibitor is used in inhibiting capsaicin-induced TRPV1 expression.

[0016] As a fourth aspect of the present invention, the cosmetic capsaicin stimulation inhibitor is used in inhibiting capsaicin-induced IL-1α secretion.

[0017] As the fifth aspect of the present invention, the use of the capsaicin irritation inhibitor for cosmetics in the preparation of external skin preparations has an anti-irritation effect.

[0018] The capsaicin irritation inhibitor for cosmetics of the present invention, and its preparation method and application, have the following beneficial effects: the capsaicin inhibitor for cosmetics can significantly inhibit the secretion of IL-1α of keratinocytes and the expression of TRPV-1 protein caused by capsaicin stimulation, and the combination of two of them has a certain synergistic effect; the capsaicin inhibitor for cosmetics can be used in external preparations to achieve an anti-irritation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the experimental result of cck-8 in Example 1; *p<0.05 vs NC; Figure 2 This is the experimental result of cck-8 in Example 2; *p<0.05 vs NC; Figure 3 This is the experimental result diagram of cck-8 in Example 3; Figure 4 This is the cck-8 experimental result diagram of Example 4; Figure 5 This is the cck-8 experimental result diagram of Example 5; Figure 6 This is the experimental result of cck-8 in Example 6; *p<0.05 vs NC; Figure 7 This is the experimental result of cck-8 in Example 7; *p<0.05 vs NC; Figure 8 This is the experimental result diagram of cck-8 in Example 8; Fig. 9 This is the experimental result of cck-8 in Example 9; *p<0.05 vs NC; Fig.10 This is a graph showing the experimental results of cck-8 in Example 10; Fig.11 This is a graph of the experimental results of cck-8 in Example 11; Fig.12 This is a graph showing the cck-8 experimental results of Comparative Example 1; Fig.13 This is a graph showing the cck-8 experimental results of Comparative Example 2; Fig.14 This is a graph showing the cck-8 experimental results of Comparative Example 3; Fig.15 This is a graph showing the cck-8 experimental results of Comparative Example 4; Fig.16 This is a graph showing the cck-8 experimental results of Comparative Example 5; Fig.17 This is a graph showing the cck-8 experimental results of Comparative Example 6; Fig.18This is the expression of TRPV1 induced by CAP inhibited by Example 1; **p<0.01 vs CAP; Fig.19 This is the inhibition of CAP-induced TRPV1 expression in Example 2; **p<0.01 vs CAP; Fig. 20 This is the inhibition of CAP-induced TRPV1 expression in Example 3; **p<0.01 vs CAP; Fig.21 This is the inhibition of CAP-induced TRPV1 expression in Example 4; **p<0.01 vs CAP; Fig. 22 This is the inhibition of TRPV1 expression induced by CAP in Example 5; **p<0.01 vs CAP; Fig.23 This is the inhibition of CAP-induced TRPV1 expression in Example 6; **p<0.01 vs CAP; Fig.24 This is the inhibition of CAP-induced TRPV1 expression in Example 7; **p<0.01 vs CAP; Fig.25 This is the inhibition of CAP-induced TRPV1 expression in Example 8; **p<0.01 vs CAP; Fig.26 This is the inhibition of CAP-induced TRPV1 expression in Example 9; **p<0.01 vs CAP; Fig. 27 This is the expression of TRPV1 induced by CAP inhibited by Example 10; **p<0.01 vs CAP; Fig.28 This is the expression of TRPV1 induced by CAP inhibited by Example 11; **p<0.01 vs CAP; Fig.29 For comparison with Example 1, the expression of TRPV1 induced by CAP was inhibited; **p<0.01 vs CAP; Fig.30 For comparison with Example 2, the expression of TRPV1 induced by CAP was inhibited; **p<0.01 vs CAP; Fig.31 For comparison with Example 3, the expression of TRPV1 induced by CAP was inhibited; **p<0.01 vs CAP; Fig.32 For comparison with Example 4, the expression of TRPV1 induced by CAP was inhibited; **p<0.01 vs CAP; Fig.33For comparison with Example 5, the expression of TRPV1 induced by CAP was inhibited; **p<0.01 vs CAP; Fig.34 For comparison with Example 6, the expression of TRPV1 induced by CAP was inhibited; *p<0.05, **p<0.01 vs CAP; Fig.35 This is the Elisa test result of inhibiting CAP-induced IL-1α secretion in Example 1; **p<0.01 vs CAP; Fig.36 This is the Elisa test result of Example 2 for inhibiting CAP-induced IL-1α secretion; **p<0.01 vs CAP; Fig.37 This is the Elisa test result of Example 3 for inhibiting CAP-induced IL-1α secretion; **p<0.01 vs CAP; Fig.38 This is the Elisa test result of Example 4 for inhibiting CAP-induced IL-1α secretion; *p<0.05, **p<0.01 vs CAP; Fig.39 This is the Elisa test result of Example 5 for inhibiting CAP-induced IL-1α secretion; *p<0.05, **p<0.01 vs CAP; Fig.40 This is the Elisa test result of Example 6 for inhibiting CAP-induced IL-1α secretion; **p<0.01 vs CAP; Fig.41 This is the Elisa test result of Example 7 for inhibiting CAP-induced IL-1α secretion; **p<0.01 vs CAP; Fig.42 This is the Elisa test result of Example 8 for inhibiting CAP-induced IL-1α secretion; **p<0.01 vs CAP; Fig.43 This is the Elisa test result of Example 9 for inhibiting CAP-induced IL-1α secretion; **p<0.01 vs CAP; Fig.44 This is the Elisa test result of Example 10 for inhibiting CAP-induced IL-1α secretion; **p<0.01 vs CAP; Fig.45 This is the Elisa test result of Example 11 for inhibiting CAP-induced IL-1α secretion; **p<0.01 vs CAP; Fig.46 The results of Elisa test for inhibiting CAP-induced IL-1α secretion in comparison with Example 1; **p<0.01 vs CAP; Fig.47 For comparison with Example 2, the Elisa test results of inhibiting CAP-induced IL-1α secretion; **p<0.01 vs CAP; Fig.48 For comparison with Example 3, the Elisa test results of inhibiting CAP-induced IL-1α secretion; **p<0.01 vs CAP; Fig.49 The results of Elisa test for inhibiting CAP-induced IL-1α secretion in comparison with Example 4; **p<0.01 vs CAP; Fig.50 The results of Elisa test for inhibiting CAP-induced IL-1α secretion in comparison with Example 5; *p<0.05, **p<0.01 vs CAP; Fig.51 The results of Elisa test for inhibiting CAP-induced IL-1α secretion in comparison with Example 6; *p<0.05, **p<0.01 vs CAP. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0021] 1. The medicinal material of the present invention 1.1 Purslane ( Portulaca oleracea L.), also known as Purslane, Grasshopper Vegetable, Five Elements Vegetable, Longevity Vegetable, Five Elements Grass, etc., is an annual fleshy herb of the genus Portulacaceae. Purslane is a plant with both medicinal and edible properties. Its tender stems, leaves, and flowers are edible. It is a healthy vegetable and also a high-quality green medicinal feed. The whole plant and seeds of Purslane can be used as Chinese medicinal materials. It has the effects of cooling blood and detoxifying, removing dampness and relieving stranguria, and stopping dysentery. It is often used to treat bacterial dysentery, sores, carbuncles, ringworm, eczema, etc.

