Composition containing Inonotus obliquus and Kava kava extract and preparation method thereof

Abstract

The present invention provides a composition containing Inonotus obliquus and Piper methysticum kava extracts and a preparation method thereof. The composition comprises the following raw materials in parts by mass: 9-11 parts of Inonotus obliquus extract; 4-8 parts of Piper methysticum kava root extract; 4-6 parts of Patchouli extract; and 5-15 parts of Gentiana davidii root extract. The Inonotus obliquus extract is cultured and extracted using different culture media to obtain different Inonotus obliquus extracts. The Inonotus obliquus extracts are compatible with the components to achieve synergistic effects, so that the composition has excellent soothing, anti-allergic and other effects. In addition, the present invention uses plant cell vesicles as packaging carriers to encapsulate the composition, so that the composition has excellent permeability.

Description

Technical Field

[0001] The invention belongs to the field of cosmetic preparation, and particularly relates to a composition containing Inonotus obliquus and Kava kava extracts and a preparation method thereof. Background Art

[0002] Sensitive skin, also known as sensitive skin syndrome, refers to skin with poor tolerance, high reactivity, or sensitization. It primarily occurs on the face and manifests as subjective symptoms such as burning, stinging, itching, and tightness in response to physical, chemical, or psychological stimuli. These symptoms are sometimes accompanied by objective signs such as erythema, scaling, and dilated capillaries.

[0003] However, the active ingredients in the anti-allergic soothing products currently on the market are not very effective. The reasons for this are:

[0004] Firstly, it may be difficult for a single substance to achieve the ideal soothing effect, and it is necessary to seek a combination that has synergistic effects on skin sensitivity and skin soothing, so as to improve the anti-allergic and soothing effects on sensitive skin.

[0005] Secondly, it may be because most of the active ingredients are oxidized in the air during storage or application.

[0006] Therefore, there is an urgent need to provide a composition with soothing and anti-allergic effects and a preparation method thereof to fill the market gap. Summary of the Invention

[0007] The invention provides a composition containing Inonotus obliquus and Piper methysticum extracts and a preparation method thereof, which can soothe and desensitize the skin through natural ingredients extracted from plants.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for extracting an Inonotus obliquus extract, and the specific preparation steps are as follows:

[0010] S1: Inonotus obliquus was inoculated into medium A and cultured on a shaker for ≥12 days. Mycelial pellets were collected and freeze-dried to obtain mycelial pellet powder A1.

[0011] S2: Inonotus obliquus was inoculated into medium B and cultured for ≥8 days. The fermentation broth was collected and freeze-dried to obtain fermentation broth powder A2.

[0012] S3: Mix A1 and A2 to get A3;

[0013] The culture medium A is composed of 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 2 wt% mulberry leaf extract, 0.01 wt% vitamin K, 0.01 wt% vitamin B2, and 0.01 wt% vitamin H;

[0014] The culture medium B is composed of 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 0.01 wt% ammonium tartrate, 4 wt% birch sap concentrate, 0.01 wt% vitamin E, and 0.01 wt% vitamin B3;

[0015] The culture temperature for S1 is 20±2°C; the culture temperature for S2 is 24±2°C;

[0016] The inoculation amount of the S1 and S2 strains is 3 wt%±0.5 wt%.

[0017] In a second aspect, the present invention provides a composition raw material configuration, the specific components of which are as follows:

[0018] 9-11 parts of Inonotus obliquus extract;

[0019] 4-8 parts of Kava Kava root extract;

[0020] 4-6 parts of patchouli extract;

[0021] 5-15 parts of gentian root extract;

[0022] In a third aspect, the present invention provides a method for preparing plant extracellular vesicles, the specific preparation steps of which are as follows:

[0023] T1: The plant tissue was completely crushed, centrifuged for 20 min, filtered, and the supernatant was collected to obtain product X1;

[0024] T2: subject X1 to gradient centrifugation, filter, and take the supernatant to obtain product X2;

[0025] T3: subject X2 to ultra-high-speed centrifugation, discard the supernatant, and take the precipitate to obtain product X3;

[0026] T4: X3 was reconstituted with PBS, and then 5-10 wt% polyethylene glycol was added, filtered, and the filtrate was stored at -80°C.

[0027] The T1 plant tissue can be selected from any plant.

[0028] The T1 centrifugal speed is 200-500g; the T2 centrifugal speeds are 2000g, 10000g, and 20000g, respectively, with each speed centrifuged for 10-20min; the T3 speed is 100000-120000g, with centrifugation for 50-90min; the T1-T3 centrifugal temperature is 4°C.

