Arctium lappa ferment liquor with soothing anti-inflammatory effect, and preparation method and application thereof

Abstract

The present invention belongs to the field of plant fermentation technology, and relates to a burdock fermentation liquid with soothing and anti-inflammatory effects, a preparation method thereof, and an application thereof. Schizosaccharomyces pombe ) JM2-16 fermentation. The present invention uses burdock as 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.

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, as well as 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:

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] In a second aspect, a method for preparing a burdock fermented liquid having soothing and anti-inflammatory effects comprises the following steps:

[0011] Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) JM2-16 was inoculated into a fermentation medium, and burdock seed powder was added to the fermentation medium for fermentation.

[0012] In some embodiments, the fermentation medium contains rice extract.

[0013] In a third aspect, a use of the burdock fruit fermentation liquid in the preparation of cosmetics.

[0014] The beneficial effects of the present invention are:

[0015] 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.

[0016] 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

[0017] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0018] It is also important to note that the terms used herein are not intended to limit the particular embodiments of the present application to the preferred embodiments described. Rather, the terms are used only to describe specific embodiments of the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0019] In view of the low bioactivity of the burdock extract in cosmetics, such as weak antioxidant capacity and low anti-inflammatory performance, it is difficult to use the burdock extract alone as an effective component of cosmetics. Therefore, the present application provides a burdock fruit ferment liquid with soothing anti-inflammatory efficacy, a preparation method thereof and an application thereof.

[0020] In a typical embodiment of the present application, a burdock fruit ferment liquid with soothing anti-inflammatory efficacy is obtained by fermenting burdock fruits with Schizochytrium limacinum (S. limacinum) JM2-16. Schizosaccharomyces pombe )JM2-16。

[0021] In some embodiments, the culture medium used for fermentation contains rice extract. After adding the rice extract to the culture medium, the activity of the burdock fruit ferment liquid can be significantly enhanced after fermentation. Specifically, the content of the rice extract in the culture medium is 4.5-5.5 g / L.

[0022] In some embodiments, the culture medium used for fermentation contains inulin and / or white water lily extract. After adding the inulin and white water lily extract to the culture medium, the antioxidant activity of the burdock fruit ferment liquid can be significantly enhanced after fermentation, and the growth of miscellaneous bacteria can also be inhibited. Specifically, the content of the inulin in the culture medium is 4.5-5.5 g / L. Specifically, the content of the white water lily extract in the culture medium is 4.5-5.5 g / L.

[0023] In some embodiments, the culture medium used for fermentation includes 10-20 g / L glucose, 1-10 g / L glycerol, 20-40 g / L soybean peptone, 1-5 g / L yeast extract, and 0.5-2 g / L potassium phosphate dibasic.

[0024] In another embodiment of the present application, a preparation method of a burdock fruit ferment liquid with soothing anti-inflammatory efficacy is provided, which includes the following steps:

[0025] S. limacinum (S. limacinum) JM2-16. Schizosaccharomyces pombe) JM2-16 is inoculated into a fermentation medium, and burdock fruit powder is added into the fermentation medium for fermentation.

[0026] In some embodiments, the fermentation medium contains rice extract. When the fermentation medium contains rice extract, the activity of the burdock fruit fermentation liquor can be significantly enhanced after fermentation. Specifically, the content of the rice extract in the fermentation medium is 4.5-5.5 g / L.

[0027] In some embodiments, the fermentation medium contains inulin and / or white water lily flower extract. Specifically, the content of the inulin in the fermentation medium is 4.5-5.5 g / L. Specifically, the content of the white water lily flower extract in the fermentation medium is 4.5-5.5 g / L.

[0028] In some embodiments, the fermentation medium comprises 10-20 g / L glucose, 1-10 g / L glycerol, 20-40 g / L soybean peptone, 1-5 g / L yeast extract, and 0.5-2 g / L potassium phosphate dibasic.

[0029] In some embodiments, the fermentation temperature is 34-37 ℃, and the fermentation time is 46-48 h.