[0022] Since the 1960s, domestic and foreign scholars have extracted a variety of chemical components from purslane, including organic acids, alkaloids, flavonoids, terpenes, polysaccharides, coumarins, steroids, anthocyanins, volatile oils, catecholamines, etc. Modern pharmacological studies have shown that purslane has good antibacterial, antioxidant, anti-aging, anti-allergic, and immune-enhancing effects.

[0023] 1.2 Marigold (scientific name: Calendula officinalisL.), also known as Calendula officinalis, Calendula officinalis, etc., is an annual herbaceous plant of the genus Calendula in the Asteraceae family. Its inflorescence can be used as medicine, and has the effects of drying and cooling, clearing the lungs and relieving cough, clearing heat and relieving asthma, etc. It is mainly used to treat damp-heat or blood-based diseases, various types of inflammation and swelling, and acne and wet sores. Its leaves can be used as salads, and the juice obtained by cold-pressing the leaves can also be used to flavor various foods such as soft drinks, iced foods, candies, and baked foods. Calendula contains a variety of active ingredients, such as flavonoids, phenolic acids, sesquiterpenes, saponins, polysaccharides and other chemical components. Modern research shows that calendula has good antibacterial and antiviral, anti-inflammatory, antioxidant, anti-aging, moisturizing, and anti-tumor effects.

