Fructus arctii fermentation liquor with soothing and anti-inflammatory effects as well as preparation method and application of fructus arctii fermentation liquor

Abstract

The invention belongs to the technical field of plant fermentation, and relates to fructus arctii fermentation liquor with soothing and anti-inflammatory effects as well as a preparation method and application of the fructus arctii fermentation liquor. The fructus arctii fermentation liquid is obtained by fermenting fructus arctii through schizosaccharomyces pombe JM2-16, and the fructus arctii fermentation liquid is obtained by fermenting fructus arctii through schizosaccharomyces pombe JM2-16. According to the invention, after the fructus arctii is used as a raw material and is fermented by the schizosaccharomyces pombe, the hyaluronidase inhibition capability, the elastase inhibition capability and the DPPH free radical scavenging capability of the fermentation product can be improved, so that the fermentation product with better soothing, anti-inflammatory and anti-oxidation capabilities is obtained, and the requirements of the market on efficient, natural and safe skin care components are met.

Description

Technical Field

[0001] The invention belongs to the technical field of plant fermentation, and relates to burdock fruit fermentation liquid with soothing and anti-inflammatory effects, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] In recent years, with improved living standards and growing awareness of skincare, the skincare industry has flourished, and the concept of "green skincare" has become increasingly popular. Consumers are increasingly favoring naturally derived, low-irritation cosmetic ingredients. Burdock, a traditional medicinal plant, exhibits potential for its extracts in cosmetics. Research has shown that burdock extract is often used as a minor additive among numerous ingredients in current cosmetic formulations, and its individual or core benefits are not fully understood and utilized. Furthermore, the direct use of burdock extract has limited biological activity, such as weak antioxidant capacity and anti-inflammatory properties, which limits its market application. Summary of the Invention

[0004] In order to solve the deficiencies of the prior art, the present invention aims to provide a burdock fermentation liquid with soothing and anti-inflammatory effects, a preparation method thereof and an application thereof. The present invention uses burdock as a raw material and is fermented with Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) After fermentation, the hyaluronidase inhibition ability, elastase inhibition ability, and DPPH free radical scavenging ability of the fermentation product can be improved, thereby obtaining a fermentation product with better soothing, anti-inflammatory and antioxidant abilities to meet the market demand for efficient, natural and safe skin care ingredients.

[0005] In order to achieve the above object, the technical solution of the present invention is: In the first aspect, a burdock fermented liquid with soothing and anti-inflammatory effects is prepared by fermenting burdock with Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) obtained by fermentation of JM2-16.

[0006] The antioxidant effect of burdock can block the chain reaction of free radicals, delay the aging process of the skin, help maintain the firmness and elasticity of the skin, and keep the skin young and healthy. The active ingredients in burdock can regulate the inflammation-related signaling pathways in skin cells, inhibit the production and release of inflammatory factors, thereby reducing the skin's inflammatory response and relieving skin discomfort. It has a good soothing effect on sensitive skin and skin types prone to inflammation. Studies have shown that burdock seeds treated with Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) The fermentation liquid obtained by fermenting JM2-16 can not only improve its soothing, anti-inflammatory and antioxidant abilities, but also has good penetration-promoting and oil-control effects.

[0007] In some embodiments, the fermentation medium used in the fermentation contains rice extract. Studies have shown that when the fermentation medium used in the fermentation contains rice extract, the activity of the burdock fruit fermentation liquid can be significantly enhanced.

[0008] Wherein, the pombe Schizosaccharomyces cerevisiae is named as Pombe Schizosaccharomyces cerevisiae ( Schizosaccharomyces pombe ) JM2-16, which was deposited in the China Center for Type Culture Collection on December 13, 2024, with the deposit number CCTCC NO: M20242810, and disclosed in the patent with publication number CN119530105A.

[0009] In a second aspect, a method for preparing a burdock fermented liquid having soothing and anti-inflammatory effects comprises the following steps: Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) JM2-16 was inoculated into a fermentation medium, and burdock seed powder was added to the fermentation medium for fermentation.