[0029] In a fourth aspect, the present invention provides a method for preparing a plant extracellular vesicle-encapsulated carrier, the specific preparation steps of which are as follows:

[0030] Q1: taking T4 plant extracellular vesicle solution and mixing it with the composition, filtering it to obtain a mixture Y1;

[0031] Q2: Place Y1 in a container, apply 120-180V voltage, pulse time 5-15ms, and shock pulse 2-3 times to obtain Y2;

[0032] Q3: Incubate Y2 in the dark for 18-24 hours, then store at 0-4°C in the dark.

[0033] In Q1, the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.02-0.1.

[0034] This invention innovatively combines mycelium or fermentation broths grown in two different culture media with Inonotus obliquus, and then adds Kava kava root extract, Patchouli extract, and Gentiana lutea root extract to form a composition. This composition is then encapsulated in plant extracellular vesicles (EVs). This results in a stable, highly permeable composition with soothing and anti-allergic effects on the skin.

[0035] Compared with the existing technology, the present invention has the following advantages: by using plant exosomes as a carrier to encapsulate the composition, the stability of the composition can be effectively maintained and the permeability of the composition can be improved to a certain extent. The special particle size of the exosomes allows the composition to penetrate deep into the skin to exert its effect. In addition, by changing the culture medium, different efficacy of Inonotus obliquus extracts can be obtained, and the addition of kava root extract, patchouli extract, and gentian root extract greatly enhances its soothing and anti-allergic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 :Electron micrograph of extracellular vesicles in wheat plants;

[0037] Figure 2 :Exosome size diagram of wheat plants;

[0038] Figure 3 : Extracellular vesicle concentration in wheat plants;

[0039] Figure 4 :Electron micrograph of extracellular vesicles of Dendrobium plants;

[0040] Figure 5 :Dendrobium plant extracellular vesicle particle size diagram

[0041] Figure 6 :Concentration of extracellular vesicles from Dendrobium officinale:

[0042] Figure 7 :Electron micrograph of extracellular vesicles of Sophora flavescens plants;

[0043] Figure 8 :Extracellular vesicles from Sophora flavescens plants

[0044] Figure 9 : Sophora flavescens plant extracellular vesicle concentration;

[0045] Figure 10 :Electron micrograph of extracellular vesicles of Patchouli plant;

[0046] Figure 11 : Diagram of the extracellular vesicles of Patchouli plant;

[0047] Figure 12 : Concentration of extracellular vesicles in Patchouli plants;

[0048] Figure 13 :Electron micrograph of extracellular vesicles of ginseng plants;

[0049] Figure 14 :Ginseng plant extracellular vesicle particle size diagram;

[0050] Figure 15 : Concentration of extracellular vesicles from ginseng plants. DETAILED DESCRIPTION

[0051] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples. The terms used in the examples are for describing specific embodiments and do not constitute a limitation on the scope of protection of the present invention.

[0052] The experimental methods in the following examples, where specific conditions are not specified, are generally based on conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0053] Some of the raw materials and their sources are as follows:

[0054] Inonotus obliquus: purchased from Ningbo Testo Biotechnology Co., Ltd., catalog number TS318940;

[0055] Kava root extract: purchased from Henan Jushikang Biotechnology Co., Ltd.

[0056] Patchouli extract: purchased from Henan Jushikang Biotechnology Co., Ltd.

[0057] Patchouli leaves: purchased from Henan Jushikang Biotechnology Co., Ltd.

[0058] Gentiana root extract: purchased from Henan Jushikang Biotechnology Co., Ltd.

[0059] Preparation, comprising the following steps:

[0060] The preparation method of Inonotus obliquus extract is as follows:

[0061] S1: Inonotus obliquus was inoculated into medium A and cultured on a shaker for ≥12 days. Mycelial pellets were collected and freeze-dried to obtain mycelial pellet powder A1.

[0062] S2: Inonotus obliquus was inoculated into medium B and cultured for ≥8 days. The fermentation broth was collected and freeze-dried to obtain fermentation broth powder A2.

[0063] S3: Mix A1 and A2 to get A3;

[0064] The culture temperature for S1 is 20±2°C; the culture temperature for S2 is 24±2°C;

[0065] The inoculation amount of the S1 and S2 strains is 3 wt%±0.5 wt%.