[0024] 1.3 Scrophulariaceae (scientific name: Scrophularia ningpoensis Hemsl. ), also known as Yuan Shen, Bei Xuan Shen, Hei Shen, etc., is a plant of the genus Scrophulariaceae. Its dried root can be used as medicine, and has the effects of clearing heat and cooling blood, nourishing yin and promoting body fluid, purging fire and detoxifying, softening and dispersing nodules. It is mainly used to treat febrile diseases with heat entering the blood, fever and thirst, red tongue spots, bone steaming and fatigue cough, restlessness and insomnia, body fluid damage and constipation, astringent eyes, sore throat, scrofula and phlegm, carbuncle and sore. Scrophularia mainly contains cyclopentane ether terpenes and phenylpropanoid glycosides, and also contains phytosterols, organic acids, flavonoids, triterpenoid saponins, volatile oils, sugars, alkaloids and trace amounts of monoterpenes and diterpenes. Modern research shows that Scrophularia has good cardiovascular protection, anti-tumor, anti-oxidation, anti-inflammatory, antibacterial, analgesic and other effects. The Scrophularia of the present invention can also be selected from SCROPHULARIA NODOSA.

[0025] Example 1 1. Add 1750 g of 0.2 mol / L sodium hydroxide solution to 100 g of calendula and soak for 16 h; 2. Extract the soaking solution at 95-105℃ for 1 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated to an extract, dried, and sterilized to obtain the capsaicin inhibitor: marigold extract.

[0026] Example 2 The difference between this example and Example 1 is that Marigold is replaced with Purslane, and the capsaicin inhibitor obtained is Purslane extract.

[0027] Example 3 The difference between this example and Example 1 is that Marigold is replaced by Scrophularia ningpoensis, and the capsaicin inhibitor obtained is Scrophularia ningpoensis extract.

[0028] Example 4 The difference between this example and Example 1 is that Marigold is replaced by Isatis indigotica root, and the capsaicin inhibitor obtained is Isatis indigotica root extract.

[0029] Example 5 The difference between this example and Example 1 is that Marigold is replaced by Honeysuckle, and the capsaicin inhibitor obtained is Honeysuckle extract.

[0030] Example 6 1. Soak 30g of marigold and 70g of purslane in 1500g of 0.2 mol / L sodium hydroxide solution for 16 h; 2. Extract the soaking solution at 95-105℃ for 1 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated until the extract is dry to obtain the capsaicin inhibitor.

[0031] Example 7 1. Add 50g of calendula and 50g of purslane into 1500g of 0.2 mol / L sodium hydroxide solution and soak for 3 h; 2. Extract the soaking solution at 95-105℃ for 1 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated until the extract is dry to obtain the capsaicin inhibitor.

[0032] Example 8 1. Soak 50g of Purslane and 50g of Scrophularia ginseng in 1700g of 0.5 mol / L arginine solution for 8 h; 2. Extract the soaking solution at 95-105℃ for 2 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated until the extract is dry to obtain the capsaicin inhibitor.

[0033] Example 9 1. Soak 60g of purslane and 40g of sphenanthera in 1700g of 0.5 mol / L arginine solution for 8 h; 2. Extract the soaking solution at 95-105℃ for 2 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated to an extract, and dried to obtain the capsaicin inhibitor.

[0034] Example 10 1. Soak 40g of marigold and 60g of scutellaria baicalensis in 1700g of 0.5 mol / L potassium hydroxide solution for 2 h; 2. Extract the soaking solution at 95-105℃ for 3 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated to an extract, and dried to obtain the capsaicin inhibitor.

[0035] Embodiment 11 1. Soak 30g of calendula, 50g of purslane and 20g of sphenanthera in 1700g of 0.5 mol / L arginine solution for 8 h; 2. Extract the soaking solution at 95-105℃ for 2 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated until the extract is dry to obtain the capsaicin inhibitor.