[0010] In some embodiments, the fermentation medium contains rice extract.

[0011] In a third aspect, a use of the burdock fruit fermentation liquid in the preparation of cosmetics.

[0012] The beneficial effects of the present invention are: The burdock fruit fermentation liquid provided by the present invention is prepared by the fermentation of Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) JM2-16. Experiments have shown that the fermentation broth obtained from burdock fruit with this strain has a higher polysaccharide content and excellent antioxidant properties. Furthermore, this strain has excellent hyaluronidase inhibitory activity in burdock fruit fermentation, and does not cause irritation or damage, indicating good safety. Furthermore, research has shown that the fermentation product of burdock fruit with this strain has excellent permeability-enhancing and oil-control effects.

[0013] Compared to traditional plant extract preparation methods, the present preparation method does not require an organic solvent extraction process, resulting in a more natural and less irritating product, while retaining more complete active ingredients beneficial to the human body and skin. The preparation method is simple and does not use organic solvents, making it more environmentally friendly and suitable for industrial production. DETAILED DESCRIPTION

[0014] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0015] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0016] Given that burdock extract in cosmetics has low biological activity such as weak antioxidant capacity and low anti-inflammatory performance, making it difficult to be used alone as an active ingredient in cosmetics, the present invention proposes a burdock fruit fermentation liquid with soothing and anti-inflammatory effects, as well as its preparation method and application.

[0017] A typical embodiment of the present invention provides a burdock fermentation liquid with soothing and anti-inflammatory effects, which is prepared by fermenting burdock with Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) obtained by fermentation of JM2-16.

[0018] In some embodiments, the fermentation medium contains rice extract. Adding rice extract to the culture medium can significantly enhance the activity of the burdock fruit fermentation liquid during fermentation. Specifically, the rice extract content in the culture medium is 4.5-5.5 g / L.

[0019] In some embodiments, the fermentation medium contains inulin and / or white water lily extract. Adding inulin and white water lily extract to the culture medium, followed by fermentation, can significantly enhance the antioxidant activity of the burdock fruit fermentation broth while also inhibiting the growth of other bacteria. Specifically, the inulin content in the culture medium is 4.5-5.5 g / L. Specifically, the white water lily extract content in the culture medium is 4.5-5.5 g / L.

[0020] In some embodiments, the culture medium used for fermentation comprises 10-20 g / L glucose, 1-10 g / L glycerol, 20-40 g / L soy peptone, 1-5 g / L yeast extract, and 0.5-2 g / L dipotassium hydrogen phosphate.

[0021] Another embodiment of the present invention provides a method for preparing burdock fruit fermented liquid with soothing and anti-inflammatory effects, comprising the following steps: Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) JM2-16 was inoculated into a fermentation medium, and burdock seed powder was added to the fermentation medium for fermentation.

[0022] In some embodiments, the fermentation medium contains rice extract. When the fermentation medium contains rice extract, the activity of the burdock fruit fermentation liquid can be significantly enhanced after fermentation. Specifically, the content of rice extract in the fermentation medium is 4.5-5.5 g / L.

[0023] In some embodiments, the fermentation medium contains inulin and / or white water lily extract. Specifically, the content of inulin in the fermentation medium is 4.5-5.5 g / L. Specifically, the content of white water lily extract in the fermentation medium is 4.5-5.5 g / L.

[0024] In some embodiments, the fermentation medium comprises 10-20 g / L glucose, 1-10 g / L glycerol, 20-40 g / L soy peptone, 1-5 g / L yeast extract, and 0.5-2 g / L dipotassium hydrogen phosphate.

[0025] In some embodiments, the fermentation temperature is 34-37° C., and the fermentation time is 46-48 h.

[0026] In some embodiments, Schizosaccharomyces pombe ( Schizosaccharomyces pombe JM2-16 was activated and cultured in batches before being inoculated into the fermentation medium for fermentation. Specifically, the activation temperature was 34-36°C for 22-26 hours. Specifically, the culture temperature was 34-38°C for 23-25 hours.

[0027] In some embodiments, fermentation is followed by centrifugation and then filtration.

