An oil-controlling, acne-removing, bacterium-inhibiting and barrier-repairing dogwood ferment, and a preparation method and application thereof

Abstract

The present application relates to a kind of oil control acne, bacteriostatic and repair barrier of service dogwood ferment and preparation method and application, belong to cosmetic technical field.Preparation method includes: the material derived from service dogwood is mixed with yeast infusion peptone glucose culture medium according to (10~15) :(7~10) Mass ratio of preparation culture medium;According to total bacterial inoculation amount 1~1.5%, yeast is inoculated into culture medium, and fermentation is carried out at 30~35 DEG C for 44~52h;The fermentation product is centrifuged to obtain service dogwood ferment;Yeast is saccharomyces cerevisiae, schizosaccharomyces pombe or pichia pastoris.The active ingredient of service dogwood treated by fermentation process can be further decomposed, transformed or enriched, so as to enhance its skin care potential, such as enhancing sensitive soothing, enhancing bacteriostatic property and promoting skin barrier repair.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a fermented product of Sanguisorba officinalis that controls oil, removes acne, inhibits bacteria, and repairs the skin barrier, as well as its preparation method and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Acne is a common chronic inflammatory disease of the pilosebaceous unit, and its onset is closely related to multiple factors. Modern lifestyles, including stress, unbalanced diets, and irregular sleep patterns, lead to abnormal sebum secretion and skin microecological imbalances, significantly increasing the risk of acne. Epidemiological studies show that over 80% of people will experience acne to varying degrees in their lifetime, with adolescents (14-19 years old) being the most affected group. Although acne is easy to diagnose, it is difficult to cure completely, prone to recurrence, and often leaves secondary skin lesions such as scars, erythema, and pigmentation.

[0004] Sanguisorba officinalis has anti-inflammatory, analgesic, antibacterial, antiviral, and intestinal function regulating effects. Based on these effects, it is often used clinically in the form of decoction pieces or compound preparations. Internally, it can treat gastrointestinal bleeding, gynecological bleeding, acute and chronic enteritis, bacterial dysentery, and other intestinal diseases. Externally, it can treat burns, eczema, dermatitis, traumatic bleeding, and oral ulcers. Topical cosmetics are a convenient means of suppressing acne. However, the large molecules contained in Sanguisorba officinalis are not easily absorbed through the skin. Applying the internally taken Sanguisorba officinalis directly to the skin makes it difficult for the active ingredients to be absorbed, resulting in very low anti-inflammatory and acne-reducing effects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a fermented product of Sanguisorba officinalis that controls oil, removes acne, inhibits bacteria, and repairs the skin barrier, as well as its preparation method and application. By using yeast fermentation, the macromolecular substances in Sanguisorba officinalis powder are decomposed into small molecules, which not only enhances the original pharmacological effects of Sanguisorba officinalis but also facilitates transdermal absorption.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: Firstly, a method for preparing a fermented product of Sanguisorba officinalis that controls oil, removes acne, inhibits bacteria, and repairs the skin barrier includes the following steps: The material derived from Sanguisorba officinalis was mixed with yeast extract peptone glucose medium at a mass ratio of (10~15):(7~10) to prepare the culture medium. Inoculate yeast onto the culture medium at a total inoculum amount of 1-1.5% and ferment at 30-35°C for 44-52 hours. The fermentation product was centrifuged and the supernatant was collected to obtain the Sanguisorba officinalis fermentation product; The yeast is Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, or Pichia pastoris. The *Schizophyllum commune* strain is *Schizophyllum commune* JM2-16 (… Schizosaccharomyces pombe JM2-16), with accession number M 20242810; The Pichia pastoris strain is Pichia pastoris JM4-20 ( Pichia kudriavzevii JM4-20 was deposited on December 13, 2024, at the China Center for Type Culture Collection, located in Wuhan, China, with accession number M 20242811.

[0007] Secondly, the fermented product of Sanguisorba officinalis obtained by the above-mentioned method for preparing Sanguisorba officinalis fermented product with oil control, acne removal, antibacterial and barrier repair properties.

[0008] Thirdly, the above-mentioned Sanguisorba officinalis fermentation products are used in the preparation of acne-removing and anti-inflammatory drugs or cosmetics.

[0009] Preservation Instructions The Pichia pastoris JM4-20 provided by this invention ( Pichia kudriavzevii JM4-20 was deposited on December 13, 2024, at the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province), with accession number CCTCC NO: M 20242811.

[0010] The beneficial effects of this invention are as follows: This invention uses Sanguisorba officinalis as a fermentation raw material, which is rich in active ingredients such as polyphenols, flavonoids, and polysaccharides. It possesses multiple skin-care benefits, including antioxidant, anti-inflammatory, astringent, and repairing properties. Through fermentation with Saccharomyces cerevisiae, the fermentable sugars in Sanguisorba officinalis are rapidly converted into ethanol, forming an ethanol-water biphasic extraction system. The active ingredients of Sanguisorba officinalis can be further decomposed, transformed, or enriched, thereby enhancing its skin-care potential, such as strengthening its soothing effect on sensitivity, enhancing its antibacterial properties, and promoting skin barrier repair. The fermented Sanguisorba officinalis not only retains the traditional natural and mild properties of Sanguisorba officinalis but also generates new active substances (such as amino acids, peptides, and fermentation-derived components) through microbial metabolism, making it more advantageous in cosmetic applications. Attached Figure Description

[0011] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0012] Figure 1The results are shown in Figure 1 of Test Example 1. Among them, A is the result of the total phenol content test, B is the result of the crude polysaccharide content test, C is the result of the flavonoid content test, and D is the result of the amino acid content test.