[0036] Comparative Example 1 1. Soak 60g of calendula and 40g of purslane in 1750g of 0.5 mol / L arginine solution for 8 h; 2. Extract the soaking solution at 95-105℃ for 2 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated until the extract is dry to obtain the capsaicin inhibitor.

[0037] Comparative Example 2 1. Soak 25g of calendula and 75g of Scrophularia ginseng in 1750g of 0.5 mol / L arginine solution for 8 h; 2. Extract the soaking solution at 95-105℃ for 2 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated until the extract is dry to obtain the capsaicin inhibitor.

[0038] Comparative Example 3 1. Soak 75g of purslane and 25g of sphenanthera in 1750g of 0.5 mol / L arginine solution for 8 h; 2. Extract the soaking solution at 95-105℃ for 2 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated until the extract is dry to obtain the capsaicin inhibitor.

[0039] Comparative Example 4 1. Add 1750g of 50° ethanol to 100g of calendula and soak for 16 hours; 2. Extract the soaking solution at 95-105℃ for 1 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated to an extract, dried, and sterilized to obtain the capsaicin inhibitor: Comparative Example Marigold Extract.

[0040] Comparative Example 5 1. Add 100g of purslane to 1750g of deionized water and soak for 16 hours; 2. Extract the soaking solution at 95-105℃ for 1 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated to an extract, dried, and sterilized to obtain the capsaicin inhibitor: Comparative Example Portulaca oleracea extract.

[0041] Comparative Example 6 1. Add 1750 g of 0.2 mol / L acetic acid solution to 100 g of Scrophularia ningpoensis and soak for 16 h; 2. Extract the soaking solution at 95-105℃ for 1 h, and filter through gauze to remove the residue; 3. Add 1wt% activated carbon to the filtrate, stir at room temperature for 1 h, and filter until clear; 4. The filtrate continues to pass through anion and cation exchange resins, and the filtrate is concentrated to an extract, dried, and sterilized to obtain the capsaicin inhibitor: Comparative Example Scrophularia ningpoensis extract.

[0042] Table 1 shows the ingredients and contents of Examples 1-11 and Comparative Examples 1-6 Note: The extraction temperature of the above examples and comparative examples is 95-105°C.

[0043] The Scrophulariae used in Example 3, Examples 8-10, Comparative Example 1 and Comparative Example 6 are Scrophulariae radiatae; the Scrophulariae used in Example 11 and Comparative Example 2 are Scrophulariae sylvaticae.

[0044] Effect Example 1 Soothing Test Based on Capsaicin Stimulation Model of Human Keratinocytes 1. Experimental Materials Hacat cells (Cell Bank of the Chinese Academy of Sciences), capsaicin (MCE), capsaicin (MCE), TRPV-1 primary antibody (cellsignaling technology), fluorescent secondary antibody (Biyuntian), DAPI (Biyuntian), IL-1α ELISA kit (Cloud Clone), inverted fluorescence microscope (olympus), multi-function microplate reader (thermo fisher), etc.

[0045] Table 2 Preparation methods of samples for cell experiments 1. Experimental Methods 1.1 Cytotoxicity test Under sterile conditions, the samples to be tested obtained in Table 1 were diluted with culture medium, and each sample to be tested was uniformly diluted to: 0.005%, 0.01%, 0.02%, and 0.05%.

[0046] Hacat cells were cultured at 5 × 10 3 Cells were inoculated into 96-well plates at a density of 100 cells / well and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h. When the cell density reached about 50%, the sample to be tested was treated. After 24 h of sample addition, cell activity was detected using the CCK-8 method, and the cell survival rate was calculated according to the following formula.

[0047] Cell survival rate = (OD value of sample group - background OD value) / (OD value of blank control group - background OD value) × 100% Note: Background OD value is not reflected in the data.

[0048] 1.2 Test of the soothing effect of samples on human keratinocytes According to the cytotoxicity test results, one concentration was selected as the soothing efficacy test concentration.