[0028] A third embodiment of the present invention provides a use of the burdock fruit fermentation liquid in the preparation of cosmetics.

[0029] In some embodiments, the cosmetic has one or more effects of inhibiting hyaluronidase activity, having an antioxidant effect, promoting penetration (permeation enhancement), and reducing skin oil secretion.

[0030] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0031] The Schizosaccharomyces pombe used in the following examples is named Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) JM2-16, which was deposited in the China Center for Type Culture Collection on December 13, 2024, with the deposit number being CCTCC NO: M 20242810.

[0032] Example 1 A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects comprises the following steps: (1) Take out the Schizosaccharomyces pombe from the -80℃ freezer ( Schizosaccharomyces pombe ) JM2-16 was inoculated at a 1% (v / v) inoculum into liquid culture medium and incubated at 35°C for 24 h for activation. This activation was repeated twice to obtain an activation solution. This activation solution was then inoculated at a 1% (v / v) inoculum into liquid culture medium and incubated at 37°C for 24 h to obtain a Schizosaccharomyces pombe culture solution. The Schizosaccharomyces pombe culture solution was then inoculated at a 1% (v / v) inoculum into liquid culture medium for expansion and incubation at 37°C for 24 h to obtain a Schizosaccharomyces pombe culture medium.

[0033] The components of the liquid culture medium include 15 g / L glucose, 5 g / L glycerol, 30 g / L soy peptone, 2.5 g / L yeast extract, and 0.75 g / L potassium dihydrogen phosphate.

[0034] (2) The burdock seed raw material is broken and then pulverized to obtain burdock seed powder.

[0035] (3) Burdock seed powder was inoculated into the S. pombe culture medium prepared in step (1) at a mass ratio of 1:20. The culture medium was fermented under the following conditions: an oscillating incubator speed of 300 r / min, a pH value of 7.5, a temperature of 35°C, and a burdock seed powder fermentation time of 48 h to obtain a crude fermentation liquid.

[0036] (4) The crude fermentation broth was centrifuged (at 3000 rpm for 15 min) to obtain a clarified fermentation product, which was then sterilized in an autoclave at 121°C for 30 min and filtered to collect the filtrate.

[0037] Example 2 A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects comprises the following steps: (1) Take out the Schizosaccharomyces pombe from the -80℃ freezer ( Schizosaccharomyces pombe ) JM2-16 was inoculated at a 1% (v / v) inoculum into liquid culture medium and incubated at 35°C for 24 h for activation. This activation was repeated twice to obtain an activation solution. This activation solution was then inoculated at a 1% (v / v) inoculum into liquid culture medium and incubated at 37°C for 24 h to obtain a Schizosaccharomyces pombe culture solution. The Schizosaccharomyces pombe culture solution was then inoculated at a 1% (v / v) inoculum into liquid culture medium for expansion and incubation at 37°C for 24 h to obtain a Schizosaccharomyces pombe culture medium.

[0038] The components of the liquid culture medium include 10 g / L glucose, 1 g / L glycerol, 20 g / L soy peptone, 1 g / L yeast extract, and 0.5 g / L potassium dihydrogen phosphate.

[0039] (2) The burdock seed raw material is broken and then pulverized to obtain burdock seed powder.

[0040] (3) Inoculate burdock seed powder into the S. pombe culture medium prepared in step (1) at a weight ratio of 1:20. Ferment the culture medium under the following conditions: an oscillating incubator speed of 300 r / min, a pH value of 7.5, a temperature of 35°C, and a burdock seed powder fermentation time of 48 h to obtain a crude fermentation liquid.

[0041] (4) The crude fermentation broth was centrifuged (at 3000 rpm for 15 min) to obtain a clarified fermentation product, which was then sterilized in an autoclave at 121°C for 30 min and filtered to collect the filtrate.