[0030] In some embodiments, the Schizochytrium sp. Schizosaccharomyces pombe JM2-16 is inoculated into a fermentation medium, and burdock fruit powder is added into the fermentation medium for fermentation. Specifically, the activation temperature is 34-36 ℃, and the activation time is 22-26 h. Specifically, the temperature of the scale-up culture is 34-38 ℃, and the time of the scale-up culture is 23-25 h.

[0031] In some embodiments, centrifugal separation is performed after fermentation, and then filtration is performed.

[0032] The third embodiment of the present application provides a use of the above burdock fruit fermentation liquor in the preparation of a cosmetic product.

[0033] In some embodiments, the cosmetic product has one or more of the following effects: inhibiting the activity of hyaluronidase, antioxidant effect, promoting penetration (pro-penetration), and reducing the amount of skin oil secretion.

[0034] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples and comparative examples.

[0035] The Schizochytrium sp. used in the following examples is named Schizochytrium sp. Schizosaccharomyces pombe) JM2-16, which was preserved in the China Center for Type Culture Collection on December 13, 2024, with a preservation number of CCTCC NO: M 20242810.

[0036] Example 1

[0037] A preparation method of a burdock fruit fermented liquid with soothing anti-inflammatory efficacy, steps as follows:

[0038] (1) Take out the Schizosaccharomyces pombe from the -80℃ refrigerator, Schizosaccharomyces pombe JM2-16 is inoculated into the liquid culture medium at an inoculation amount of 1% (v / v), activated at 35℃ for 24 h, continuously activated for 2 times, and an activated liquid is obtained. The activated liquid is inoculated into the liquid culture medium at an inoculation amount of 1% (v / v), and cultured at 37℃ for 24 h to obtain a Schizosaccharomyces pombe bacterial liquid. The Schizosaccharomyces pombe bacterial liquid is continuously inoculated into the liquid culture medium at an inoculation amount of 1% (v / v) for expansion culture, and cultured at 37℃ for 24 h to obtain a Schizosaccharomyces pombe culture medium.

[0039] The components of the liquid culture medium include glucose 15 g / L, glycerol 5 g / L, soybean peptone 30 g / L, yeast extract 2.5 g / L, and potassium phosphate dibasic 0.75 g / L.

[0040] (2) The burdock fruit raw material is broken and crushed to obtain burdock fruit powder.

[0041] (3) The burdock fruit powder is inoculated into the Schizosaccharomyces pombe culture medium prepared in step (1) at a solid-liquid mass ratio of 1:20. It is fermented under the following conditions: the shaking incubator speed is 300 r / min, the pH value is 7.5, the temperature is 35℃, and the burdock fruit powder fermentation culture time is 48 h to obtain a crude fermentation liquid.

[0042] (4) The crude fermentation liquid is centrifuged (at a speed of 3000 rpm for 15 min) to obtain a clear fermentation product, which is then sterilized in a 121℃ high-temperature sterilization pot for 30 min, and the filtrate is collected after filtration.

[0043] Example 2

[0044] A preparation method of a burdock fruit fermented liquid with soothing anti-inflammatory efficacy, steps as follows:

[0045] (1) Take out the Schizosaccharomyces pombe from the -80℃ refrigerator, 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.

[0046] 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.

[0047] (2) The burdock seed raw material is broken and then pulverized to obtain burdock seed powder.

[0048] (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.

[0049] (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.

[0050] Example 3

[0051] A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects comprises the following steps:

[0052] (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.

[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, and 2 g / L potassium dihydrogen phosphate.

[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 4

[0058] A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects comprises the following steps:

[0059] (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.

[0060] 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).

[0061] (2) The burdock seed raw material is broken and then pulverized to obtain burdock seed powder.

[0062] (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.

[0063] (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.

[0064] Example 5

[0065] A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects comprises the following steps:

[0066] (1) Take out the Schizosaccharomyces pombe from the -80℃ freezer ( Schizosaccharomyces pombe ) JM2-16 was inoculated into liquid culture 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 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 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.

