A soothing and anti-inflammatory plant extract composition and its application

Abstract

The present invention relates to the field of A61K36 / 67, specifically a soothing and anti-inflammatory plant extract composition and its application, comprising at least two of kanuka extract, kava extract, daisy flower extract, purslane extract, rosemary extract, astragalus membranaceus extract, gardenia fruit extract, and gardenia flower extract. The composition can be effectively added and applied to skin care products, sunscreen products, and cleansing products to achieve soothing and anti-inflammatory effects, and the composition has high formula compatibility and safety, good stability, and is easy to add and use.

Description

Technical Field

[0001] The present invention relates to the field of A61K36 / 67, and in particular to a soothing and anti-inflammatory plant extract composition and application thereof. Background Art

[0002] At present, the incidence of sensitive skin in countries around the world is increasing year by year. The mechanism of sensitive skin is a complex process of cumulative skin barrier, nerve blood vessels, and immune inflammation. Under the interaction of internal and external factors, the skin barrier function is damaged, causing an increase in sensory nerve afferent signals, resulting in increased skin reactivity to external stimuli, and triggering skin immune inflammatory reactions. Specifically, subjectively, people will feel burning, stinging, itching, tightness, etc., and objectively, they may be accompanied by symptoms such as erythema, peeling, and capillary dilation.

[0003] According to market data such as beauty and cosmetics filings, active substances with anti-inflammatory and soothing effects developed from natural plants and added to daily skin products are more easily accepted and recognized by consumers. Chinese patent application CN113144039A discloses a plant anti-allergic soothing composition and its preparation method and application, which specifically uses a combination of alpine edelweiss or white flower oldenlandia diffusa with Rehmannia glutinosa or Andrographis paniculata with pine mouth abrasive bacteria. The anti-allergic and soothing effects of the composition need to be obtained by the three components in a specific proportion. However, on the one hand, the cost of alpine edelweiss is relatively high, and the cost of adding and using it in daily skin products is too high, which limits its large-scale application to a certain extent. On the other hand, white flower oldenlandia diffusa will have certain side effects after long-term use, which is not conducive to human health. As an affordable alternative to high-cost anti-allergic active ingredients, kava extract is gradually used in cosmetics to achieve anti-allergic effects. However, due to the limitations of the kava extract extraction process, the anti-inflammatory and soothing effects of kava are not significant. Chinese patent application CN115006314A discloses a kava extract and a preparation method and application thereof. The kava raw material needs to be crushed and then subjected to high temperature and high pressure treatment. However, the main active ingredients in the kava extract are easily oxidized and discolored at high temperatures and become ineffective, and even the oxidized products produced can form a source of irritation. Therefore, it is limited to use it alone or in combination with other active components in cosmetics to achieve excellent anti-allergic and soothing effects. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a soothing and anti-inflammatory plant extract composition, which is obtained by compounding a Kava Piper methysticum extract prepared by extraction with a Kanuka leukoplakia extract. The soothing and anti-inflammatory plant extract composition can be effectively added and applied to skin care products, sunscreen products, and cleansing products to achieve soothing and anti-inflammatory effects, and the composition has high formula compatibility and safety, good stability, and is easy to add and use.

[0005] On one hand, the present invention provides a soothing and anti-inflammatory plant extract composition, which includes at least two of kanuka extract, kava extract, daisy flower extract, purslane extract, rosemary extract, astragalus membranaceus extract, gardenia fruit extract, and gardenia flower extract.

[0006] Preferably, the soothing and anti-inflammatory plant extract composition is a combination of Kunzea Ericoides Leaf Extract and Piper methysticum Extract;

[0007] Preferably, the mass ratio of the kanuka extract to the kava extract is 1:(0.1-5);

[0008] Preferably, the mass ratio of the kanuka extract to the kava extract is 1:(0.5-2); preferably 1:1.