[0066] Example 1: Following the preparation methods S1-S3, the medium A is prepared by comprising 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 2 wt% mulberry leaf extract, 0.01 wt% vitamin K, 0.01 wt% vitamin B2, and 0.01 wt% vitamin H;

[0067] The culture medium B is composed of 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 0.01 wt% ammonium tartrate, 4 wt% birch sap concentrate, 0.01 wt% vitamin E, and 0.01 wt% vitamin B3;

[0068] The culture temperature of S1 is 20°C; the culture temperature of S2 is 24°C;

[0069] The inoculation amount of the S1 and S2 strains is 3 wt%.

[0070] Example 2: Following the preparation methods S1-S3, the medium A is prepared by comprising 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 2 wt% mulberry leaf extract, 0.01 wt% vitamin K, 0.01 wt% vitamin B2, and 0.01 wt% vitamin H;

[0071] The culture medium B is composed of 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 0.01 wt% ammonium tartrate, 4 wt% birch sap concentrate, 0.01 wt% vitamin E, and 0.01 wt% vitamin B3;

[0072] The culture temperature of S1 is 22°C; the culture temperature of S2 is 26°C;

[0073] The inoculation amount of the S1 and S2 strains is 3.5 wt%.

[0074] Example 3: Following the preparation methods S1-S3, the medium A is prepared by using the following raw materials: 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 2 wt% mulberry leaf extract, 0.01 wt% vitamin K, 0.01 wt% vitamin B2, and 0.01 wt% vitamin H;

[0075] The culture medium B is composed of 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 0.01 wt% ammonium tartrate, 4 wt% birch sap concentrate, 0.01 wt% vitamin E, and 0.01 wt% vitamin B3;

[0076] The culture temperature of S1 is 18°C; the culture temperature of S2 is 22°C;

[0077] The inoculation amount of the S1 and S2 strains is 2.5 wt%.

[0078] Comparative Example 1: The difference from the example is that the medium A is configured with raw materials of 2wt% glucose, 0.03wt% magnesium sulfate, 0.1wt% potassium nitrate, 0.5wt% yeast extract, 0.01wt% vitamin K, 0.01wt% vitamin B2, and 0.01wt% vitamin H;

[0079] The culture medium B is configured with raw materials including 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 0.01 wt% ammonium tartrate, 0.01 wt% vitamin E, and 0.01 wt% vitamin B3; the remaining steps are the same as those in Example 1.

[0080] The mass ratio of the components of the composition is as follows:

[0081] Example 4: 10 parts of Inonotus obliquus extract, 6 parts of Piper methysticum root extract, 5 parts of Patchouli extract, and 10 parts of Gentiana root extract; the Inonotus obliquus extract was prepared in Example 1.

[0082] Example 5: 9 parts of Inonotus obliquus extract, 4 parts of Piper methysticum root extract, 6 parts of Patchouli extract, and 15 parts of Gentiana root extract; the Inonotus obliquus extract was prepared in Example 1.

[0083] Example 6: 11 parts of Inonotus obliquus extract, 8 parts of Piper methysticum root extract, 4 parts of Patchouli extract, and 5 parts of Gentiana scabra root extract; the Inonotus obliquus extract was prepared in Example 1.

[0084] Example 7: 10 parts of Inonotus obliquus extract, 6 parts of Piper methysticum root extract, 5 parts of Patchouli extract, and 10 parts of Gentiana root extract; the Inonotus obliquus extract was prepared in Example 2.

[0085] Example 8: 10 parts of Inonotus obliquus extract, 6 parts of Piper methysticum root extract, 5 parts of Patchouli extract, and 10 parts of Gentiana root extract; the Inonotus obliquus extract was prepared in Example 3.

[0086] Comparative Example 2: 10 parts of Inonotus obliquus extract, 6 parts of Piper methysticum root extract, 5 parts of Patchouli extract, and 10 parts of Gentiana root extract; the Inonotus obliquus extract was prepared in Comparative Example 1.

[0087] Comparative Example 3: 10 parts of Inonotus obliquus extract, 6 parts of Piper methysticum root extract, 5 parts of Patchouli extract, and 10 parts of Gentiana root extract; the Inonotus obliquus extract is commercially available.

[0088] Comparative Example 4: 6 parts of Kava Piper methysticum root extract, 5 parts of Patchouli extract, and 10 parts of Gentiana dahurica root extract.

[0089] Comparative Example 5: 10 parts of Inonotus obliquus extract, 5 parts of Patchouli extract, and 10 parts of Gentiana root extract; the Inonotus obliquus extract was prepared in Example 1.