[0067] 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.).

[0068] (2) The burdock seed raw material is broken and then pulverized to obtain burdock seed powder.

[0069] (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.

[0070] (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.

[0071] Example 6

[0072] A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects comprises the following steps:

[0073] (1) Take out the Schizosaccharomyces pombe from the -80℃ freezer ( Schizosaccharomyces pombe) JM2-16, inoculated in liquid medium according to an inoculation amount of 1% (v / v), activated at 35°C for 24 h, continuously activated for 2 times, to obtain an activated liquid, the activated liquid inoculated in liquid medium according to an inoculation amount of 1% (v / v), cultured at 37°C for 24 h, to obtain a Schizochytrium limacinum liquid culture. The Schizochytrium limacinum liquid culture is continuously inoculated in liquid medium according to an inoculation amount of 1% (v / v) for scale-up culture, cultured at 37°C for 24 h, to obtain a Schizochytrium limacinum culture medium.

[0074] The components of the liquid culture medium include glucose 20 g / L, glycerol 10 g / L, soybean peptone 40 g / L, yeast extract 5 g / L, potassium phosphate dibasic 2 g / L, rice extract 5 g / L, inulin 5 g / L, and white water lily flower extract 5 g / L.

[0075] (2) The burdock fruit raw material is broken and crushed to obtain burdock fruit powder.

[0076] (3) The burdock fruit powder is inoculated into the Schizochytrium limacinum culture medium prepared in step (1) according to a solid-liquid weight ratio of 1:20. The burdock fruit powder is fermented under the following conditions: a shaking incubator speed of 200 r / min, a pH value of 7.5, a temperature of 35°C, and a burdock fruit powder fermentation culture time of 48 h, to obtain a crude fermentation liquid.

[0077] (4) The crude fermentation liquid is centrifuged (at a speed of 3000 rpm for 15 min) to obtain a clarified fermentation product, which is then sterilized in a 121°C high-temperature sterilization pot for 30 min, and the filtrate is collected after filtration.

[0078] Comparative Example 1

[0079] A method for preparing a burdock fruit alcohol extract, comprising the following steps:

[0080] (1) The burdock fruit powder and 95% ethanol solution are mixed according to a solid-liquid weight ratio of 1:20.

[0081] (2) The ethanol extract is obtained by heating and refluxing.

[0082] (3) After cooling, centrifugation is performed (at a speed of 3000 rpm for 15 min), and the supernatant is combined.

[0083] (4) The supernatant is concentrated to 30% of the original volume to obtain a burdock fruit alcohol extract concentrate.

[0084] Comparative Example 2

[0085] This comparative example is the same as Example 1, except that the burdock fruit is replaced by burdock root to obtain a burdock root fermentation liquid.

[0086] Performance test

[0087] 1. Antioxidant test - DPPH radical scavenging experiment

[0088] The samples of Examples 1-6 and Comparative Examples 1-2 were respectively prepared into 3%, 2%, 1%, and 0.5% Arctium lappa product sample solutions, and were respectively placed in transparent PET bottles to observe the DPPH radical scavenging effect and evaluate the antioxidant performance of the Arctium lappa fruit fermentation liquor.

[0089] 0.1 mM DPPH alcohol solution was mixed with different concentrations of Arctium lappa product sample solutions at a volume ratio of 1:1, and was placed on a 96-well plate, 3 replicates per group. After 30 min of reaction at 37°C in a water bath under dark conditions, the absorbance at 517 nm was measured, and the DPPH scavenging rate of each sample was calculated.

[0090] Scavenging rate = [A 对照组 -(A 样品组 -A 空白组 ) / A 对照组 ] x 100%;

[0091] A 样品组 : absorbance of sample solution and DPPH alcohol solution;

[0092] A 空白组 : absorbance of sample solution and anhydrous ethanol;

[0093] A 对照组 : absorbance of DPPH alcohol solution and dimethyl sulfoxide (DMSO).