[0009] The main active ingredients of Kava extract are unsaturated lactone components, including active ingredients of capsaicin. The active ingredients of capsaicin mainly achieve the soothing effect by acting on the skin sensory nervous system, specifically by inhibiting nerve excitation (inhibiting TRPV1 protein expression) and blocking nerve conduction after excitation (inhibiting action potential), and blocking the burning, pain and itching of the skin through two targets at the same time, thereby achieving an immediate soothing effect. However, Kava extract is easily oxidized, resulting in a short effective period of use. The inventor accidentally discovered during the actual research and development process that by compounding the canuka extract with the kava extract, especially when the mass ratio of the canuka extract to the kava extract is 1: (0.1-5), the control can produce a synergistic effect on the efficacy on the one hand, avoiding the single efficacy and expanding the scope of application; on the other hand, it helps to enhance the stability of the shelf life of the active ingredients in the kava extract, and the compounded composition has a longer effective period of use. The inventors analyzed that the reason may be that while the kava extract and kanuka extract achieve the soothing effect by inhibiting the expression of pro-inflammatory factors and the secretion of inflammatory mediators, it promotes the storage and use stability of the kava extract, establishes a four-stage inhibition pathway from skin damage to actual skin problems, namely, preventing the invasion of stimuli - clearing reactive oxygen - inhibiting cell damage - inhibiting low cell activity, and achieves soothing and anti-inflammatory effects that are better than the same amount of a single raw material.

[0010] Preferably, the extraction process of the kanuka extract and kava extract comprises at least the following steps: crushing, extracting, separating, concentrating, drying, and crushing.

[0011] Preferably, the extraction process of the kanuka extract and kava extract specifically comprises the following steps:

[0012] (1) grinding the kanuka raw material and / or kava raw material into 40-80 meshes, adding the alcohol compound diluted with deionized water, and performing reflux extraction to obtain an extract;

[0013] (2) adding a concentrator to the extract, mixing the mixture, and performing column chromatography separation to obtain an extract filtrate;

[0014] (3) The extract filtrate is concentrated and then recrystallized, and then dried and pulverized to obtain the product.

[0015] As a preferred technical solution, the kanuka raw material is kanuka leaves, which come from the North Island of New Zealand; the kava raw material is kava roots, which come from island countries in the South Pacific.

[0016] As a preferred technical solution, the alcohol compound is diluted with deionized water to a solute concentration of 50-90wt%; preferably, the alcohol compound is at least one of methanol, ethanol, isopropanol, propylene glycol, 1,3-butanediol, ethylene glycol, and 1,2-pentanediol; preferably, the alcohol compound is a combination of ethanol and 1,3-butanediol; the mass ratio of ethanol to 1,3-butanediol is (3-4):1.

[0017] As a preferred technical solution, the temperature of the reflux extraction is 40-60°C, and the time of the reflux extraction is 1-2h. Preferably, the temperature of the reflux extraction is 50-60°C, and the time of the reflux extraction is 1-2h.

[0018] As a preferred technical solution, the mass ratio between the kanuka raw material and / or kava raw material and the alcohol compound diluted with deionized water is 1:(3-5).

[0019] The kanuka extract and kava extract of the present invention are prepared by crushing the raw materials into 40-80 meshes separately or after mixing, and then performing medium-temperature reflux extraction with a mixed alcohol solution of ethanol and 1,3-butanediol, thereby improving the utilization rate of the raw materials and fully extracting the fat-soluble active ingredients in the raw materials, while avoiding the oxidation discoloration and failure of kava lactone under high temperature conditions, and improving the activity retention rate of the effective ingredients in the extract.

[0020] As a preferred technical solution, the concentrator is at least one of chitosan and hydroxypropyl-β-cyclodextrin; preferably, the concentrator is hydroxypropyl-β-cyclodextrin. Preferably, the mass ratio between the extract and the concentrator is (40-60):0.1.

[0021] The kanuka extract and kava extract used in the present invention are concentrated with hydroxypropyl-β-cyclodextrin before the column chromatography molecular process, which, on the one hand, effectively enhances the stability of the effective active ingredients in the extract and prevents the effective active ingredients from being oxidized and ineffective in the subsequent process; on the other hand, the finally prepared extract composition has excellent subsequent application and use effects, and reduces the stimulation of organic molecules in cosmetics to skin mucosal tissues.