[0049] Hacat cells were cultured at 2 × 10 5The cells were inoculated in 6-well plates at a density of 100 cells / well and cultured in a cell culture incubator at 37 ℃ and 5% CO2 for 24 h before treatment. As shown in Table 3, the cells were divided into a blank control group (NC), a CAP-induced group (CAP), a positive control group (PC) and a sample group. The positive control and sample were added to the positive control group and the sample group at corresponding concentrations and cultured in a cell culture incubator at 37 ℃ and 5% CO2 for 4 h. Capsaicin was added to the model group, the positive control group and the sample group and cultured in a cell culture incubator at 37 ℃ and 5% CO2 for 20 h. At the end of the time action, the culture supernatant of each well was collected, and the IL-1α content was detected by ELISA according to the instructions of the kit, and the inhibition rate was calculated; after the cells were fixed, the expression of TRPV1 protein was detected by immunofluorescence. After taking pictures, the average fluorescence intensity of TRPV1 protein in the picture was calculated using ImageJ software and the inhibition rate was calculated.

[0050] Table 3 Overview of soothing efficacy experimental groups Inhibition rate % = (1-expression level of sample group / expression level of CAP-induced group) × 100% 1.3 Calculation of Combination Index (CI) value Combination Index (CI) is a method for evaluating the combined effects of drugs, proposed by Chou and Talalay in 1984. This method is based on the dose-effect curve of the drug, and determines whether the combined drug has a synergistic effect, additive effect, or antagonistic effect by calculating the ratio of the actual effect to the theoretical effect during combined use. A CI value less than 0.9 indicates a synergistic effect, and the smaller the value, the stronger the synergistic effect; a CI value between 0.9-1.1 indicates an additive effect; a CI value greater than 1.1 indicates an antagonistic effect, and the larger the value, the stronger the antagonistic effect.

[0051] The calculated inhibition rate was analyzed using CompuSyn software to obtain the CI value.

[0052] 1.4 Statistical analysis of experimental data All data were expressed as mean ± standard deviation. One-way ANOVA was used to compare the groups. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0053] 1.5 Experimental Results 1.5.1 Cytotoxicity test results Table 4 Experimental results of Example 1-Calendula officinalis extract cck-8 Table 5 Experimental results of Example 2-Purslane extract cck-8 Table 6 Experimental results of Example 3-Scrophularia ningpoensis extract cck-8 Table 7 Example 4 - Isatis indigotica root extract cck-8 experimental results Table 8 Example 5 - Experimental results of honeysuckle extract cck-8 Table 9 Experimental results of Example 6 cck-8 Table 10 Experimental results of Example 7 cck-8 Table 11 Experimental results of Example 8 cck-8 Table 12 Experimental results of Example 9 cck-8 Table 13 Experimental results of cck-8 in Example 10 Table 14 Experimental results of cck-8 in Example 11 Table 15 Comparative Example 1 cck-8 experimental results Table 16 Comparative Example 2 cck-8 experimental results Table 17 Comparative Example 3 cck-8 experimental results Table 18 Comparative Example 4 cck-8 experimental results Table 19 Comparative Example 5 cck-8 experimental results Table 20 Comparative Example 6 cck-8 experimental results Result analysis: According to the results of the cck-8 toxicity experiment, a concentration of 0.005-0.02% was selected for subsequent experiments.

[0054] 2.4.2 Soothing efficacy test 2.4.2.1 TRPV-1 protein expression Table 21 Example 1 - Calendula officinalis extract inhibits CAP-induced TRPV1 expression Result analysis: As shown in the results, the calendula extract at a concentration of 0.005-0.02% can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 32.67%±4.90%, 39.69%±3.04%, and 44.49%±4.76%, respectively.

[0055] Table 22 Example 2 - Purslane inhibits CAP-induced TRPV1 expression Result analysis: As shown in the results, purslane extract at a concentration of 0.005-0.02% can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 34.27%±4.19%, 39.71%±4.24%, and 46.54%±4.06%, respectively.

[0056] Table 23 Example 3 - Scrophularia ningpoensis extract inhibits CAP-induced TRPV1 expression Result analysis: As shown in the results, the extract of Scrophularia ningpoensis at a concentration of 0.005-0.02% can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 39.80%±0.72%, 46.79%±0.75%, and 53.46%±0.13%, respectively.

[0057] Table 24 Example 4 - Isatis indigotica root extract inhibits CAP-induced TRPV1 expression Result analysis: As shown in the results, the extract of Isatis indigotica root at a concentration of 0.01-0.02% can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin ( ** p<0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 9.92%±1.67% and 12.71%±3.28%, respectively.