[0013] Figure 2 The results are for test example 2; where A is the test result for Staphylococcus aureus, B is the test result for Malassezia, and C is the test result for Propionibacterium acnes.

[0014] Figure 3 The results are shown in Figure 3 of Test Example 3. Among them, A is the result of the test on the effect of hyaluronidase, B is the result of the effect of different concentrations of Sanguisorba officinalis ferment on cell viability, C is the result of the test on cell migration ability, D is the result of the statistical results of wound healing rate, E is the result of the test on TNF-α inflammatory factor, F is the result of the test on IL-1 inflammatory factor, and G is the result of the test on IL-6 inflammatory factor.

[0015] Figure 4 The results are shown in Figure 4 of Test Example 4. Figure A shows the test results for inhibition of 5α-reductase, Figure B shows the test results for tyrosinase, Figure C shows the test results for the effect on B16 cell viability, and Figure D shows the test results for melanin production.

[0016] Figure 5 This is the result graph for test example 5; where A is the test result graph for DPPH free radical scavenging rate, and B is the result graph for O 2- The graphs show the test results for scavenging rates. C represents the test results for hydroxyl radical scavenging rate, and D represents the test results for ROS inhibition. Detailed Implementation

[0017] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 invention pertains.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0019] Current research on the fermentation of Sanguisorba officinalis mainly focuses on lactic acid bacteria (LAB) fermentation and the optimization of fermentation processes, while research on the fermentation of Sanguisorba officinalis by Saccharomyces cerevisiae is almost non-existent.

[0020] One or more embodiments of the present invention provide a method for preparing a fermented product of Sanguisorba officinalis that controls oil, removes acne, inhibits bacteria, and repairs the skin barrier, comprising the following steps: S1. Prepare a culture medium by mixing powder derived from Sanguisorba officinalis root with yeast extract peptone glucose medium at a mass ratio of (10~15):(7~10); S2. Inoculate the yeast onto the culture medium at a total inoculum amount of 1-1.5% and ferment at 30-35℃ for 44-52 hours. S3. Centrifuge the fermentation product and collect the supernatant to obtain the Sanguisorba officinalis fermentation product; The yeast is Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, or Pichia pastoris. The *Schizophyllum commune* strain is *Schizophyllum commune* JM2-16 (… Schizosaccharomyces pombe JM2-16), with accession number M 20242810; The Pichia pastoris strain is Pichia pastoris JM4-20 ( Pichia kudriavzevii JM4-20 was deposited on December 13, 2024, at the China Center for Type Culture Collection, located in Wuhan, China, with accession number M 20242811.

[0021] In the above process, the material derived from Sanguisorba officinalis is first mixed with yeast extract peptone glucose medium to form a medium containing the active ingredients in Sanguisorba officinalis. Then, the medium is used for fermentation to transform the active ingredients into new active substances, thereby improving its acne-removing and anti-inflammatory effects.

[0022] Sanguisorba officinalis, the dried root of Sanguisorba officinalis or Sanguisorba longifolia, both belonging to the Rosaceae family, is an irregularly cylindrical or spindle-shaped medicinal material. Its surface is grayish-brown to dark brown, hard and brittle, with a pink or pale yellow cross-section. It has a faint odor and a slightly bitter and astringent taste. Sanguisorba officinalis has a complex chemical composition, containing tannins, triterpenoid saponins, flavonoids, etc. Among these, tannins and triterpenoid saponins are the core components exerting its pharmacological effects. The former can constrict blood vessels, promote platelet aggregation to stop bleeding, and form a protective film to protect the wound surface; the latter can inhibit the release of inflammatory mediators and promote fibroblast proliferation.

[0023] Optionally, in S1, the material derived from Sanguisorba officinalis is selected from dried Sanguisorba officinalis or Sanguisorba officinalis extract, and can be added in the form of dried Sanguisorba officinalis powder.

[0024] Optionally, in S1, the yeast extract peptone glucose medium (also known as YPD medium) is a commonly used culture medium for culturing yeast in the art, comprising the following components by weight percentage: 1% yeast extract, 2% peptone and 2% glucose.

[0025] Optionally, in S1, the powder derived from Sanguisorba officinalis root is mixed with yeast extract peptone glucose medium and then 10-15 times the weight of water is added to prepare the culture medium.

[0026] Optionally, in S1, the total inoculum amount is the ratio of the seed liquid volume to the fermentation medium volume, and the OD600 of the seed liquid is 0.8~1.2.

[0027] Optionally, in S1, the culture medium is subjected to autoclaving.

[0028] Optionally, in S2, multiple layers of gauze are used to seal the fermentation process to ensure aseptic conditions and meet the oxygen requirements of the yeast.

[0029] Optionally, in S2, the fermentation process is shake-flask fermentation at 160~180 rpm, which allows the substrate and the inoculum to come into full contact and mix evenly, so as to give full play to the advantages of the inoculum used.