[0022] As a preferred technical solution, the column chromatography separation is specifically as follows: adding the extract filtrate mixed with the concentrator to a macroporous resin column, first eluting with 1-3BV / h of deionized water, and then eluting with 4-6BV / h of a mixed alcohol aqueous solution, collecting the mixed alcohol aqueous solution eluate as the extract filtrate. Preferably, the total concentration of the mixed alcohol in the mixed alcohol aqueous solution is 80-90wt%, the mixed alcohol is a combination of ethanol and 1,3-butanediol, and the mass ratio of ethanol to 1,3-butanediol is (3-4): 1. Preferably, the macroporous resin is at least one of HPD-100, HPD-300, D-101, X-5, H103, AB-8, DA-201, and HPD-400. Preferably, HPD-300.

[0023] As a preferred technical solution, the concentration is vacuum reduced pressure distillation concentration, and the conditions of the vacuum reduced pressure distillation concentration are: vacuum degree is 60-80Pa, and temperature is 40-50°C.

[0024] As a preferred technical solution, the concentration specifically comprises: concentrating the extraction filtrate by vacuum distillation to 1 / 10-1 / 15 of the original volume.

[0025] The present invention uses a vacuum reduced pressure distillation concentration process to concentrate the filtrate after column chromatography separation, especially controls the vacuum degree to be 60-80Pa and the temperature to be 40-50°C to concentrate the extraction filtrate to 1 / 10-1 / 15 of the original volume, so that the prepared kanuka extract and kava extract retain the characteristic smell while ensuring the activity of the oily components in the extract, thereby ensuring the common soothing and anti-inflammatory effects of the kanuka extract and the kava extract after compounding.

[0026] As a preferred technical solution, the recrystallization is specifically as follows: drying the concentrated extraction filtrate, re-dissolving it with an equal mass of ethanol, and drying it again, and repeating this process 2-3 times.

[0027] Based on the system of the present invention, the purity of the kanuka extract and the kava extract is ensured by drying the concentrated extraction filtrate and then recrystallizing it with an equal mass of ethanol.

[0028] The preparation method of the soothing and anti-inflammatory plant extract composition is specifically as follows: diluting the prepared kanuka extract and kava extract with a 1,3-butanediol aqueous solution and mixing them according to a mass ratio. The mass concentration of 1,3-butanediol in the 1,3-butanediol aqueous solution is 20-40%.

[0029] Another aspect of the present invention provides an application of a soothing and anti-inflammatory plant extract composition, which is added to skin care products, sunscreen products, and cleansing products to achieve soothing and anti-inflammatory effects, wherein the addition amount of the soothing and anti-inflammatory plant extract composition is 0.1-2wt%.

[0030] Beneficial effects:

[0031] 1. The present invention provides a soothing and anti-inflammatory plant extract composition, which is obtained by compounding a Kava extract prepared by extraction with a Kanuka extract. The soothing and anti-inflammatory plant extract composition can be effectively added to skin care products, sunscreen products, and cleansing products to achieve soothing and anti-inflammatory effects, and the composition has high formula compatibility and safety, good stability, and is easy to add and use.

[0032] 2. The present invention uses a kanuka extract and a kava extract for compounding, especially when the mass ratio of the kanuka extract to the kava extract is 1: (0.1-5), on the one hand, it can produce a synergistic effect on efficacy, avoid the single efficacy, and expand the scope of application; on the other hand, it helps to enhance the shelf life stability of the active ingredients in the kava extract, and the compounded composition has a longer effective service life.

[0033] 3. The kanuka extract and kava extract of the present invention are prepared by crushing the raw materials into 40-80 meshes separately or in combination, and then performing medium-temperature reflux extraction with a mixed alcohol solution of ethanol and 1,3-butanediol, thereby improving the utilization rate of the raw materials and fully extracting the fat-soluble active ingredients in the raw materials, while avoiding the oxidation discoloration and failure of kava lactone under high temperature conditions, thereby improving the activity retention rate of the effective ingredients in the extract.

[0034] 4. The kanuka extract and kava extract used in the present invention are concentrated with hydroxypropyl-β-cyclodextrin before column chromatography molecular process, which can effectively enhance the stability of the effective active ingredients in the extracts and prevent the effective active ingredients from being oxidized and ineffective in subsequent processes. On the other hand, the extract composition finally prepared has excellent subsequent application and use effects, and reduces the stimulation of organic molecules in cosmetics to skin and mucous membrane tissues.