[0090] Comparative Example 6: 10 parts of Inonotus obliquus extract, 6 parts of Piper mesenteriae root extract, and 10 parts of Gentiana scabra root extract; the Inonotus obliquus extract was prepared in Example 1.

[0091] A method for preparing plant extracellular vesicles, the specific steps of which are as follows:

[0092] T1: The plant tissue was completely crushed, centrifuged for 20 min, filtered, and the supernatant was collected to obtain product X1;

[0093] T2: subject X1 to gradient centrifugation, filter, and take the supernatant to obtain product X2;

[0094] T3: subject X2 to ultra-high-speed centrifugation, discard the supernatant, and take the precipitate to obtain product X3;

[0095] T4: X3 was re-dissolved in PBS, and then 8 wt% polyethylene glycol was added, filtered, and the filtrate was stored at -80°C.

[0096] The T1 plant tissue can be selected from any plant, and this application uses patchouli leaf tissue.

[0097] The T1 centrifugal speed is 500g; the T2 centrifugal speeds are 2000g, 10000g, and 20000g, respectively, and each speed is centrifuged for 20 minutes; the T3 speed is 120000g, and the centrifugation is for 90 minutes; the T1-T3 centrifugal temperature is 4°C.

[0098] A method for preparing a plant extracellular vesicle-encapsulated carrier, the specific preparation steps of which are as follows:

[0099] Q1: taking T4 plant extracellular vesicle solution and mixing it with the composition, filtering it to obtain a mixture Y1;

[0100] Q2: Place Y1 in a container, apply 120-180V voltage, pulse time 5-15ms, and shock pulse 2-3 times to obtain Y2;

[0101] Q3: Incubate Y2 in the dark for 18-24 hours, then store at 0-4°C in the dark.

[0102] In Q1, the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.02-0.1.

[0103] Example 9: The composition is that of Example 4, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.07.

[0104] Example 10: The composition is that of Example 4, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.02.

[0105] Example 11: The composition is that of Example 4, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.1.

[0106] Example 12: The composition is that of Example 5, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.07.

[0107] Example 13: The composition is that of Example 6, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.07.

[0108] Example 14: The composition is that of Example 7, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.07.

[0109] Example 15: The composition is that of Example 8, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.07.

[0110] Comparative Example 7: The composition is that of Comparative Example 2, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.07.

[0111] Comparative Example 8: The composition is that of Comparative Example 3, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.07.

[0112] Comparative Example 9: The composition is that of Comparative Example 4, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.07.

[0113] Comparative Example 10: The composition is that of Comparative Example 5, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.07.

[0114] Comparative Example 11: the composition is that of Comparative Example 6, and the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.07.

[0115] Comparative Example 12: The composition is the same as that of Example 4, except that only the composition is used without using plant extracellular vesicles for encapsulation, and the final concentration of the composition used is the same as that of Example 4.

[0116] A preparation method of a leave-on skin topical preparation, wherein the specific mass proportions are as follows:

[0117] Example 16: 5 wt% of encapsulating carrier, 10 wt% of sodium alginate, 1 wt% of sodium lauryl sulfate, and 5 wt% of honey. The encapsulating carrier is the one prepared in Example 9.

[0118] Example 17: 1 wt% of encapsulating carrier, 10 wt% of sodium alginate, 1 wt% of sodium lauryl sulfate, and 5 wt% of honey. The encapsulating carrier is the one prepared in Example 9.

[0119] Example 18: 8 wt% of encapsulating carrier, 10 wt% of sodium alginate, 1 wt% of sodium lauryl sulfate, and 5 wt% of honey. The encapsulating carrier is the one prepared in Example 9.

[0120] Example 19: 5 wt% of encapsulating carrier, 10 wt% of sodium alginate, 1 wt% of sodium lauryl sulfate, and 5 wt% of honey. The encapsulating carrier is the one prepared in Example 10.

[0121] Example 20: 5 wt% of encapsulating carrier, 10 wt% of sodium alginate, 1 wt% of sodium lauryl sulfate, and 5 wt% of honey. The encapsulating carrier is the one prepared in Example 11.

[0122] Example 21: 5 wt% of encapsulating carrier, 10 wt% of sodium alginate, 1 wt% of sodium lauryl sulfate, and 5 wt% of honey. The encapsulating carrier is the one prepared in Example 12.