[0030] Optionally, in S3, the fermentation product is sterilized by high-pressure steam and then centrifuged at 10,000-12,000 rpm for 8-12 minutes to remove any remaining Sanguisorba officinalis powder after fermentation.

[0031] Optionally, in S3, the supernatant is subjected to vacuum filtration to further remove impurities.

[0032] Optionally, the process may also include the following steps: decolorizing the fermented product of Sanguisorba officinalis with activated carbon, centrifuging the activated carbon to obtain a second supernatant, filtering the second supernatant with a filter membrane to obtain a transparent liquid; and further separating the incompletely fermented Sanguisorba officinalis powder.

[0033] One or more embodiments of the present invention provide a method for preparing the above-mentioned Sanguisorba officinalis fermented product.

[0034] One or more embodiments of the present invention provide the application of the above-mentioned Sanguisorba officinalis ferment in acne treatment and anti-inflammatory.

[0035] The present invention will be further described below with reference to specific embodiments.

[0036] The fermentation bacteria involved in the following examples and comparative examples include: Saccharomyces cerevisiae Y17, also known as brewer's yeast (Y17), was purchased from Jinan Ruibeike Biotechnology Co., Ltd.

[0037] Saccharomyces cerevisiae Y21, also known as brewer's yeast (Y21), was purchased from Jinan Ruibeike Biotechnology Co., Ltd.

[0038] Schizosaccharomyces cerevisiae (JM2-16), accession number: CCTCC NO: M20242810, and disclosed in patent publication number CN119530105A.

[0039] Pichia pastoris (JM4-20), accession number: CCTCC NO:M20242811.

[0040] Example 1 A fermented product of Sanguisorba officinalis, the preparation method includes the following steps: S1. Take 10g of dried Sanguisorba officinalis root powder (the dried Sanguisorba officinalis root powder is processed to 40 mesh powder, with a fineness of less than 425μm), mix it with 10g of yeast extract powder peptone glucose medium, add 200mL of deionized water and stir evenly. Sterilize the resulting liquid in a high-pressure steam sterilizer at 121℃ for 20min to obtain the culture medium.

[0041] S2. Take Saccharomyces cerevisiae (Y21) and inoculate the seed culture with an OD600 value of 0.8 onto the culture medium obtained in S1 at a ratio of 1.5% of the total culture medium volume. Ferment at 160 rpm in a shake flask at 30°C for 44 hours.

[0042] S3. Place the fermented liquid in a high-pressure steam sterilizer and sterilize at 115℃ for 20 minutes. Then place it in a centrifuge and centrifuge at 10000 rpm for 12 minutes. Take the supernatant and place it in a vacuum filter for filtration to obtain the fermented product of Sanguisorba officinalis.

[0043] S4. After decolorizing the Sanguisorba officinalis ferment with activated carbon, centrifuge to obtain the supernatant, filter with a 0.22μm filter membrane to obtain the Sanguisorba officinalis ferment product.

[0044] The obtained Sanguisorba officinalis fermentation product does not contain live bacteria.

[0045] Yeast extract peptone glucose medium (YPD medium) comprises the following components by weight percentage: 1% yeast extract, 2% peptone and 2% glucose.

[0046] Example 2 A fermented product of Sanguisorba officinalis, the preparation method includes the following steps: S1. Take 15g of dried Sanguisorba officinalis powder (dried Sanguisorba officinalis root, processed to 40 mesh powder, fineness less than 425μm), mix with 7g of yeast extract peptone glucose medium, add 300mL of deionized water and stir evenly. Sterilize the resulting liquid in a high-pressure steam sterilizer at 121℃ for 20min to obtain the culture medium.

[0047] S2. Take Saccharomyces cerevisiae (Y21) and inoculate the seed culture with an OD600 of 1.2 onto the culture medium obtained in S1 at a ratio of 1.0% of the total volume of the inoculated culture liquid. Ferment at 35℃ and 180 rpm for 52 hours.

[0048] S3. Place the fermented liquid in a high-pressure steam sterilizer and sterilize at 115℃ for 20 minutes. Then place it in a centrifuge and centrifuge at 12000 rpm for 8 minutes. Take the supernatant and place it in a vacuum filter for filtration to obtain the fermented product of Sanguisorba officinalis.

[0049] S4. After decolorizing the Sanguisorba officinalis ferment with activated carbon, centrifuge to obtain the supernatant, filter with a 0.22μm filter membrane to obtain the Sanguisorba officinalis ferment product.

[0050] The obtained Sanguisorba officinalis fermentation product does not contain live bacteria.

[0051] The yeast extract peptone glucose medium used in this embodiment is the same as that in Example 1.

[0052] Example 3 A fermented product of Sanguisorba officinalis, the preparation method includes the following steps: S1. Take 10g of dried Sanguisorba officinalis powder, mix it with 10g of yeast extract peptone glucose medium, add 200mL of deionized water and stir well. Sterilize the resulting liquid in a high-pressure steam sterilizer at 121℃ for 20min to obtain the culture medium.

[0053] S2. Take Saccharomyces cerevisiae (Y17) and inoculate it onto the culture medium obtained in S1 at a total inoculum of 1.5%. Ferment at 30°C and 160 rpm for 52 hours.