[0035] 5. The present invention uses a vacuum reduced pressure distillation concentration process to concentrate the filtrate after column chromatography separation, especially controlling the vacuum degree to 60-80Pa and the temperature to 40-50°C to concentrate the extraction filtrate to 1 / 10-1 / 15 of the original volume, so that the prepared kanuka extract and kava extract retain the characteristic smell while ensuring the activity of the oily components in the extract, thereby ensuring the common soothing and anti-inflammatory effects of the kanuka extract and kava extract after compounding. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 MTT test results for sample A;

[0037] Figure 2 This is the MTT test result of test sample B. DETAILED DESCRIPTION

[0038] Example 1

[0039] Example 1 of the present invention provides a soothing and anti-inflammatory plant extract composition, which is a combination of a kanuka extract and a kava extract; the mass ratio of the kanuka extract to the kava extract is 1:1.

[0040] The extraction process of the kanuka extract specifically comprises the following steps:

[0041] (1) grinding the raw material of Kanuka eruca into 40 meshes, adding an alcohol compound diluted with deionized water, and performing reflux extraction to obtain an extract;

[0042] (2) adding a concentrator to the extract, mixing the mixture, and performing column chromatography separation to obtain an extract filtrate;

[0043] (3) The extract filtrate is concentrated and then recrystallized, and then dried and pulverized to obtain the product.

[0044] The alcohol compound is diluted with deionized water to a solute concentration of 80 wt %; the alcohol compound is a combination of ethanol and 1,3-butanediol; the mass ratio of ethanol to 1,3-butanediol is 3:1.

[0045] The temperature of the reflux extraction is 55° C., and the time of the reflux extraction is 1.5 h.

[0046] The mass ratio between the Kanuka raw material and the alcohol compound diluted with deionized water is 1:4.

[0047] The concentrate is hydroxypropyl-β-cyclodextrin. The mass ratio of the extract to the concentrate is 50:0.1.

[0048] The column chromatography separation is specifically as follows: adding the extract filtrate mixed with the concentrator to a macroporous resin column, first eluting with 2BV / h of deionized water, then eluting with 5BV / h of a mixed alcohol aqueous solution, collecting the mixed alcohol aqueous solution eluate as the extract filtrate. The total concentration of the mixed alcohol in the mixed alcohol aqueous solution is 80wt%, the mixed alcohol is a combination of ethanol and 1,3-butanediol, and the mass ratio of ethanol to 1,3-butanediol is 3:1. The macroporous resin is HPD-300 type.

[0049] The concentration is vacuum reduced pressure distillation concentration, and the conditions of the vacuum reduced pressure distillation concentration are: vacuum degree of 70Pa, temperature of 45°C.

[0050] The concentration specifically comprises: concentrating the extraction filtrate to 1 / 10 of the original volume by vacuum distillation.

[0051] The recrystallization is specifically as follows: drying the concentrated extraction filtrate, re-dissolving it with an equal mass of ethanol, and drying it again, and repeating this process twice.

[0052] The specific implementation method of the extraction process of the kava extract is the same as that of the kanuka extract, except that the kanuka raw material is replaced by the kava raw material. The kanuka raw material is kanuka leaves, which come from the North Island of New Zealand; the kava raw material is kava roots, which come from island countries in the South Pacific.

[0053] The preparation method of the soothing and anti-inflammatory plant extract composition is specifically as follows: diluting the prepared kanuka extract and kava extract by 1 time with a 1,3-butanediol aqueous solution respectively and mixing them according to a mass ratio. The mass concentration of 1,3-butanediol in the 1,3-butanediol aqueous solution is 30%.

[0054] Example 2

[0055] Example 2 of the present invention provides a Kanuka eruca extract.

[0056] The extraction process of the kanuka extract specifically comprises the following steps:

[0057] (1) grinding the raw material of Kanuka eruca into 40 meshes, adding an alcohol compound diluted with deionized water, and performing reflux extraction to obtain an extract;

[0058] (2) adding a concentrator to the extract, mixing the mixture, and performing column chromatography separation to obtain an extract filtrate;

[0059] (3) The extract filtrate is concentrated and then recrystallized, and then the dried and crushed Kanuka keiskei extract is diluted twice with a 1,3-butanediol aqueous solution to obtain a 1,3-butanediol aqueous solution having a mass concentration of 30%.