[0058] Table 25 Example 5 - Honeysuckle extract inhibits CAP-induced TRPV1 expression Result analysis: As shown in the results, honeysuckle extract at a concentration of 0.01-0.02% can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 11.74%±1.35% and 16.93%±1.50%, respectively.

[0059] Table 26 Example 6 Inhibition of CAP-induced TRPV1 expression Result analysis: As shown in the results, Example 6 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.005-0.02% (**p<0.01 vs CAP), and has a dose-effect relationship; the inhibition rates are: 34.14%±0.65%, 39.44%±0.24%, 49.98%±3.68%, respectively.

[0060] Table 27 Example 7 Inhibition of CAP-induced TRPV1 expression Result analysis: As shown in the results, Example 7 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 39.38%±1.99%, 41.44%±2.82%, and 49.79%±1.43%, respectively.

[0061] Table 28 Example 8 Inhibition of CAP-induced TRPV1 expression Result analysis: As shown in the results, Example 8 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 45.44%±0.92%, 51.49%±1.06%, and 59.63%±1.10%, respectively.

[0062] Table 29 Example 9 Inhibition of CAP-induced TRPV1 expression Result analysis: As shown in the results, Example 9 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 44.21%±1.11%, 50.79%±0.62%, and 57.97%±0.96%, respectively.

[0063] Table 30 Example 10 Inhibition of CAP-induced TRPV1 expression Result analysis: As shown in the results, Example 10 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 41.39%±0.89%, 47.86%±0.23%, and 55.82%±0.23%, respectively.

[0064] Table 31 Example 11 Inhibition of CAP-induced TRPV1 expression Result analysis: As shown in the results, Example 11 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 45.64%±3.13%, 52.63%±2.16%, and 60.23%±1.29%, respectively.

[0065] Table 32 Comparative Example 1 Inhibition of CAP-induced TRPV1 expression Result analysis: As shown in the results, Example 5 at a concentration of 0.005-0.02% can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 33.49%±3.52%, 39.82%±4.48%, and 45.99%±1.75%, respectively.

[0066] Table 33 Comparative Example 2 Inhibition of CAP-induced TRPV1 expression Result analysis: As shown in the results, comparative example 1 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 38.38%±2.70%, 44.04%±4.52%, and 52.93%±1.03%, respectively.

[0067] Table 34 Comparative Example 3 Inhibition of CAP-induced TRPV1 expression Result analysis: As shown in the results, comparative example 3 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 37.53%±3.27%, 41.26%±2.69%, and 50.40%±2.85%, respectively.

[0068] Table 35 Comparative Example 4 inhibits the expression of TRPV1 induced by CAP Result analysis: As shown in the results, comparative example 4 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.01-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 11.09%±2.50% and 17.67%±1.30%, respectively.

[0069] Table 36 Comparative Example 5 inhibits the expression of TRPV1 induced by CAP Result analysis: As shown in the results, comparative example 5 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.01-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 10.49%±2.04% and 16.76%±1.36%, respectively.

[0070] Table 37 Comparative Example 6 inhibits the expression of TRPV1 induced by CAP Result analysis: As shown in the results, comparative example 6 can significantly inhibit the expression of TRPV1 in Hacat cells induced by capsaicin at a concentration of 0.01-0.02% ( * p <0.05, ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 7.38%±2.46% and 14.86%±1.97%, respectively.

[0071] Table 38 Inhibition rate and CI value of samples on CAP-induced TRPV1 expression Analysis of results: As shown in the results, the inhibition rates of the combinations of two or three samples on capsaicin-induced TRPV1 expression were higher than those of the single samples, and the CI values ​​were between 0.3 and 0.8, indicating that the samples had a synergistic effect. The capsaicin irritation inhibitors for cosmetics prepared using the process of comparative examples 4-6 were significantly less effective than the capsaicin irritation inhibitors for cosmetics prepared in examples 1-3.

[0072] 2.4.2.2 IL-1α secretion Table 39 Example 1 - Calendula officinalis extract inhibits CAP-induced IL-1α secretion Result analysis: As shown in the results, calendula extract at a concentration of 0.005-0.02% can significantly inhibit the secretion of IL-1α by Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 18.69%±1.41%, 34.65%±0.31%, and 42.96%±0.90%, respectively.