[0054] S3. Place the fermented liquid in a high-pressure steam sterilizer and sterilize at 115℃ for 20 minutes. Then place it in a centrifuge and centrifuge at 10000 rpm for 12 minutes. Take the supernatant and place it in a vacuum filter for filtration to obtain the fermented product of Sanguisorba officinalis.

[0055] S4. After decolorizing the Sanguisorba officinalis ferment with activated carbon, centrifuge to obtain the supernatant, filter with a 0.22μm filter membrane to obtain the Sanguisorba officinalis ferment product.

[0056] The obtained Sanguisorba officinalis fermentation product does not contain live bacteria.

[0057] The difference between this comparative example and Example 1 is that the fermentation uses Saccharomyces cerevisiae (Y17). Other raw materials and preparation methods are the same as in Example 1.

[0058] Example 4 A fermented product of Sanguisorba officinalis, the preparation method includes the following steps: S1. Take 10g of dried Sanguisorba officinalis powder, mix it with 10g of yeast extract peptone glucose medium, add 200mL of deionized water and stir well. Sterilize the resulting liquid in a high-pressure steam sterilizer at 121℃ for 20min to obtain the culture medium.

[0059] S2. Take Saccharomyces cerevisiae (JM2-16) and inoculate it onto the culture medium obtained in S1 at a total inoculum of 1.5%. Ferment at 30°C and 160 rpm for 52 hours.

[0060] S3. Place the fermented liquid in a high-pressure steam sterilizer and sterilize at 115℃ for 20 minutes. Then place it in a centrifuge and centrifuge at 10000 rpm for 12 minutes. Take the supernatant and place it in a vacuum filter for filtration to obtain the fermented product of Sanguisorba officinalis.

[0061] S4. After decolorizing the Sanguisorba officinalis ferment with activated carbon, centrifuge to obtain the supernatant, filter with a 0.22μm filter membrane to obtain the Sanguisorba officinalis ferment product.

[0062] The obtained Sanguisorba officinalis fermentation product does not contain live bacteria.

[0063] The difference between this comparative example and Example 1 is that fermentation was performed using *Schizosaccharomyces cerevisiae* (JM2-16). Other raw materials and preparation methods were the same as in Example 1.

[0064] Example 5 A fermented product of Sanguisorba officinalis, the preparation method includes the following steps: S1. Take 10g of dried Sanguisorba officinalis powder, mix it with 10g of yeast extract peptone glucose medium, add 200mL of deionized water and stir well. Sterilize the resulting liquid in a high-pressure steam sterilizer at 121℃ for 20min to obtain the culture medium.

[0065] S2. Take Pichia pastoris (JM4-20) and inoculate it onto the culture medium obtained in S1 at a total inoculum of 1.5%. Ferment at 30℃ and 160 rpm for 52 h.

[0066] S3. Place the fermented liquid in a high-pressure steam sterilizer and sterilize at 115℃ for 20 minutes. Then place it in a centrifuge and centrifuge at 10000 rpm for 12 minutes. Take the supernatant and place it in a vacuum filter for filtration to obtain the fermented product of Sanguisorba officinalis.

[0067] S4. After decolorizing the Sanguisorba officinalis ferment with activated carbon, centrifuge to obtain the supernatant, filter with a 0.22μm filter membrane to obtain the Sanguisorba officinalis ferment product.

[0068] The obtained Sanguisorba officinalis fermentation product does not contain live bacteria.

[0069] The difference between this comparative example and Example 1 is that Pichia pastoris (JM4-20) was used for fermentation. Other raw materials and preparation methods are the same as in Example 1.

[0070] Comparative Example 1 A method for preparing a Sanguisorba officinalis aqueous extract includes: S1. Process the root of Sanguisorba officinalis into powder that passes through a 40-mesh sieve. Mix 13g of Sanguisorba officinalis root powder with 250mL of water in a container. S2. Place the container in a magnetic heating stirrer and extract for 35 min at 165 rpm and 85°C. Centrifuge the resulting liquid at 8000 rpm for 5 min to separate the first supernatant and precipitate. S3. Add 100 mL of deionized water to the precipitate obtained by centrifugation in S2, and perform a second extraction according to step S2 to separate and obtain the second supernatant. S4. After combining the first and second supernatants, the mixture is concentrated under reduced pressure to 2 / 3 of its original volume at 60℃, 60 rpm, and 0.09 MPa. Then, it is placed in a vacuum filter for filtration to obtain the Sanguisorba officinalis aqueous extract.

[0071] Comparative Example 2 A method for preparing a sanguisorba officinalis alcohol extract includes: S1. Process the root of Sanguisorba officinalis into powder that passes through a 40-mesh sieve. Mix 13g of Sanguisorba officinalis root powder with 350mL of ethanol solution (concentration of 70% v / v) in a container. S2. Place the container in an ultrasonic water bath constant temperature oscillator and extract for 35 minutes at 600W, 40kHz, and 85±5℃. Concentrate the obtained liquid to 2 / 3 of its original volume in a 90℃ constant temperature water bath, and then filter it in a vacuum filter to obtain the sanguisorbent extract.