[0123] Example 22: 5 wt% of encapsulating carrier, 10 wt% of sodium alginate, 1 wt% of sodium lauryl sulfate, and 5 wt% of honey. The encapsulating carrier is the one prepared in Example 13.

[0124] Example 23: 5 wt% of encapsulating carrier, 10 wt% of sodium alginate, 1 wt% of sodium lauryl sulfate, and 5 wt% of honey. The encapsulating carrier is the one prepared in Example 14.

[0125] Example 24: 5 wt% of encapsulating carrier, 10 wt% of sodium alginate, 1 wt% of sodium lauryl sulfate, and 5 wt% of honey. The encapsulating carrier is the one prepared in Example 15.

[0126] Comparative Example 13: The difference from Example 16 is that the packaging carrier is that of Comparative Example 7, and the other components are the same as those of Example 16.

[0127] Comparative Example 14: The difference from Example 16 is that the packaging carrier is that of Comparative Example 8, and the other components are the same as those of Example 16.

[0128] Comparative Example 15: The difference from Example 16 is that the packaging carrier is that of Comparative Example 9, and the other components are the same as those of Example 16.

[0129] Comparative Example 16: The difference from Example 16 is that the packaging carrier is Comparative Example 10, and the other components are the same as Example 16.

[0130] Comparative Example 17: The difference from Example 16 is that the packaging carrier is Comparative Example 11, and the other components are the same as Example 16.

[0131] Comparative Example 18: The difference from Example 16 is that the packaging carrier is Comparative Example 12, and the other components are the same as Example 16.

[0132] Comparative Example 19: The difference from Example 16 is that no carrier is included, and the other components are the same as Example 16.

[0133] Safety trials

[0134] The 2015 "Technical Specifications for Safety of Cosmetics" was used as a reference standard to conduct a cosmetic irritation evaluation on the skin preparations of Examples 16-24. The test method was a skin patch test, and the test was conducted on 90 randomly distributed people aged 16-65 years.

[0135] Test Method: Place the test substance in a patch tester at a dosage of 0.020-0.025g. Cover the patch tester with non-irritating cloth-based tape on the subject's back or forearm. Use the palm of your hand to gently press the patch tester to evenly adhere to the skin surface. Continue for 24 hours. Remove the patch tester and observe the skin reaction 30 minutes after the indentation disappears. If the result is negative, observe again 24 hours and 48 hours after the patch test.

[0136] Evaluation criteria:

[0137] Grade 0: negative reaction;

[0138] Grade 1: Suspicious reaction, only slight erythema;

[0139] Grade 2: weak positive reaction, erythema, infiltration, edema, and papules may be present;

[0140] Grade 3: Strong positive reaction, with erythema, infiltration, edema, papules, and the reaction may extend beyond the test area;

[0141] Grade 4: Very strong positive reaction, obvious erythema, severe infiltration, edema, confluent blister, and reaction beyond the test area.

[0142] Test results: All subjects had negative skin reactions.

[0143] The above test results show that the leave-on skin external preparation containing the composition-encapsulated carrier provided by the present invention is mild and non-irritating to the skin and is safe to use.

[0144] Skin retention test

[0145] Take appropriate rat skin (effective skin area 0.752cm 2 ) was fixed in a Faranz cell and placed in a receiving solution (a mixture of physiological saline and ethanol (4:1)). The receiving cell was maintained in a constant temperature water bath at 32±1°C and stirred at 400 rpm. After equilibration for 20 minutes, the receiving solution was refreshed. Subsequently, 1g of Examples 9-15 and Comparative Example 12 was evenly applied to the skin surface of rats. Comparative Example 12 was used as a reference reagent.

[0146] After 36 hours of in vitro testing, isolated rat skin was obtained, and the inner and outer surfaces of the skin were rinsed with physiological saline. The skin moisture was absorbed, and the basal layer of skin was cut and minced, and then homogenized. 3 mL of extraction solvent (acetone: methanol = 3:1) was added, vortexed for 2 minutes, and centrifuged at 1500 rpm for 10 minutes. The supernatant was transferred to a centrifuge tube. This was repeated twice, and the extracts were combined, dried with nitrogen, re-dissolved in methanol, vortexed for 2 minutes, centrifuged, and the supernatant was aspirated. The retention of the drug in the basal layer of skin was determined by HPLC.