[0073] Table 40 Example 2 - Purslane extract inhibits CAP-induced IL-1α secretion Result analysis: As shown in the results, purslane extract at a concentration of 0.005-0.02% can significantly inhibit the secretion of IL-1α by Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 19.54%±1.38%, 35.86%±1.50%, and 43.55%±2.25%, respectively.

[0074] Table 41 Example 3 - Scrophularia ningpoensis extract inhibits CAP-induced IL-1α secretion Result analysis: As shown in the results, the extract of Scrophularia ningpoensis at a concentration of 0.005-0.02% can significantly inhibit the secretion of IL-1α by Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 28.56%±0.62%, 40.38%±2.05%, and 47.67%±0.68%, respectively.

[0075] Table 42 Example 4 - Isatis indigotica root extract inhibits CAP-induced IL-1α secretion Result analysis: As shown in the results, the Isatis indigotica root extract of Example 4 at a concentration of 0.01-0.02% can inhibit the secretion of IL-1α by Hacat cells induced by capsaicin ( * p <0.05, ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 9.12%±0.13% and 15.40%±3.67%, respectively.

[0076] Table 43 Example 5 - Honeysuckle extract inhibits CAP-induced IL-1α secretion Result analysis: As shown in the results, the honeysuckle extract of Example 5 at a concentration of 0.02% can inhibit the secretion of IL-1α by Hacat cells induced by capsaicin ( * p <0.05 vs CAP); inhibition rate: 15.93%±0.66%.

[0077] Table 44 Example 6 Inhibition of CAP-induced IL-1α secretion Result analysis: As shown in the results, Example 6 can significantly inhibit the secretion of IL-1α by Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 33.74%±3.76%, 44.05%±1.30%, and 54.00%±0.88%, respectively.

[0078] Table 45 Example 7 Inhibition of CAP-induced IL-1α secretion Result analysis: As shown in the results, Example 7 at a concentration of 0.005-0.02% can significantly inhibit the secretion of IL-1α by Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 31.37%±1.75%, 43.54%±1.95%, and 52.84%±0.12%, respectively.

[0079] Table 46 Example 8 Inhibition of CAP-induced IL-1α secretion Result analysis: As shown in the results, Example 8 at a concentration of 0.005-0.02% can significantly inhibit the secretion of IL-1α by Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 36.57%±1.72%, 47.40%±4.00%, and 59.38%±3.99%, respectively.

[0080] Table 47 Example 9 Inhibition of CAP-induced IL-1α secretion Result analysis: As shown in the results, Example 9 at a concentration of 0.005-0.02% can significantly inhibit the secretion of IL-1α by Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 36.22%±2.55%, 46.50%±0.49%, and 58.46%±1.43%, respectively.

[0081] Table 48 Example 10 Inhibition of CAP-induced IL-1α secretion Result analysis: As shown in the results, Example 10 at a concentration of 0.005-0.02% can significantly inhibit the secretion of IL-1α by Hacat cells induced by capsaicin ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 36.01%±7.06%, 45.52%±4.12%, and 57.78%±5.25%, respectively.

[0082] Table 49 Example 11 Inhibition of CAP-induced IL-1α secretion Result analysis: As shown in the results, Example 11 can inhibit the secretion of IL-1α by Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 38.38%±3.03%, 48.46%±4.98%, and 60.12%±3.23%, respectively.

[0083] Table 50 Comparative Example 1 Inhibition of CAP-induced IL-1α secretion Result analysis: As shown in the results, comparative example 1 can inhibit the secretion of IL-1α by Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 22.85%±2.47%, 37.82%±3.51%, and 45.65%±3.39%, respectively.

[0084] Table 51 Comparative Example 2 Inhibition of CAP-induced IL-1α secretion Result analysis: As shown in the results, comparative example 2 can inhibit the secretion of IL-1α by Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 24.65%±4.13%, 39.53%±0.51%, and 47.07%±4.26%, respectively.

[0085] Table 52 Comparative Example 3 Inhibition of CAP-induced IL-1α secretion Result analysis: As shown in the results, comparative example 3 can inhibit the secretion of IL-1α by Hacat cells induced by capsaicin at a concentration of 0.005-0.02% ( ** p <0.01 vs CAP), and had a dose-effect relationship; the inhibition rates were 24.80%±7.82%, 39.67%±3.11%, and 46.65%±3.14%, respectively.