[0042] Example 3 A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects comprises the following steps: (1) Take out the Schizosaccharomyces pombe from the -80℃ freezer ( Schizosaccharomyces pombe ) JM2-16 was inoculated at a 1% (v / v) inoculum into liquid culture medium and incubated at 35°C for 24 h for activation. This activation was repeated twice to obtain an activation solution. This activation solution was then inoculated at a 1% (v / v) inoculum into liquid culture medium and incubated at 37°C for 24 h to obtain a Schizosaccharomyces pombe culture solution. The Schizosaccharomyces pombe culture solution was then inoculated at a 1% (v / v) inoculum into liquid culture medium for expansion and incubation at 37°C for 24 h to obtain a Schizosaccharomyces pombe culture medium.

[0043] The components of the liquid culture medium include 20 g / L glucose, 10 g / L glycerol, 40 g / L soy peptone, 5 g / L yeast extract, and 2 g / L potassium dihydrogen phosphate.

[0044] (2) The burdock seed raw material is broken and then pulverized to obtain burdock seed powder.

[0045] (3) Burdock seed powder was inoculated into the S. pombe culture medium prepared in step (1) at a weight ratio of 1:20. The culture medium was fermented under the following conditions: an oscillating incubator speed of 200 r / min, a pH value of 7.5, a temperature of 35°C, and a burdock seed powder fermentation time of 48 h to obtain a crude fermentation liquid.

[0046] (4) The crude fermentation broth was centrifuged (at 3000 rpm for 15 min) to obtain a clarified fermentation product, which was then sterilized in an autoclave at 121°C for 30 min and filtered to collect the filtrate.

[0047] Example 4 A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects comprises the following steps: (1) Take out the Schizosaccharomyces pombe from the -80℃ freezer ( Schizosaccharomyces pombe ) JM2-16 was inoculated into a liquid culture medium at a 1% (v / v) inoculum size and incubated at 35°C for 24 h for activation. This activation was repeated twice to obtain an activation solution. This activation solution was then inoculated into a liquid culture medium at a 1% (v / v) inoculum size and incubated at 37°C for 24 h to obtain a Schizosaccharomyces pombe culture solution. The Schizosaccharomyces pombe culture solution was further inoculated into a liquid culture medium at a 1% (v / v) inoculum size for expansion and incubation at 37°C for 24 h to obtain a Schizosaccharomyces pombe culture medium.

[0048] The components of the liquid culture medium include 20 g / L glucose, 10 g / L glycerol, 40 g / L soy peptone, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, and 5 g / L rice extract (purchased from Xi'an Fenghe Biotechnology Co., Ltd., named rice oligopeptide, also known as rice extract).

[0049] (2) The burdock seed raw material is broken and then pulverized to obtain burdock seed powder.

[0050] (3) Inoculate burdock seed powder into the S. pombe culture medium prepared in step (1) at a weight ratio of 1:20. Ferment the culture medium under the following conditions: an oscillating incubator speed of 300 r / min, a pH value of 7.5, a temperature of 35°C, and a burdock seed powder fermentation time of 48 h to obtain a crude fermentation liquid.

[0051] (4) The crude fermentation broth was centrifuged (at 3000 rpm for 15 min) to obtain a clarified fermentation product, which was then sterilized in an autoclave at 121°C for 30 min and filtered to collect the filtrate.

[0052] Example 5 A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects comprises the following steps: (1) Take out the Schizosaccharomyces pombe from the -80℃ freezer ( Schizosaccharomyces pombe ) JM2-16 was inoculated into liquid culture medium at a 1% (v / v) inoculum size and incubated at 35°C for 24 h for activation. This activation was repeated twice to obtain an activation solution. This activation solution was then inoculated into liquid culture medium at a 1% (v / v) inoculum size and incubated at 37°C for 24 h to obtain a Schizosaccharomyces pombe culture solution. The Schizosaccharomyces pombe culture solution was then inoculated into liquid culture medium at a 1% (v / v) inoculum size and expanded. The culture was then incubated at 37°C for 24 h to obtain a Schizosaccharomyces pombe culture medium.

[0053] The components of the liquid culture medium include 20 g / L glucose, 10 g / L glycerol, 40 g / L soy peptone, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 5 g / L inulin (Orafti HSI), and 5 g / L white water lily extract (Jinan Ruibeike Biotechnology Co., Ltd.).