[0094] Table 1 DPPH radical scavenging rate

[0095]

[0096] As can be seen from the data in Table 1, compared with the product of the comparative example, the Arctium lappa fruit fermentation liquor involved in the present application has excellent DPPH radical scavenging activity, that is, excellent skin antioxidant property, and the antioxidant property is enhanced with the increase of the concentration of the fermentation liquor.

[0097] Table 1 also shows that when rice extract or inulin and white calla flower medium is added to the culture medium, it is beneficial to improve the antioxidant property of the Arctium lappa fruit fermentation liquor, and when rice extract, inulin, and white calla flower medium are simultaneously added to the culture medium, the antioxidant property of the Arctium lappa fruit fermentation liquor is stronger.

[0098] 2. Soothing test - hyaluronidase inhibition rate experiment

[0099] (1) Reagent preparation

[0100] Hyaluronidase solution: 500 U / mL, prepared immediately before use, using acetic acid buffer as solvent;

[0101] Sodium hyaluronate solution: 0.5 mg / mL, using acetic acid buffer as solvent;

[0102] Acetic acid buffer: 0.2 mol / L acetic acid 4.8 mL and 0.2 mol / L sodium acetate 45.2 mL, mixed and diluted to 100 mL to prepare a pH = 5.6 acetic acid buffer;

[0103] Acetylacetone solution: 50 mL 1.0 mol / L sodium carbonate solution and 3.5 mL acetylacetone are mixed evenly (prepared immediately before use);

[0104] Ehrlich reagent: 0.8 g p-dimethylaminobenzaldehyde is dissolved in a mixture of 15 mL concentrated hydrochloric acid and 15 mL anhydrous ethanol, and mixed evenly;

[0105] The mass concentration of the concentrated hydrochloric acid is 37%;

[0106] CaCl2solution: 2.5 mol / L;

[0107] NaOH solution: 5 mol / L.

[0108] Detection method: the samples prepared in each example and the samples prepared in each comparative example are respectively prepared into 3%, 2%, 1%, and 0.5% burdock product sample solutions. 0.1 mL of 0.25 mol / L CaCl2solution and 0.5 mL of hyaluronidase solution are taken and incubated at 37°C for 20 min; 0.5 mL of burdock product sample solution of different concentrations is added, and incubation is continued at 37°C for 20 min; 0.5 mL of sodium hyaluronate solution is added and incubated at 37°C for 30 min, and then placed at room temperature for 5 min; 0.1 mL of 0.4 mol / L NaOH solution and 0.5 mL of acetylacetone solution are added, and heated in a boiling water bath for 15 min, immediately cooled with ice water for 5 min; 1.0 mL of Ehrlich reagent is added, and diluted with 3.0 mL of anhydrous ethanol, and color development is performed for 20 min, during which wavelength scanning in the range of 450-700 nm is performed to determine the maximum absorption wavelength, and the absorbance value is measured at the maximum absorption wavelength by using a spectrophotometer. The inhibition rate of the sample on hyaluronidase is calculated.

[0109] Inhibition rate = [(A1-A2)-(B1-B2) / (A1-A2)];

[0110] A1: reference solution absorbance, using deionized water instead of the obtained sample;

[0111] A2: The absorbance value of the reference blank solution, which is prepared by using deionized water instead of the obtained sample and using an acetic acid buffer solution instead of the hyaluronidase and sodium hyaluronate solution;

[0112] B1: The absorbance value of the sample solution;

[0113] B: The absorbance value of the sample blank solution, which is prepared by using an acetic acid buffer solution instead of the hyaluronidase and sodium hyaluronate solution.

[0114] Table 2: Inhibition rate of hyaluronidase

[0115]

[0116] As can be seen from the data in Table 2, compared with the product of the comparative example, the burdock fruit fermentation liquid involved in the present application has excellent hyaluronidase inhibitory activity, reduces the diffusion of inflammatory mediators, and thus plays an anti-inflammatory role, i.e., has excellent skin soothing potential. Moreover, as the concentration of the fermentation liquid increases, the soothing effect is enhanced.