[0147] Table 1

[0148] experimental group Retention rate (%) Example 9 21.85±0.96 Example 10 20.17±1.17 Example 11 22.41±1.03 Example 12 20.74±1.42 Example 13 20.38±0.96 Example 14 20.07±1.36 Example 15 20.52±1.19 Comparative Example 12 3.17±0.41

[0149] According to the results of Examples 9-15 and Comparative Example 12 in Table 1, the carrier contained in the plant extracellular vesicle solution provided in the embodiments of the present invention has a good penetration and retention effect, especially the effects of Examples 9-11 are better, indicating that in the composition, when the mass ratio of the added amount of the plant extracellular vesicle solution to the composition is 1:0.07-0.1, the skin retention rate of the carrier is the best in the embodiments.

[0150] Inflammatory factor inhibition test

[0151] TNF-α is the most common inflammatory factor in inflammatory responses. It can induce an inflammatory phenotype in vascular endothelial cells, leading to metabolic and hemodynamic changes and promoting the production of inflammatory mediators. Inflammatory mediators, in turn, increase vascular permeability, causing localized congestion and edema. They can also stimulate nerve endings, leading to itching and pain. Therefore, inhibiting TNF-α can help soothe and repair the skin.

[0152] RAW264.7 cells grown to the logarithmic phase were cultured at 1.5 × 10 5 Cells were seeded at a density of 100 μg / mL in a 24-well plate, with 800 μL of cell suspension per well. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. When the cells reached near confluence, 50 μL of a 10 mg / L sample was administered, with three replicates per group. The positive control was 50 μL of 10 μg / mL dexamethasone. One hour later, the cells were stimulated with 1 μg / mL lipopolysaccharide (LPS). After 24 hours of incubation in a cell culture incubator, the cell culture medium was transferred to a centrifuge tube and centrifuged at 5000 rpm for 10 minutes to collect the cell supernatant. The assay was performed according to the instructions of the mouse TNF-α enzyme-linked immunosorbent assay kit.

[0153]

[0154] Table 2

[0155] experimental group Inhibition rate Positive group 100％ Blank group 0.00％ Example 16 78.43％ Example 17 71.17％ Example 18 79.31％ Example 19 74.17％ Example 20 74.36％ Example 21 75.23％ Example 22 72.51％ Example 23 74.23％ Example 24 73.87％ Comparative Example 13 31.42％ Comparative Example 14 28.23％ Comparative Example 15 22.42％ Comparative Example 16 53.41％ Comparative Example 17 55.36％ Comparative Example 18 74.36％ Comparative Example 19 6.37％

[0156] Analysis of the results in Table 2: It can be seen from the results of Examples 16-24 that the compositions provided by the embodiments of the present invention have good TNF-α inhibitory efficacy, especially Examples 16 and 18 have better effects, indicating that using Example 1 as the culture process parameter can effectively improve the inhibitory effect of the composition on TNF-α.

[0157] From the results of Example 16 and Example 18, it can be seen that the inhibitory effect on TNF-α is not linearly positively correlated with the amount of carrier added. Therefore, for cost-effectiveness considerations, 5 wt% was used as the addition amount for subsequent experiments.

[0158] From the results of Example 16 and Comparative Example 13, it can be seen that during the cultivation of Inonotus obliquus, the mulberry leaf extract and birch sap concentrate respectively added to the culture medium can significantly enhance the inhibitory effect of the composition on TNF-α.

[0159] The results of Example 16 and Comparative Examples 14-17 show that there is a certain synergistic effect when using the components defined in the present invention, and the effect achieved by the present invention cannot be achieved if any one of them is omitted.

[0160] The results of Example 16 and Comparative Example 19 show that the carrier prepared by the composition of the present invention has a significant effect of improving the inhibitory effect of TNF-α.

[0161] Skin allergy test

[0162] The anti-allergic emulsions of Examples 16-24 and Comparative Examples 13-19 were evaluated for skin allergy suppression using a xylene-induced mouse ear swelling model.

[0163] Experimental animals, mice, SPF grade, Shanghai Shuyuan Biotechnology Co., Ltd., use license SYXK (Shanghai) 2024-0006, weighing 18-20 g, half male and half female, a total of 180, were randomly divided into 18 groups, with 10 mice in each group. The 18 groups of animals were:

[0164] Blank group: 0.05 ml of xylene was evenly applied to the right ears of mice. After 1.5 hours, the mice were killed by cervical dislocation, and the left and right ears were punched and weighed.