[0054] (2) The burdock seed raw material is broken and then pulverized to obtain burdock seed powder.

[0055] (3) Burdock seed powder was inoculated into the S. pombe culture medium prepared in step (1) at a weight ratio of 1:20. The culture medium was fermented under the following conditions: an oscillating incubator speed of 200 r / min, a pH value of 7.5, a temperature of 35°C, and a burdock seed powder fermentation time of 48 h to obtain a crude fermentation liquid.

[0056] (4) The crude fermentation broth was centrifuged (at 3000 rpm for 15 min) to obtain a clarified fermentation product, which was then sterilized in an autoclave at 121°C for 30 min and filtered to collect the filtrate.

[0057] Example 6 A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects comprises the following steps: (1) Take out the Schizosaccharomyces pombe from the -80℃ freezer ( Schizosaccharomyces pombe ) JM2-16 was inoculated into a liquid culture medium at a 1% (v / v) inoculum size and incubated at 35°C for 24 hours for activation. This activation was repeated twice to obtain an activation solution. This activation solution was then inoculated into a liquid culture medium at a 1% (v / v) inoculum size and incubated at 37°C for 24 hours to obtain a Schizosaccharomyces pombe culture solution. The Schizosaccharomyces pombe culture solution was then inoculated into a liquid culture medium at a 1% (v / v) inoculum size and expanded. The culture was then incubated at 37°C for 24 hours to obtain a Schizosaccharomyces pombe culture medium.

[0058] The components of the liquid culture medium include 20 g / L glucose, 10 g / L glycerol, 40 g / L soy peptone, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 5 g / L rice extract, 5 g / L inulin, and 5 g / L white water lily extract.

[0059] (2) The burdock seed raw material is broken and then pulverized to obtain burdock seed powder.

[0060] (3) Burdock seed powder was inoculated into the S. pombe culture medium prepared in step (1) at a weight ratio of 1:20. The culture medium was fermented under the following conditions: an oscillating incubator speed of 200 r / min, a pH value of 7.5, a temperature of 35°C, and a burdock seed powder fermentation time of 48 h to obtain a crude fermentation liquid.

[0061] (4) The crude fermentation broth was centrifuged (at 3000 rpm for 15 min) to obtain a clarified fermentation product, which was then sterilized in an autoclave at 121°C for 30 min and filtered to collect the filtrate.

[0062] Comparative Example 1 A method for preparing burdock fruit alcohol extract comprises the following steps: (1) Mix burdock seed powder and 95% ethanol solution in a material-liquid weight ratio of 1:20.

[0063] (2) Heat and reflux to obtain ethanol extract.

[0064] (3) After cooling, centrifuge (3000 rpm, 15 min), and combine the supernatants.

[0065] (4) Concentrate the supernatant to 30% of the original volume to obtain the burdock fruit alcohol extract concentrate.

[0066] Comparative Example 2 This comparative example is the same as Example 1, except that burdock seeds are replaced with burdock roots to obtain burdock root fermentation liquid.

[0067] Performance Testing 1. Antioxidant test - DPPH free radical scavenging experiment The samples of Examples 1 to 6 and Comparative Examples 1 to 2 were prepared into burdock product sample solutions with concentrations of 3%, 2%, 1%, and 0.5%, respectively, and placed in transparent PET bottles. The DPPH free radical scavenging effects were observed and the antioxidant properties of the burdock fermentation solution were evaluated.

[0068] A 0.1 mM DPPH alcohol solution was mixed with burdock product sample solutions of different concentrations in a volume ratio of 1:1. The mixture was plated on a 96-well plate with 3 replicates per group. The mixture was kept in a 37°C water bath in the dark for 30 min. The absorbance at 517 nm was measured and the DPPH scavenging rate of each sample was calculated.

[0069] Clearance rate = [A 对照组 -(A 样品组 -A 空白组 ) / A 对照组 ]×100%; A 样品组 : absorbance of sample solution and DPPH alcohol solution; A 空白组 : absorbance of sample solution and anhydrous ethanol; A 对照组 : Absorbance values of DPPH alcohol solution and dimethyl sulfoxide (DMSO).