[0072] Test Example 1: Determination of Active Ingredient Content The active ingredient content of products obtained by different extraction processes was determined. The test objects included: the fermented product of Sanguisorba officinalis prepared in Example 1 (referred to as Y21-1), the fermented product of Sanguisorba officinalis prepared in Example 2 (referred to as Y21-2), the fermented product of Sanguisorba officinalis prepared in Example 3 (referred to as Y17), the fermented product of Sanguisorba officinalis prepared in Example 4 (referred to as JM2-16), the fermented product of Sanguisorba officinalis prepared in Example 5 (referred to as JM4-20), the aqueous extract of Sanguisorba officinalis prepared in Comparative Example 1 (referred to as aqueous extract), and the alcoholic extract of Sanguisorba officinalis prepared in Comparative Example 2 (referred to as alcoholic extract).

[0073] The test content includes: 1. The method for determining the total phenol content includes: preparing a 60% ethanol solution and a 5 mg / mL gallic acid solution; preparing gallic acid standards with concentrations of 0.625, 0.15625, 0.078125, 0.0391, 0.0191, 0.0098, and 0.0049 mg / mL using a serial dilution method; setting up four experimental groups, labeled as control group, assay group, standard group, and blank group, where the assay group is used to add one of the test objects; conducting the reaction according to the instructions of the plant total phenol (TP) content detection kit (catalog number: BC1345, Beijing Solarbio Science & Technology Co., Ltd.); vortexing the assay group, allowing it to stand at room temperature for 10 min, and measuring the absorbance at 760 nm in a 96-well plate; plotting a standard curve, obtaining the concentration of each sample based on the standard curve, and calculating the total phenol content per unit mass (gram weight) of each sample using the sample mass.

[0074] Total phenol content (mg / mL) = x / W; x is the sample concentration calculated from the standard curve, in mg / mL; W is the sample mass in g.

[0075] 2. The method for detecting crude polysaccharide content includes: preparing a glucose solution with a concentration of 10 mg / mL; preparing glucose standards with concentrations of 0.2, 0.16, 0.08, 0.04, 0.02, and 0.01 mg / mL using a gradient dilution method; conducting the reaction according to the instructions of the plant crude polysaccharide content detection kit (catalog number: BC6165, Beijing Solarbio Science & Technology Co., Ltd.), with the sample solution being one of the test objects; vortexing the experimental group, reacting in a boiling water bath for 20 min, removing it and immediately cooling it to room temperature, and measuring the absorbance at 490 nm after mixing; plotting a standard curve, obtaining the concentration of each sample based on the standard curve, and calculating the crude polysaccharide content per unit mass (gram weight) of each sample using the sample mass.

[0076] The content of crude polysaccharide (mg / mL) = x / W×F; x is the sample concentration calculated from the standard curve, in mg / mL; W is the sample mass in g; and F is the dilution factor.

[0077] 3. The method for detecting flavonoid content includes: preparing a 60% v / v ethanol solution and a 10 mg / mL rutin solution; preparing rutin standards with concentrations of 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078, 0.039, and 0.02 mg / mL using a gradient dilution method, mixing thoroughly after each dilution; conducting the reaction according to the instructions of the flavonoid content detection kit (catalog number: BC1335, Beijing Solarbio Science & Technology Co., Ltd.); using one of the test substances as the sample solution; vortexing the experimental group, reacting in a 37℃ water bath for 45 min, centrifuging, and measuring the absorbance at 470 nm in a 96-well plate; plotting a standard curve, obtaining the concentration of each sample based on the standard curve, and calculating the content of plant flavonoids per unit mass (gram weight) of each sample using the sample mass.

[0078] Plant flavonoid content (mg / mL) = x / W×F; x is the sample concentration (mg / mL) calculated from the standard curve; W is the sample mass in g; F: dilution factor.

[0079] 4. Amino acid content detection method includes: preparing a 1.25 μmol / mL glutamic acid standard, mixing thoroughly, and preparing fresh before use; performing the reaction according to the instructions of the amino acid content detection kit (catalog number: BC1575, Beijing Solarbio Science & Technology Co., Ltd.); the sample solution to be tested is one of the test objects; vortexing the experimental group, incubating in a boiling water bath for 15 min, cooling, repeatedly inverting, centrifuging, and taking the supernatant to measure the absorbance at 570 nm in a 96-well plate; calculating the amino acid content.

[0080] Amino acid content (μmol / mL) = 2.5 × ΔA (determined) / ΔA (standard) × F; ΔA measurement = A measurement tube - A blank tube; ΔAstandard = Astandard tube - Ablank tube; F is the dilution factor.

[0081] The test results of Examples 1-5, Comparative Examples 1 and 2 are as follows: Figure 1 As shown, Figure 1 Figure A in the figure shows the total phenol content in different examples and comparative examples; Figure 1 Figure B in the figure shows the content of crude polysaccharides in the plants in different embodiments and comparative examples; Figure 1 Figure C in the figure shows the content of Sanguisorba officinalis flavonoids after fermentation of Sanguisorba officinalis root by different strains in different embodiments and comparative examples; Figure 1Figure D in the table shows the amino acid content of *Sanguisorba officinalis* in different examples and comparative examples. The results show that the total phenolic, flavonoid, polysaccharide, and amino acid contents of Y21-1 and Y21-2 are significantly better than those of Y17, JM2-16, and JM4-20, and the fermentation effect of these four strains is significantly better than that of water extraction and alcohol extraction processes; among them, the fermentation effect of Y21-2 is better than that of Y21-1. In summary, based on Example 2, which has the highest content of active ingredients in the fermentation broth of *Sanguisorba officinalis* root and the best economic benefits, further research will be conducted.