[0060] The alcohol compound is diluted with deionized water to a solute concentration of 80 wt %; the alcohol compound is a combination of ethanol and 1,3-butanediol; the mass ratio of ethanol to 1,3-butanediol is 3:1.

[0061] The temperature of the reflux extraction is 55° C., and the time of the reflux extraction is 1.5 h.

[0062] The mass ratio between the Kanuka raw material and the alcohol compound diluted with deionized water is 1:4.

[0063] The concentrate is hydroxypropyl-β-cyclodextrin. The mass ratio of the extract to the concentrate is 50:0.1.

[0064] The column chromatography separation is specifically as follows: adding the extract filtrate mixed with the concentrator to a macroporous resin column, first eluting with 2BV / h of deionized water, then eluting with 5BV / h of a mixed alcohol aqueous solution, collecting the mixed alcohol aqueous solution eluate as the extract filtrate. The total concentration of the mixed alcohol in the mixed alcohol aqueous solution is 80wt%, the mixed alcohol is a combination of ethanol and 1,3-butanediol, and the mass ratio of ethanol to 1,3-butanediol is 3:1. The macroporous resin is HPD-300 type.

[0065] The concentration is vacuum reduced pressure distillation concentration, and the conditions of the vacuum reduced pressure distillation concentration are: vacuum degree of 70Pa, temperature of 45°C.

[0066] The concentration specifically comprises: concentrating the extraction filtrate to 1 / 10 of the original volume by vacuum distillation.

[0067] The recrystallization is specifically as follows: drying the concentrated extraction filtrate, re-dissolving it with an equal mass of ethanol, and drying it again, and repeating this process twice.

[0068] The kanuka raw material is kanuka leaves, which come from the North Island of New Zealand.

[0069] Example 3

[0070] Example 3 of the present invention provides a kava extract.

[0071] The extraction process of the Piper methysticum extract specifically comprises the following steps:

[0072] (1) grinding the Piper methysticum raw material into 40 meshes, adding the alcohol compound diluted with deionized water, and performing reflux extraction to obtain an extract;

[0073] (2) adding a concentrator to the extract, mixing the mixture, and performing column chromatography separation to obtain an extract filtrate;

[0074] (3) The extraction filtrate is concentrated and then recrystallized, and then the dried and crushed Kava pepper extract is diluted twice with a 1,3-butanediol aqueous solution to obtain a 1,3-butanediol aqueous solution having a mass concentration of 30%.

[0075] The alcohol compound is diluted with deionized water to a solute concentration of 80 wt %; the alcohol compound is a combination of ethanol and 1,3-butanediol; the mass ratio of ethanol to 1,3-butanediol is 3:1.

[0076] The temperature of the reflux extraction is 55° C., and the time of the reflux extraction is 1.5 h.

[0077] The mass ratio between the kava raw material and the alcohol compound diluted with deionized water is 1:4.

[0078] The concentrate is hydroxypropyl-β-cyclodextrin. The mass ratio of the extract to the concentrate is 50:0.1.

[0079] The column chromatography separation is specifically as follows: adding the extract filtrate mixed with the concentrator to a macroporous resin column, first eluting with 2BV / h of deionized water, then eluting with 5BV / h of a mixed alcohol aqueous solution, collecting the mixed alcohol aqueous solution eluate as the extract filtrate. The total concentration of the mixed alcohol in the mixed alcohol aqueous solution is 80wt%, the mixed alcohol is a combination of ethanol and 1,3-butanediol, and the mass ratio of ethanol to 1,3-butanediol is 3:1. The macroporous resin is HPD-300 type.

[0080] The concentration is vacuum reduced pressure distillation concentration, and the conditions of the vacuum reduced pressure distillation concentration are: vacuum degree of 70Pa, temperature of 45°C.

[0081] The concentration specifically comprises: concentrating the extraction filtrate to 1 / 10 of the original volume by vacuum distillation.

[0082] The recrystallization is specifically as follows: drying the concentrated extraction filtrate, re-dissolving it with an equal mass of ethanol, and drying it again, and repeating this process twice.