[0070] Table 1 DPPH free radical scavenging rate

[0071] As shown in Table 1, the burdock fermentation liquid of the present invention has excellent DPPH free radical scavenging activity compared with the comparative example product, that is, it has excellent skin antioxidant properties, and the antioxidant properties increase with the increase of the fermentation liquid concentration.

[0072] Table 1 also shows that when rice extract or inulin and white water lily culture medium are added to the culture medium, the antioxidant activity of the burdock fruit fermentation liquid is improved. When rice extract, inulin and white water lily culture medium are added to the culture medium at the same time, the antioxidant activity of the burdock fruit fermentation liquid is stronger.

[0073] 2. Soothing test - Hyaluronidase inhibition rate experiment (1) Reagent preparation Hyaluronidase solution: Concentration is 500 U / mL, prepared immediately for use, using acetate buffer as solvent; Sodium hyaluronate solution: concentration 0.5 mg / mL, using acetate buffer as solvent; Acetate buffer: Mix 4.8 mL of 0.2 mol / L acetic acid and 45.2 mL of 0.2 mol / L sodium acetate and dilute to 100 mL to prepare an acetate buffer solution with a pH of 5.6. Acetylacetone solution: Mix 50 mL of 1.0 mol / L sodium carbonate solution and 3.5 mL of acetylacetone (prepare immediately before use). Ehrlich reagent: Dissolve 0.8 g of p-dimethylaminobenzaldehyde in a mixture of 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol and mix well. The mass concentration of the concentrated hydrochloric acid is 37%; CaCl2 solution: concentration is 2.5 mol / L; NaOH solution: concentration is 5 mol / L.

[0074] Detection method: The samples prepared in each embodiment and the samples prepared in each comparative example were respectively prepared into burdock product sample solutions with concentrations of 3%, 2%, 1%, and 0.5%. Take 0.1 mL of 0.25 mol / L CaCl₂ solution and 0.5 mL of hyaluronidase solution and incubate at 37°C for 20 minutes. Then add 0.5 mL of burdock product sample solution of varying concentrations and continue incubating at 37°C for another 20 minutes. Then add 0.5 mL of sodium hyaluronate solution and incubate at 37°C for 30 minutes. Then let it stand at room temperature for 5 minutes. Then add 0.1 mL of 0.4 mol / L NaOH solution and 0.5 mL of acetylacetone solution. Heat in a boiling water bath for 15 minutes and immediately cool with ice water for 5 minutes. Then add 1.0 mL of Ehrlich's reagent and dilute with 3.0 mL of anhydrous ethanol. Let the solution develop color for 20 minutes. During this time, scan the sample from 450 to 700 nm to determine the maximum absorption wavelength. The absorbance at this wavelength is measured using a spectrophotometer. The hyaluronidase inhibition rate of the sample is calculated.

[0075] Inhibition rate = [(A1-A2)-(B1-B2) / (A1-A2)]; A1: Absorbance of reference solution, using deionized water instead of the obtained sample; A2: Reference blank solution absorbance value, replace the obtained sample with deionized water, and replace the hyaluronidase and sodium hyaluronate solutions with acetate buffer solution; B1: absorbance value of sample solution; B: is the absorbance value of the blank solution of the sample, with acetate buffer solution replacing the hyaluronidase and sodium hyaluronate solution.

[0076] Table 2 Hyaluronidase inhibition rate

[0077] As shown in Table 2, the burdock fruit fermented liquid of the present invention exhibits superior hyaluronidase inhibitory activity compared to the comparative example product, reducing the spread of inflammatory mediators and thus exerting anti-inflammatory effects. This indicates excellent skin-soothing potential. Furthermore, the soothing effect increases with increasing fermented liquid concentration.

[0078] Meanwhile, Table 2 shows that the addition of rice extract to the culture medium is beneficial to further enhance the inhibitory activity of hyaluronidase, thereby further improving the anti-inflammatory effect and soothing efficacy.