[0082] Test Example 2: Evaluation of Antibacterial Efficacy In the entire process of acne (acne vulgaris) development, from microcomedones to inflammatory papules to pustules / nodules, *Propionibacterium acnes* is the core driving factor, *Malassezia* is a co-aggravating factor, and *Staphylococcus aureus* mainly plays the role of secondary infection / inflammation amplifier. The effect of *Sanguisorba officinalis* ferment filtrate on antibacterial and acne-reducing effects was evaluated by detecting its inhibition rate against *Staphylococcus aureus*, *Propionibacterium acnes*, and *Malassezia*.

[0083] 1. The inhibition rate of Staphylococcus aureus was tested using conventional methods in the field, including: preparing LB liquid medium: weighing 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl, adding approximately 900 mL of deionized water, magnetically stirring until completely dissolved, adjusting the pH to 7.0±0.2 (25 ℃) dropwise with 5 mol / L NaOH, autoclaving at 121 ℃ for 20 min, and then aliquoting and storing; taking 100 μL of Staphylococcus aureus inoculum from the cryopreservation tube and inoculating it into 5 mL of the corresponding liquid medium, and incubating the Staphylococcus aureus aerobicly at 37 ℃ for 21±3 h; taking the activated bacterial solution and transferring it to fresh medium at a ratio of 1:100, repeating the culture once to obtain the logarithmic-phase bacterial strain (to ensure the stability of the inhibition experiment); taking the logarithmic-phase bacterial solution, diluting it with the corresponding medium, incubating for 2.5±0.5 h, and then measuring the OD value... 600 The absorbance was measured at a wavelength to make its OD 600 =0.5±0.05 (corresponding to a bacterial count of approximately 10) 6 -10 7 CFU / mL); In the experimental group, 5 mL of calibrated bacterial culture and 200 μL of different concentrations of Sanguisorba officinalis ferment (concentrations of 1 vol%, 3 vol%, 5 vol%, 10 vol%, or 20 vol%) were added to sterile centrifuge tubes, i.e., a volume ratio of 5 mL:0.2 mL = 25:1 were used to construct the system; in the control group, 5 mL of calibrated bacterial culture and 200 μL of sterile culture medium (used to replace the fermentation broth to eliminate the influence of the culture medium itself) were added to sterile centrifuge tubes, and the mixture was gently mixed. Three parallel samples were set up for each group to avoid experimental error; the samples were incubated on an aerobic, 37°C shaker, and samples were taken after 120 min; immediately after sampling, the OD was measured. 600Measure the absorbance at the specified wavelength and record the OD value of each parallel sample (mix the bacterial solution gently before measurement to avoid precipitation affecting the results); calculate the inhibition rate.

[0084] Antibacterial rate (%) = [(average OD value of control group - average OD value of sample group) / average OD value of control group] ×100%.

[0085] 2. The inhibition rate test against Malassezia was conducted using conventional methods in the field, including: dissolving 57.5 g of Dixon culture in 1 L of distilled water to prepare a culture medium; inoculating 100 μL of bacterial culture from a cryopreservation tube into 5 mL of the corresponding liquid culture medium, and incubating Malassezia aerobically at 32°C for 48 h; transferring the activated bacterial solution to fresh culture medium at a 1:100 ratio, repeating the culture once to obtain the logarithmic-phase bacterial strain (to ensure the stability of the inhibition experiment); diluting the logarithmic-phase bacterial solution with the corresponding culture medium, incubating for 2.5 ± 0.5 h, and then measuring the inhibition rate at OD0.05. 600 The absorbance was measured at a wavelength to make its OD 600 =0.5±0.05 (corresponding to a bacterial count of approximately 10) 6 -10 7 CFU / mL); In the experimental group, 5 mL of calibrated bacterial culture and 200 μL of different concentrations of Sanguisorba officinalis ferment broth (concentrations of 1 vol%, 3 vol%, 5 vol%, 10 vol%, or 20 vol%) were added to sterile centrifuge tubes, i.e., a volume ratio of 5 mL:0.2 mL = 25:1 were used to construct the system; in the control group, 5 mL of calibrated bacterial culture and 200 μL of sterile culture medium (used to replace the fermentation broth to eliminate the influence of the culture medium itself) were added to sterile centrifuge tubes, and gently mixed. Three parallel samples were set up for each group to avoid experimental errors; the samples were incubated on an aerobic, 37°C shaker, and samples were taken after 120 min of incubation; immediately after sampling, the OD was measured. 600 Measure the absorbance at the specified wavelength and record the OD value of each parallel sample (mix the bacterial solution gently before measurement to avoid precipitation affecting the results); calculate the inhibition rate.

[0086] Antibacterial rate (%) = [(average OD value of control group - average OD value of sample group) / average OD value of control group] ×100%.