[0083] The kava raw material is kava root, which comes from Vanuatu.

[0084] Comparative Example 1

[0085] Comparative Example 1 of the present invention provides a soothing and anti-inflammatory plant extract composition, and its specific implementation is the same as that of Example 1, except that the temperature of the reflux extraction is 55° C., and the time of the reflux extraction is 1.5 h.

[0086] Comparative Example 2

[0087] Comparative Example 2 of the present invention provides a soothing and anti-inflammatory plant extract composition, and its specific implementation method is the same as that of Example 1, except that step (2) in the extraction process of the Kava Piper methysticum extract and the Kanuka oleracea extract is specifically to obtain an extraction filtrate after column chromatography separation of the extract.

[0088] Comparative Example 3

[0089] Comparative Example 3 of the present invention provides a soothing and anti-inflammatory plant extract composition, and its specific implementation manner is the same as that of Example 1, except that the concentrate is chitosan (product number C3646), CAS number 9012-76-4, sourced from Sigma-Aldrich.

[0090] Performance Testing Methods

[0091] 1. Stability: The soothing and anti-inflammatory plant extract compositions prepared in the examples and comparative examples were placed at constant temperatures of 4° C., 25° C. and 40° C. for three months, and the appearance of the compositions was observed. The results are recorded in Table 1.

[0092] Table 1

[0093]

[0094]

[0095] 2. Efficacy test:

[0096] 1. Test Purpose

[0097] This test is divided into two parts; the first part is based on macrophages, conducting cytotoxicity tests to determine the dosage concentration of the sample on the macrophages; the second part uses 1μg / mL LPS to stimulate macrophages, and evaluates the soothing effect of the sample by detecting the changes in the content of proinflammatory factors (IL-1β, IL-6, TNF-α) and inflammatory mediators (PGE2) after the sample is acted on.

[0098] 2. Test materials

[0099] 2.1 Test system

[0100] The cells used in this test were macrophages, batch number: 210622, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0101] 2.2 Main reagents

[0102] High glucose DMEM (Gibco), PBS (Solebao), MTT (Sigma), DMSO (Sigma), fetal bovine serum (Lanzhou Rongye), lipopolysaccharide LPS (E.Coli.Sigma), dexamethasone (Sigma), IL-1β ELISA kit (Abcam), IL-6 ELISA kit (Abcam), TNF-α ELISA kit (Abcam), PGE2 ELISA kit (Abcam).

[0103] 2.3 Main Equipment

[0104] CO2 incubator (Thermo, 150I), clean bench (Suzhou Antai, SW-CJ-1F), inverted microscope (Olympus, CKX41), and microplate reader (BioTek, Epoch).

[0105] 2.4 Sample Information

[0106] Test sample A: Example 2;

[0107] Test Sample B: Example 1.

[0108] 3. Cytotoxicity

[0109] 3.1 Test Method

[0110] 1) Cell inoculation: 9×10 macrophages 3 Cells were inoculated into 96-well plates at a seeding density of 100 cells / well and incubated in an incubator (37°C, 5% CO2).

[0111] Incubate overnight.

[0112] 2) Experimental grouping: The experiment set up a zero adjustment group, a solvent control group, a positive control group and a sample group. In the sample group, each sample was set with 8 concentration gradients, and 3 replicate wells were set under each concentration gradient.

[0113] 3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table (Table 2).

[0114] Table 2. Test concentration setting table

[0115]

[0116] 4) Drug administration: When the cell plating rate in the 96-well plate reaches 50%, drug administration is performed. Add 200 μL of culture medium to each well of the solvent control group; add 200 μL of culture medium containing 10% DMSO to each well of the positive control group; add 200 μL of culture medium containing the corresponding concentration of sample to each well of the sample group; the zero adjustment group has no cell inoculation and only adds 200 μL of cell culture medium. After drug administration, place the 96-well plate in an incubator (37°C,

[0117] 5% CO2) for 24 h.

[0118] 5) Detection: After incubating the cells for 24 hours, discard the supernatant, add MTT working solution (0.5 mg / mL), and incubate at 37°C in the dark for 4 hours. After the incubation, discard the supernatant, add 150 μL DMSO to each well, and read the OD value at 490 nm.