[0117] Meanwhile, Table 2 shows that the addition of rice extract to the culture medium is conducive to further improving the inhibitory activity of hyaluronidase, thereby further improving the anti-inflammatory effect and soothing effect.

[0118] 3. Safety test - HaCaT cell toxicity test

[0119] HaCaT cell model establishment: The keratinocytes (HaCaT) were cultured in a DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). The cells were incubated in a constant temperature incubator containing 5% CO2 at 37°C.

[0120] Cell inoculation: The keratinocytes (HaCaT) were inoculated in a 96-well plate at a seeding density of 6 x 10 3 cells / well, and were incubated in an incubator (37°C, 5% CO2) overnight.

[0121] Experimental grouping: A blank group, a control group, and a sample group were set up. In the sample group, three concentration gradients were set up, and three repeated wells were set up under each concentration gradient.

[0122] Liquid preparation: The fermentation liquids prepared using the examples and the comparative example were used to prepare working solutions of the test substances at different concentrations according to the set test concentrations.

[0123] Drug administration: When the plating rate of the cells in the 96-well plate reached 40%-60%, drug administration was performed. The blank group was not inoculated with cells, and only 200 μL of cell culture solution was added; the control group was added with 200 μL of culture solution per well; and the sample group was added with 200 μL of culture solution containing the corresponding concentration of samples per well. After the drug administration was completed, the 96-well plate was placed in an incubator (37°C, 5% CO2) for culture.

[0124] Detection: After the cell culture was incubated for 24 h, 10 μL CCK-8 was added, and the incubation was carried out at 37 °C in the dark for 2-4 h. After the incubation, the OD value was read at 490 nm. The cell survival rate was calculated according to the following formula:

[0125] Cell survival rate = (OD 样品组 - OD 空白组 ) / (OD 对照组 - OD 空白组 ) x 100%;

[0126] In the formula, OD 样品组 is the OD value of the sample group, OD 空白组 is the OD value of the blank group, and OD 对照组 is the OD value of the control group.

[0127] Table 3 CCK-8 detection of cell toxicity results

[0128]

[0129] According to the results in Table 3, the samples prepared in each example did not show cytotoxicity to keratinocytes at a concentration of 3%. This result indicates that the fermented Arctium lappa L. liquid has little toxic effect on cells and does not cause irritation or damage, and has good safety.

[0130] 4. In vitro transdermal absorption experiment

[0131] Preparation of isolated pig skin: Take the intact fresh pig skin, first remove the residual pig hair with tweezers, then scrape off the subcutaneous tissue and fat with a small knife, and wash with PBS solution, wrap with tin paper, freeze in a-20 °C refrigerator, and store for use. Before use, it needs to be thawed in PBS solution for 45 min before the experiment can be carried out.

[0132] The treated pig skin stratum corneum was fixed upward on the Franz diffusion cell, 5 mL of 30% ethanol-saline was added to each receiving cell, and the intelligent transdermal tester was placed 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 1 h of equilibrium, the bubbles in the receiving cell were exhausted, and the drug was administered. 1 mL of sample of each example and comparative example was added to the supply cell to prepare Arctium lappa L. product sample liquid with a concentration of 3%, 2%, 1%, and 0.5%, each group was repeated 3 times, and the sample was taken after 3 h.

[0133] Treatment of skin sample: After the test skin was taken off, it was washed with physiological saline, dried with filter paper, put into 1.5 mL centrifuge tube, cut into pieces, 1 mL methanol was added, sealed and ultrasonically treated for 30 min, centrifuged at 10000 r·min -1 for 5 min, the supernatant was filtered with 0.22 μm microporous filter membrane, and then analyzed by sample injection. The cumulative permeation amount was calculated according to the following formula.