[0087] 3. The inhibition rate of Sanguisorba officinalis against Propionibacterium acnes was tested using conventional methods in the field. The method included: dissolving 13.5 g of enhanced carboxylic acid bacteria culture medium in 1 L of water to prepare the culture medium; inoculating 100 μL of bacterial culture from a cryopreservation tube into 5 mL of the corresponding liquid culture medium and anaerobically incubating it at 37℃ for 36 ± 12 h; transferring the activated bacterial solution to fresh culture medium at a 1:100 ratio and repeating the incubation once to obtain the logarithmic-phase bacterial strain (to ensure the stability of the antibacterial experiment); diluting the logarithmic-phase bacterial solution with the corresponding culture medium and incubating for 2.5 ± 0.5 h before measuring the OD value.600 The absorbance was measured at a wavelength to make its OD 600 =0.5±0.05 (corresponding to a bacterial count of approximately 10) 6 -10 7 CFU / mL); In the experimental group, 5 mL of calibrated bacterial solution and 200 μL of different concentrations of Sanguisorba officinalis ferment (0.5 vol%, 1 vol%, 2 vol%, 4 vol%, or 6 vol%) were added to sterile centrifuge tubes to obtain sterile Sanguisorba officinalis ferment, i.e., a system was constructed at a volume ratio of 5 mL:0.2 mL = 25:1; In the control group, 5 mL of calibrated bacterial solution and 200 μL of sterile culture medium (used to replace the fermentation broth to eliminate the influence of the culture medium itself) were added to sterile centrifuge tubes and gently mixed. Three parallel samples were set up for each group to avoid experimental error; the samples were cultured on an aerobic, 37°C shaker, and samples were taken after 24 h of culture; immediately after sampling, the samples were analyzed by OD. 600 Measure the absorbance at the specified wavelength and record the OD value of each parallel sample group (gently mix the bacterial solution before measurement to avoid precipitation affecting the results); calculate the inhibition rate. Inhibition rate (%) = [(average OD value of control group - average OD value of sample group) / average OD value of control group] × 100%.

[0088] Antibacterial efficacy test results are as follows Figure 2 As shown, where, Figure 2 In this context, A represents the test result for Staphylococcus aureus. Figure 2 The B in the figure represents the test result for Malassezia. Figure 2 The "C" in the figure represents the test results for Propionibacterium acnes. The results show that the Sanguisorba officinalis ferment filtrate inhibited the growth of Staphylococcus aureus, Malassezia, and Propionibacterium acnes in a concentration-dependent manner. Specifically, after 2 hours of treatment with Sanguisorba officinalis ferment filtrate against Staphylococcus aureus and Malassezia, a concentration of approximately 5% (50 mg / ml) achieved the half-inhibition concentration against these bacteria. After 24 hours of treatment with Sanguisorba officinalis ferment filtrate, a concentration of approximately 2% (20 mg / ml) achieved the half-inhibition effect against Propionibacterium acnes. In summary, Sanguisorba officinalis ferment filtrate exhibits certain initial inhibitory effects against Staphylococcus aureus, Malassezia, and Propionibacterium acnes, indicating its good antibacterial effect.

[0089] Test Example 3: Evaluation of Soothing and Repairing Efficacy Hyaluronidase regulates the extracellular matrix by degrading hyaluronic acid (HA), exhibiting multiple roles in skin repair, including accelerating healing, soothing, and promoting collagen production. TNF-α (tumor necrosis factor-α) is an important pro-inflammatory cytokine closely related to skin sensitivity. When the skin comes into contact with irritants or allergens, immune cells are activated and release various inflammatory mediators, including TNF-α. TNF-α increases vascular permeability, attracting more immune cells to the site of inflammation, further exacerbating the inflammatory response and leading to increased skin sensitivity. Furthermore, TNF-α can also act on nerve endings, synergistically interacting with neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) to cause symptoms such as itching. LPS-induced RAW264.7 is a classic cell model for studying inflammatory factors. By comparing the difference in TNF-α secretion levels in RAW264.7 cells after administration of the test substance to negative controls and RAW264.7 cells, the soothing effect of the raw material on cells was evaluated. This test case evaluated the soothing effect of Sanguisorba officinalis ferment on hyaluronidase inhibition rate, keratinocyte repair capacity, and inflammatory factor release levels at both biochemical and cellular levels. Cellular repair capacity is crucial for skin repair; this test case used a cell scratch assay to reflect the cell repair or migration capacity.

[0090] 1. The effect on hyaluronidase was tested using conventional methods in the art, including: adding hyaluronidase, sodium hyaluronate, and a buffer solution to the reaction system to ensure suitable pH and temperature; establishing a blank control group (without hyaluronidase) and a negative control group (without sample); adding a pre-determined concentration of *Sanguisorba officinalis* fermentation solution to the reaction system, making the concentration of *Sanguisorba officinalis* fermentation solution in the reaction system 1 mg / mL, 10 mg / mL, or 50 mg / mL, respectively, to ensure sufficient contact between the sample and hyaluronidase; incubating the reaction system at 37°C for 30 min to allow sufficient reaction between hyaluronidase and hyaluronic acid; adding acetylacetone and p-dimethylaminobenzaldehyde for a colorimetric reaction under alkaline conditions; measuring the absorbance of the reaction system at 530 nm using a UV spectrophotometer; calculating the hyaluronidase inhibition rate; and comparing the hyaluronidase inhibition rate of the sample group with that of the negative control group to determine the soothing efficacy of different concentrations of *Sanguisorba officinalis* fermentation. If the hyaluronidase inhibition rate of the sample is significantly higher than that of the negative control group (P<0.05), the sample is considered to have anti-allergic activity.