[0079] 3. Safety Testing - HaCaT Cytotoxicity Assay HaCaT cell model establishment: Keratinocytes (HaCaT) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). The cells were incubated at 37°C in a constant temperature incubator containing 5% CO2.

[0080] Cell seeding: 6×10 3 Keratinocytes (HaCaT) were seeded in a 96-well plate at a seeding density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0081] Experimental grouping: set up a blank group, a control group and a sample group. In the sample group, set up three concentration gradients, and set up three replicate wells under each concentration gradient.

[0082] Preparation of liquid: According to the set test concentration, the fermentation liquid prepared in each example and comparative example was used to prepare working liquids of the test substance of different concentrations.

[0083] Dosing: Dosing was performed when the cell plating rate in the 96-well plate reached 40%-60%. The blank group received no cells and only 200 μL of cell culture medium was added; the control group received 200 μL of culture medium per well; and the sample group received 200 μL of culture medium containing the corresponding sample concentration per well. After dosing, the 96-well plate was placed in an incubator (37°C, 5% CO2).

[0084] Detection: After incubating cells for 24 hours, add 10 μL of CCK-8 and incubate at 37°C in the dark for 2-4 hours. After incubation, read the OD value at 490 nm. The cell viability is calculated as follows: Cell viability = (OD 样品组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%; Where, OD 样品组 is the OD value of the sample group, OD 空白组 is the OD value of the blank group, OD 对照组 is the OD value of the control group.

[0085] Table 3 Results of CCK-8 cytotoxicity assay

[0086] According to the results in Table 3, the samples prepared in each example showed no cytotoxicity to keratinocytes at a concentration of 3%. This result indicates that the burdock fruit fermented liquid has almost no toxic effect on cells, does not cause irritation or damage, and has good safety.

[0087] 4. In vitro transdermal absorption experiment Preparation of ex vivo pigskin: Prepare intact fresh pigskin. Remove any remaining hair with tweezers, then scrape off the subcutaneous tissue and fat with a knife. Wash with PBS, wrap in tin foil, and freeze at -20°C until ready for use. Thaw in PBS for 45 minutes before use.

[0088] The treated pig skin stratum corneum was fixed upward on the Franz diffusion cell, 5 mL of 30% ethanol-normal saline was added to each receiving cell, and the cells were placed in an intelligent transdermal tester in a constant temperature water bath (the transdermal area was 0.5 cm 2 The experimental temperature was maintained at 35 ± 0.5 °C and the stirring speed was maintained at 350 ± 10 r·min -1 After equilibration for 1 h, the air bubbles in the receiving cell were exhausted before administration. 1 mL of the sample from each example and comparative example was added to the supply cell to prepare burdock product sample solutions with concentrations of 3%, 2%, 1%, and 0.5%, respectively. Each group was replicated three times, and sampling was performed after 3 h.

[0089] Skin sample treatment: The test skin was removed, rinsed with an appropriate amount of physiological saline, dried with filter paper, and placed in a 1.5 mL centrifuge tube. The skin was cut into pieces, 1 mL of methanol was added, and the tube was sealed and ultrasonicated for 30 min at 10,000 r / min. -1 Centrifuge for 5 minutes, take the supernatant and filter it with a 0.22 μm microporous filter membrane, inject the sample for analysis, and calculate the cumulative permeation according to the following formula.

[0090] Calculation method of cumulative transdermal permeation: Calculation of the cumulative permeation per unit area of burdock product sample liquid (μg·cm -2 ) and transdermal rate Js (μg·cm -2 ·h -1 ).

[0091] = / S = / T Where: C is the concentration measured at time t, mg / mL; V is the volume of each sample, mL; S is the effective area of the diffusion cell, cm 2 ; T is the total sampling time interval, h.

[0092] Table 4 In vitro percutaneous absorption results

[0093] As shown in Table 4, the transdermal rate of the burdock fruit fermented extract was greater than that of the burdock fruit alcohol extract and the burdock root fermented liquid. The transdermal rate was the highest when the penetration enhancer concentration was 3%, indicating that the burdock fruit fermented extract had a good penetration enhancing effect.