[0134] Cumulative permeation amount calculation method:

[0135] Cumulative permeation amount per unit area of arctium lappa product sample liquid (μg·cm -2 ) and transdermal rate Js (μg·cm -2 ·h -1 ).

[0136] = / S

[0137] = / T

[0138] In the formula, C is the concentration measurement value at t time, 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.

[0139] Table 4 In vitro transdermal absorption results

[0140]

[0141] As can be seen from Table 4, the transdermal rate of arctium lappa fruit fermented extract is greater than that of arctium lappa fruit alcohol extract and arctium lappa root fermented liquid. When the concentration of penetration enhancer is 3%, the transdermal rate is the largest. It shows that arctium lappa fruit fermented extract has good penetration effect.

[0142] 5. Skin oil content test

[0143] The amount of skin surface sebum was measured by measuring the transparency of oil absorption tape after absorbing sebum.

[0144] Experimental sample: the experimental group is arctium lappa fruit fermented liquid of each example, and the control group is arctium lappa fruit alcohol extract of comparative example 1 and arctium lappa root fermented liquid of comparative example 2.

[0145] Experimental method:

[0146] Select 30 subjects aged 18-45 years old, male and female, all oily and mixed skin, no history of skin disease and skin allergy, each group is applied with different concentration of 3% test sample. The same part of the face is tested 3 times with a skin oil test probe (Sebumeter, Germany CK), and the average value is used to evaluate the oil control effect of the composition of the application. The set measurement time interval should not be less than 1 h, and the whole test period should not exceed 24 h.

[0147] Table 5

[0148]

[0149] From Table 5, it can be seen that the fermented extract of Arctium lappa fruit can significantly reduce the secretion of skin oil, and the effect is better than that of the alcohol extract of Arctium lappa fruit and the fermented liquid of Arctium lappa root. The fermented extract of Arctium lappa fruit can regulate the metabolism of the stratum corneum, improve the skin barrier function, and indirectly affect the balance of oil secretion. During the experiment, no irritation reactions such as redness and itching occurred in the experimental group, indicating that the extract of Arctium lappa fruit is safe at this concentration and can be used in oil control formulations.

[0150] The results show that the antioxidant, soothing performance, and penetration promoting effect and oil control effect of the fermented extract of Arctium lappa fruit are better than those of the alcohol extract of Arctium lappa fruit and the fermented liquid of Arctium lappa root. The single fermentation of Schizosaccharomyces pombe can significantly improve the antioxidant and soothing performance of Arctium lappa fruit. Therefore, the fermented liquid of Arctium lappa fruit of the application has significant anti-inflammatory and antioxidant capacity, can promote penetration and control oil, and has high biological safety, can be used for repairing skin barrier, and has the effects of moisturizing, soothing and oil control.

[0151] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing burdock fermented liquid with soothing and anti-inflammatory effects, characterized in that: The steps include: Inoculating Schizosaccharomyces pombe JM2-16 into a fermentation medium, and adding burdock seed powder to the fermentation medium for fermentation; wherein the deposit number of Schizosaccharomyces pombe JM2-16 is CCTCC NO: M 20242810; 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; the fermentation medium comprises rice extract; the fermentation medium comprises inulin and white water lily extract; the content of the rice extract in the fermentation medium is 4.5-5.5 g / L; the content of the inulin in the fermentation medium is 4.5-5.5 g / L, and the content of the white water lily extract in the fermentation medium is 4.5-5.5 g / L; The fermentation temperature is 34~37 ℃ and the fermentation time is 46~48 h.

2. The preparation method according to claim 1, characterized in that: The Schizosaccharomyces pombe JM2-16 was activated and expanded in sequence, and then inoculated into the fermentation medium for fermentation.

3. The burdock fruit fermentation liquid obtained by the preparation method according to any one of claims 1-2.

4. Use of the burdock fruit fermentation liquid according to claim 3 in the preparation of cosmetics.

5. The use according to claim 4, 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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