[0119] 6) Calculation of relative cell viability: Calculated according to the formula, relative cell viability (%) = sample well OD - zero adjustment well OD / solvent control well OD - zero adjustment well OD * 100%.

[0120] 3.2 Test Results

[0121] (1) Test sample AMTT test results (see Figure 1 ), according to the MTT results, it was considered that the test sample A showed no obvious cytotoxicity based on macrophages within the concentration range of 25 mg / mL.

[0122] (2) MTT test results of sample B (see Figure 2 ), according to the MTT results, it was considered that the test sample B showed no obvious cytotoxicity within the concentration range of 10 mg / mL based on macrophages.

[0123] 4. Soothing effect test

[0124] 4.1 Test Method

[0125] 4.1.1 Test Grouping

[0126] Table 3. Test groups

[0127]

[0128] 4.1.2 Working fluid configuration

[0129] Positive control group (dexamethasone) working solution preparation: 2 μL of 10% mother solution was dissolved in 2 mL of culture medium to prepare 0.01% dexamethasone.

[0130] 4.1.3 Operation steps

[0131] 1) Cell inoculation: Inoculate cells into 6-well plates at an inoculation density of 2.2×10 5 / well, add 2 mL of cell suspension to each well, and place the inoculated cell culture plate in an incubator for 24 hours (5% CO2, 37°C). 2) Aspirate and discard the old cell culture medium in the wells. Add 1.8 mL of culture medium containing the test substance at the corresponding concentration to each well of the sample group; add 1.8 mL of solvent control culture medium to each well of the negative control group; add 1.8 mL of positive control culture medium to each well of the positive control group; and add 1.8 mL of normal culture medium to each well of the blank control group.

[0132] 3) After administration, the 6-well plate was placed in a cell culture incubator (5% CO2, 37°C) for 2 h.

[0133] 4) LPS stimulation: 2 hours after administration, 200 μL of the prepared LPS-containing working solution was added to each well of each group and the cells were placed in a cell culture incubator (5% CO 2 , 37° C.) and cultured for 22 hours.

[0134] 5) ELISA test: Collect the cell culture supernatant and perform ELISA test according to the instructions of the ELISA kit.

[0135] 6) Statistical analysis of results: GraphPad Prism was used for plotting, and the results were expressed as Mean ± SD. The t-test was used for statistical analysis for comparison between groups. All statistical analyses were two-tailed. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0136] 4.2 Test Results

[0137] 4.2.1 IL-1β test results are shown in Table 7.

[0138] Table 4. Summary of IL-1β content results.

[0139]

[0140]

[0141] Note: When the t-test method is used for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **; compared with the test sample A group, the significance is indicated by ◆, P-value < 0.05 is indicated by ◆, and P-value < 0.01 is indicated by ◆◆.

[0142] Compared with the BC group, the IL-1β content in the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the IL-1β content in the PC group decreased significantly, indicating that the positive control of this test was effective. Compared with the NC group, the IL-1β content of test sample A-10.2mg / mL and test sample B-7.6mg / mL decreased significantly, with inhibition rates of 45.73% and 57.82%, respectively; compared with test sample A-10.2mg / mL, the IL-1β content of test sample B-7.6mg / mL decreased significantly.

[0143] 4.2.2 IL-6 test results

[0144] Table 5. Summary of IL-6 content results

[0145]

[0146] Note: When the t-test method is used for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **; compared with the test sample A group, the significance is indicated by ◆, P-value < 0.05 is indicated by ◆, and P-value < 0.01 is indicated by ◆◆.

[0147] Compared with the BC group, the IL-6 content in the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the IL-6 content in the PC group decreased significantly, indicating that the positive control of this test was effective. Compared with the NC group, the IL-6 content of test sample A-10.2mg / mL and test sample B-7.6mg / mL decreased significantly, with inhibition rates of 16.69% and 34.76%, respectively; compared with test sample A-10.2mg / mL, the IL-6 content of test sample B-7.6mg / mL decreased significantly.

[0148] 4.2.3 TNF-α

[0149] Table 6. Summary of TNF-α content results

[0150]

[0151] Note: When the t-test method is used for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **; compared with the test sample A group, the significance is indicated by ◆, P-value < 0.05 is indicated by ◆, and P-value < 0.01 is indicated by ◆◆.