[0086] Table 53 Comparative Example 4 inhibits CAP-induced IL-1α secretion Result analysis: As shown in the results, comparative example 4 can inhibit the secretion of IL-1α by Hacat cells induced by capsaicin at a concentration of 0.02% ( ** p <0.01 vs CAP; inhibition rate: 17.18%±2.92%.

[0087] Table 54 Comparative Example 5 Inhibition of CAP-induced IL-1α secretion Result analysis: As shown in the results, comparative example 5 at a concentration of 0.02% can inhibit the secretion of IL-1α by Hacat cells induced by capsaicin ( * p <0.05 vs CAP); inhibition rate: 11.33%±3.87%.

[0088] Table 55 Comparative Example 6 Inhibition of CAP-induced IL-1α secretion Analysis of results: As shown in the results, comparative example 6 can inhibit the secretion of IL-1α by Hacat cells induced by capsaicin at a concentration of 0.01-0.02% (*p<0.05, **p<0.01 vs CAP), and has a dose-effect relationship; the inhibition rates are 9.65%±3.73% and 14.50%±2.52%, respectively.

[0089] Table 56 Inhibition rate and CI value of samples on CAP-induced IL-1α secretion Analysis of results: As shown in the results, the inhibition rates of the combinations of two or three samples on capsaicin-induced IL-1α secretion were higher than those of the single samples, and the CI values ​​were between 0.4 and 0.7, indicating that the samples had a synergistic effect. The capsaicin stimulation inhibitor for cosmetics prepared using the process of comparative examples 4-6 was significantly less effective than that of examples 1-3.

[0090] In summary, the capsaicin stimulation inhibitor for cosmetics provided by the present invention can effectively inhibit the expression of TRPV1 and the secretion of IL-1α.

[0091] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing a capsaicin irritation inhibitor for cosmetics, characterized in that: The steps include: A. Soaking the medicinal material in 15-20 times the weight of an extraction solvent for 2-16 hours to obtain an immersion solution; the extraction solvent is a 0.2 mol / L-0.8 mol / L alkaline aqueous solution, and the medicinal material is at least one of calendula, purslane, and Scrophularia; B. Extract the soaked solution at 95-105℃ for 1-3h.

2. The method for preparing the capsaicin irritation inhibitor for cosmetics as claimed in claim 1, characterized in that: After step B, the following steps are also included: C. removing the filter residue, decolorizing, filtering, and desalting to prepare a capsaicin irritation inhibitor for cosmetics.

3. The method for preparing the capsaicin irritation inhibitor for cosmetics as claimed in claim 1, characterized in that: The concentration of the alkaline aqueous solution is 0.2 mol / L-0.5 mol / L.

4. The method for preparing the capsaicin irritation inhibitor for cosmetics according to any one of claims 1 to 3, characterized in that: The medicinal materials are two of marigold, purslane and Scrophularia ningpoensis, and the weight ratio of marigold to purslane is 30-50:50-70; or, The weight ratio of the marigold to the Scrophularia ningpoensis is 40:60; or, The weight ratio of the purslane to the Scrophularia ningpoensis is 50-60:40-50.

5. The method for preparing the capsaicin irritation inhibitor for cosmetics according to any one of claims 1 to 3, characterized in that: The medicinal materials are marigold, purslane and Scrophularia ningpoensis, and the weight ratio of marigold, purslane and Scrophularia ningpoensis is 30:50:

20.

6. The method for preparing the capsaicin irritation inhibitor for cosmetics as claimed in claim 1, characterized in that: The alkaline aqueous solution in step A is one or more combinations of sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, sodium carbonate aqueous solution and arginine aqueous solution.

7. A capsaicin irritation inhibitor for cosmetics, which is prepared by the preparation method of the capsaicin irritation inhibitor for cosmetics according to any one of claims 1 to 6.

8. Use of the capsaicin stimulation inhibitor for cosmetics as claimed in claim 7 in inhibiting capsaicin-induced TRPV1 expression.

9. Use of the capsaicin stimulation inhibitor for cosmetics as claimed in claim 7 in inhibiting capsaicin-induced IL-1α secretion.

10. Use of the capsaicin irritation inhibitor for cosmetics as claimed in claim 7 in the preparation of external skin preparations.

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