[0165] Positive group: 0.05 ml of xylene was evenly applied to the right ear of the mice. After 30 minutes, 0.1 g of Piyanping was applied to the right ear. One hour later, the mice were killed by cervical dislocation, and the left and right ears were punched and weighed.

[0166] Experimental group: 0.05 ml of xylene was evenly applied to the right ears of mice. 30 minutes later, 0.1 g of the antiallergic emulsions of Examples 5 to 8 and Comparative Examples 7 to 15 were applied to the right ears. One hour later, the mice were killed by cervical dislocation, and the left and right ears were punched and weighed.

[0167] Experimental Method: Xylene applied to the ears of mice can cause local tissue inflammation, release inflammatory substances, and result in acute exudative edema of the ears. Mice were randomly divided into 18 groups: blank, positive, and experimental, with ten mice in each group. 0.05 ml of xylene was evenly applied to the right ears of each group. Thirty minutes later, 0.1 g of the corresponding drug was applied to the right ears of each group, except the blank group. The drugs were applied for 60 minutes, and the mice were sacrificed by cervical dislocation. Ear pieces were punched from the same part of both ears using a 7 mm punch and weighed. The difference in weight between the two ear pieces was used to determine the degree of swelling. The average degree of swelling for each group was calculated. The results are shown in Table 3.

[0168] Table 3

[0169] experimental group Swelling degree / mg blank 10.22 Positive 0.27 Example 16 1.75 Example 17 2.98 Example 18 1.73 Example 19 2.05 Example 20 2.24 Example 21 2.18 Example 22 2.85 Example 23 2.91 Example 24 3.02 Comparative Example 13 6.05 Comparative Example 14 4.96 Comparative Example 15 4.94 Comparative Example 16 4.57 Comparative Example 17 5.08 Comparative Example 18 4.26 Comparative Example 19 9.91

[0170] Analysis of the results in Table 3 shows that the compositions provided by Examples 16-24 have good TNF-α inhibitory efficacy, especially those in Examples 16 and 18, indicating that using Example 1 as the culture process parameter can effectively improve the anti-sensitization effect of the composition.

[0171] From the results of Example 16 and Example 18, it can be seen that the inhibitory effect on TNF-α is not linearly positively correlated with the amount of carrier added. Therefore, for cost-effectiveness considerations, 5 wt% was used as the addition amount for subsequent experiments.

[0172] From the results of Example 16 and Comparative Example 13, it can be seen that during the cultivation of Inonotus obliquus, the mulberry leaf extract and birch sap concentrate respectively added to the culture medium have significantly improved the anti-allergic effect of the composition.

[0173] The results of Example 16 and Comparative Examples 14-17 show that there is a certain synergistic effect when using the components defined in the present invention, and the effect achieved by the present invention cannot be achieved if any one of them is omitted.

[0174] From the results of Example 16 and Comparative Example 18, it can be seen that the anti-allergic effect of the composition can be effectively improved after being wrapped with plant vesicles.

[0175] The results of Example 16 and Comparative Example 19 show that the carrier prepared by the composition of the present invention has a significant anti-allergic effect.

[0176] Human body 30s instant soothing effect test

[0177] Volunteer selection criteria: a) Healthy volunteers aged 18 to 60 years; b) No skin lesions or dermatitis at the test site, which could affect measurement; c) Those with sensitive or dry faces; d) Those who test positive for lactic acid stinging.

[0178] The number of volunteers is guaranteed to be more than 30, and the subjects should mainly engage in indoor activities to avoid long-term exposure to light and high temperature or cold environments.

[0179] 110 volunteers aged 22-55 years with sensitive facial skin (lactic acid stinging test score ≥ 3 points on both sides of the nose) and dry East Asian skin were selected. A sample group and a blank group (i.e., model control group: no sample was applied) were set up, and the test areas were randomly distributed on the left and right sides of the volunteers' faces. After using the essence of the present invention for 30 seconds, the red image was collected using the skin facial image analyzer VISIA 7, and the skin redness a* value was analyzed using Image-Pro Plus (IPP) analysis software to evaluate its moisturizing and soothing effects. The laboratory temperature was 21±1°C; the relative humidity was 50%±10%.

[0180] The experiment used the self-before and after control method and the blank group control method. The test cycle was 30s, and the skin image collection times were 2 times, 0s and 30s respectively. A sample group and a blank group (i.e., a model control group: no sample was applied) were set up, and the test areas were randomly distributed on the left and right faces of the volunteers. The sample was applied at a dosage of 0.25g / half face. Qualified subjects were selected according to the requirements. When the volunteers visited, they signed an informed consent form with them, collected skin images, and completed questionnaire data 30s after using the product. When visiting, after washing the face with the designated cleanser, they sat quietly in the test environment for about 20-30 minutes. After the sitting was over, the test began.