[0094] 5. Skin oil content test The amount of sebum on the skin surface was measured by measuring the transparency of the oil-absorbing tape after absorbing sebum.

[0095] Experimental samples: the experimental group was the burdock fruit fermentation liquid of each example, and the control group was the burdock fruit alcohol extract of Comparative Example 1 and the burdock root fermentation liquid of Comparative Example 2.

[0096] Experimental methods: Thirty subjects aged 18-45 years, both male and female, with oily or combination skin and no history of skin diseases or allergies were selected. Each group was treated with a 3% test sample at varying concentrations. Three tests were performed on the same facial area using a Sebumeter (CK, Germany), and the average was used to evaluate the oil-control effectiveness of the composition of the present invention. The interval between measurement points should be no less than 1 hour, and the entire testing period should not exceed 24 hours.

[0097] Table 5

[0098] Table 5 shows that burdock fruit fermented extract significantly reduced skin oil secretion, with a greater effect than burdock fruit alcohol extract and burdock root fermented extract. This extract indirectly affects oil secretion balance by regulating stratum corneum metabolism and improving skin barrier function. During the experiment, no irritation reactions, such as redness, swelling, or itching, occurred in the experimental group. This indicates that burdock fruit extract is safe at this concentration and can be used in oil-control formulations.

[0099] The results showed that the burdock fruit fermented extract had superior antioxidant and soothing properties, as well as its penetration-promoting and oil-controlling effects, compared to burdock fruit alcohol extract and burdock root fermented liquid. Fermentation with Schizosaccharomyces pombe alone significantly enhanced the antioxidant and soothing properties of burdock fruit. Therefore, the burdock fruit fermented liquid of the present invention possesses significant anti-inflammatory and antioxidant properties, promotes penetration and oil control, is highly biosafe, and can be used to repair the skin barrier, providing moisturizing, soothing, and oil-control effects.

[0100] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A burdock fruit fermented liquid with soothing and anti-inflammatory effects, characterized in that: From burdock seeds to Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) JM2-16 fermentation; Among them, Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) The deposit number of JM2-16 is CCTCC NO: M 20242810.

2. The burdock fruit fermented liquid according to claim 1, wherein The fermentation medium contains rice extract.

3. The burdock fruit fermented liquid according to claim 1 or 2, wherein: The fermentation medium contains inulin and / or white water lily extract.

4. A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects, characterized in that: The steps include: Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) JM2-16 is inoculated into a fermentation medium, and burdock seed powder is added to the fermentation medium for fermentation; wherein, pombe Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) The deposit number of JM2-16 is CCTCC NO: M 20242810.

5. The preparation method according to claim 4, wherein: The fermentation medium contains rice extract, and the content of the rice extract in the fermentation medium is 4.5-5.5 g / L; And / or, the fermentation medium contains inulin and / or white water lily extract, the content of inulin in the fermentation medium is 4.5-5.5 g / L, and the content of white water lily extract in the fermentation medium is 4.5-5.5 g / L.

6. The preparation method according to claim 4, characterized in that: The fermentation medium comprises 10-20 g / L glucose, 1-10 g / L glycerol, 20-40 g / L soy peptone, 1-5 g / L yeast extract, and 0.5-2 g / L dipotassium hydrogen phosphate.

7. The preparation method according to claim 4, wherein: The fermentation temperature is 34~37 ℃ and the fermentation time is 46~48 h.

8. The preparation method according to claim 4, wherein: Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) JM2-16 is activated and expanded in sequence, and then inoculated into the fermentation medium for fermentation.

9. Use of the burdock fruit fermented liquid according to any one of claims 1 to 3 or the burdock fruit fermented liquid obtained by the preparation method according to any one of claims 4 to 8 in the preparation of cosmetics.

10. The use according to claim 9, characterized in that: The cosmetic has one or more of the following effects: inhibiting hyaluronidase activity, anti-oxidation, promoting penetration, and reducing skin oil secretion.

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