[0152] Compared with the BC group, the TNF-α content in the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the TNF-α content in the PC group decreased significantly, indicating that the positive control of this test was effective. Compared with the NC group, the TNF-α content of test sample A-10.2mg / mL and test sample B-7.6mg / mL decreased significantly, with inhibition rates of 31.68% and 49.48%, respectively; compared with test sample A-10.2mg / mL, test sample B-7.6mg / mL decreased significantly.

[0153] 4.2.4 PGE2 test results

[0154] Table 10. Summary of PGE2 content results

[0155]

[0156] Note: When the t-test method is used for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **; compared with the test sample A group, the significance is indicated by ◆, P-value < 0.05 is indicated by ◆, and P-value < 0.01 is indicated by ◆◆.

[0157] Compared with the BC group, the PGE2 content of the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the PGE2 content of the PC group decreased significantly, indicating that the positive control of this test was effective. Compared with the NC group, the PGE2 content of the test sample B-7.6mg / mL decreased significantly, with an inhibition rate of 70.80%; compared with the test sample A-10.2mg / mL, the PGE2 content of the test sample B-7.6mg / mL decreased significantly;

[0158] 5. Conclusion

[0159] Based on macrophages, compared with the control group, the contents of pro-inflammatory factors IL-1β, IL-6, and TNF-α in test sample A at a concentration of 10.2 mg / mL were significantly reduced, with inhibition rates of 45.73%, 16.69%, and 31.68%, respectively, indicating that the sample, at this concentration, achieved a soothing effect by inhibiting the secretion of pro-inflammatory factors (IL-1β, IL-6, and TNF-α);

[0160] The contents of pro-inflammatory factors IL-1β, IL-6, TNF-α and inflammatory mediator PGE2 in test sample B at a concentration of 10.2 mg / mL were significantly decreased, with inhibition rates of 57.82%, 34.76%, 49.48% and 70.80%, respectively, indicating that the sample, at this concentration, achieved a soothing effect by inhibiting the secretion of pro-inflammatory factors (IL-1β, IL-6, TNF-α) and inflammatory mediators (PGE2);

[0161] The comparison results between sample groups showed that:

[0162] Compared with test sample A-10.2 mg / mL, the contents of IL-1β, IL-6, TNF-α, and PGE2 in test sample B at a concentration of 10.2 mg / mL were significantly decreased.

Claims

1. A soothing and anti-inflammatory plant extract composition, characterized in that: The soothing and anti-inflammatory plant extract composition is a combination of kanuka extract and kava extract, wherein the mass ratio of the kanuka extract to the kava extract is 1:(0.1-5); the extraction process of the kanuka extract and the kava extract specifically comprises the following steps: (1) grinding the kanuka raw material and / or kava raw material into 40-80 meshes, adding the alcohol compound diluted with deionized water, and performing reflux extraction to obtain an extract; (2) adding a concentrator to the extract, mixing the mixture, and performing column chromatography separation to obtain an extract filtrate; (3) The extract filtrate is concentrated and then recrystallized, and then dried and crushed to obtain; The alcohol compound is a combination of ethanol and 1,3-butanediol; the temperature of the reflux extraction is 40-60° C., and the time of the reflux extraction is 1-2 hours; and the concentrator is hydroxypropyl-β-cyclodextrin.

2. The soothing and anti-inflammatory plant extract composition according to claim 1, characterized in that: The column chromatography separation is specifically as follows: adding the extract mixed with the concentrator into a macroporous resin column, first eluting with 1-3BV / h of deionized water, then eluting with 4-6BV / h of a mixed alcohol aqueous solution, and collecting the mixed alcohol aqueous solution eluate as the extraction filtrate.

3. An application of the soothing and anti-inflammatory plant extract composition according to any one of claims 1 to 2, characterized in that: The invention is applied to the preparation of skin care products, wherein the addition amount of the soothing and anti-inflammatory plant extract composition is 0.1-2wt%.

4. The use of the soothing and anti-inflammatory plant extract composition according to claim 3, characterized in that: The skin care products include sunscreen products and facial cleansing products.

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