[0181] With the help of facial image analyzer VISIA 7 ( Company, USA), the instrument uses three different light sources to capture facial images, which can intuitively observe changes in facial skin condition.

[0182] The a* values ​​of the red images were analyzed using Image-Pro Plus software.

[0183] Skin redness a* value: The a* value represents the redness of the skin. An increase in the a* value indicates increased vascular reactivity of the skin.

[0184] The data were analyzed using SPSS19.0 data analysis software.

[0185] Table 4

[0186]

[0187]

[0188] From the analysis of the results in Table 4: Comparison of Examples 16-24 and Comparative Example 19, the results show that the skin redness a* of Examples 16-24 decreased, and the degree of decrease was the highest in Example 16, indicating that the skin soothing effect of the essence prepared using the composition of the present invention does not show a linear trend with the addition amount.

[0189] Comparative Example 16, Comparative Example 19 and the blank show that the use of the composition prepared by the present invention can achieve the maximum effect of the plant-encapsulated carrier composition on reducing the skin redness a* value.

[0190] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of the present application.

Claims

1. A composition with soothing and anti-allergic effects, characterized in that: The composition is composed of the following raw materials in parts by weight: 9-11 parts of Inonotus obliquus extract; 4-8 parts of Kava Kava root extract; 4-6 parts of patchouli extract; 5-15 parts of gentian root extract; The preparation method of the Inonotus obliquus extract is as follows: S1: Inonotus obliquus was inoculated into medium A and cultured on a shaker for ≥12 days. Mycelial pellets were collected and freeze-dried to obtain mycelial pellet powder A1. S2: Inonotus obliquus was inoculated into medium B and cultured for ≥8 days. The fermentation broth was collected and freeze-dried to obtain fermentation broth powder A2. S3: Mix A1 and A2 to obtain A3; The culture medium A is composed of 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 2 wt% mulberry leaf extract, 0.01 wt% vitamin K, 0.01 wt% vitamin B2, and 0.01 wt% vitamin H; The culture medium B is composed of 2 wt% glucose, 0.03 wt% magnesium sulfate, 0.1 wt% potassium nitrate, 0.5 wt% yeast extract, 0.01 wt% ammonium tartrate, 4 wt% birch sap concentrate, 0.01 wt% vitamin E, and 0.01 wt% vitamin B3; The culture temperature of S1 is 20±2°C; For S2, the culture temperature is 24±2°C; The inoculation amount of the S1 and S2 strains is 3±0.5wt%.

2. A plant extracellular vesicle carrier, characterized in that: The carrier consists of the composition according to claim 1 and plant extracellular vesicles; The specific preparation steps of the plant extracellular vesicles are as follows: T1: Completely crush the plant tissue, centrifuge for 20 min, filter, and collect the supernatant to obtain product X1; T2: subject X1 to gradient centrifugation, filter, and take the supernatant to obtain product X2; T3: subject X2 to ultra-high-speed centrifugation, discard the supernatant, and take the precipitate to obtain product X3; T4: Redissolve X3 in PBS, then add 8 wt% polyethylene glycol, filter, and store the filtrate at -80°C; The T1 centrifugal speed is 500g; the T2 centrifugal speeds are 2000g, 10000g, and 20000g, respectively, for 20 minutes each; the T3 speed is 120000g, for 90 minutes; the T1-T3 centrifugal temperature is 4°C; The T1 plant tissue is Patchouli leaf tissue; The specific preparation steps of the plant extracellular vesicle-encapsulated carrier are as follows: Q1: Mix the plant extracellular vesicles and the composition, and filter to obtain a mixture Y1; Q2: Place Y1 in a container, apply 120-180V voltage, pulse time 5-15ms, and shock pulse 2-3 times to obtain Y2; Q3: Incubate Y2 in the dark for 18-24 hours, then store at 0-4°C in the dark. In Q1, the mass ratio of the plant extracellular vesicle solution to the composition is 1:0.02-0.

1.

3. A leave-on skin preparation, characterized in that: The invention further comprises 1-8 wt % of the carrier according to claim 2.

4. The leave-on skin preparation according to claim 3, characterized in that: The skin preparation further comprises sodium alginate, sodium lauryl sulfate and honey.

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