[0091] Hyaluronidase inhibition rate (%) = { 1 – (B1-B2) / (A1-A2)}×100%, where: A1 is the absorbance of the reaction solution without sample; A2 is the absorbance of the reaction solution without sample and enzyme; B1 is the absorbance of the reaction solution containing sample and enzyme; B2 is the absorbance of the reaction solution containing sample and without enzyme.

[0092] 2. The effect on cell viability was tested using conventional methods in the field, including: selecting Hacat cells in good growth condition and in the logarithmic growth phase, digesting them with trypsin, centrifuging them, discarding the supernatant, and gently pipetting the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension; diluting the cell suspension to a concentration of 4 × 10⁻⁶. 4 Cells were seeded at a density of 1 / mL into 96-well plates (with sterile PBS in the edge wells), and three replicates were set up. After the cells had fully adhered to the plate and entered the logarithmic growth phase, the control group was replaced with fresh culture medium, while the experimental groups were added with 200 μL of culture medium containing different concentrations of Sanguisorba officinalis fermentation material, and cultured for another 24 h. After the culture was completed, 10% CCK-8 solution was added to each well for another 2.5 ± 0.5 h. The absorbance (OD value) of each well was measured using 490 nm as the detection wavelength, and the cell viability was calculated.

[0093] Cell viability (%) = (OD experimental group - OD zero-adjustment group) / (OD blank control group - OD zero-adjustment group) × 100%. Results are as follows: Figure 3 As shown in B, the Sanguisorba officinalis ferment broth at concentrations of 0.1–6 mg / mL showed no cytotoxicity to Hacat cells, while 8 mg / mL produced slight cytotoxicity. In contrast, 1 mg / mL significantly promoted cell viability. Therefore, a concentration of 1 mg / mL of Sanguisorba officinalis ferment broth was selected for further research.

[0094] 3. The effect on cell repair capacity was tested using conventional methods in the field, including: selecting cells in good growth condition, digesting with trypsin, preparing a cell suspension, and counting cells. (The cell count was performed at a concentration of 1×10⁻⁶ cells / cells). 5 Cells were seeded at a density of 100 cells / well in 24-well plates (the back of the 24-well plates were marked with a pencil for easy photography), shaken to mix, and placed in an incubator. The next day, when the cell growth density reached 80-90%, the culture medium was removed, and each well was scratched with a 200 μL sterile pipette tip, ensuring that the scratch area and area in each well were as consistent as possible. The cells were washed three times with PBS buffer to remove floating cells. The scratched area of ​​each well was randomly selected for photography, i.e., the scratch width at 0 hours, and the photography position was recorded. Cell culture medium containing 1% serum was added to the control group (CTR), and cell culture medium containing 1% serum and Sanguisorba officinalis ferment broth were added to the sample group. The concentration of Sanguisorba officinalis ferment broth in the sample group was 1 mg / mL. Each group had 3 replicates. After 20 hours of treatment, photos were taken at the original positions, and the migration rate was calculated.

[0095] Migration rate = (0-hour scratch width - 20-hour scratch width) / 0-hour scratch width × 100%.

[0096] 4. The effect of Sanguisorba officinalis fermentation product on inflammatory factors was tested using conventional methods in this field. The methods included: selecting RAW264.7 cells in good growth condition and in the logarithmic growth phase, digesting them with trypsin, centrifuging, discarding the supernatant, and gently pipetting the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension; thoroughly mixing the cell suspension, taking 10 μL for cell counting using a hemocytometer, and diluting the cell suspension to a concentration of 4 × 10⁻⁶. 4 Cells / mL; mix the cell suspension thoroughly, then seed it into six-well plates, setting up three replicates for each concentration gradient. Note that after adding a few wells, the cells should be resuspended by pipetting. The next day, discard the old culture medium, add 1 mg / mL of Sanguisorba officinalis ferment to the sample group, and replace the culture medium with fresh medium for the LPS and CTR groups. Incubate in a cell culture incubator for 24 h. After 24 h, discard the original culture medium, add 20 μL (1 μg / mL) of LPS to the sample group and LPS group, and incubate at 37°C for 2 h. After incubation, perform the experiment using an ELISA kit. Remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 min, seal the remaining strips in a self-sealing bag and return to 4°C. Set up standard wells and sample wells. Add 50 μL of different concentrations of standard to each standard well. Add 10 μL of the sample to be tested to the sample well, followed by 40 μL of sample diluent (i.e., sample diluted 5 times). Do not add anything to the blank wells. Except for the blank wells, add 10 μL of the sample to be tested to each standard well and 40 μL of sample diluent to the sample wells. Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well, seal the wells with sealing film, and incubate at 37°C in a water bath or incubator for 60 min. Discard the liquid, pat dry on absorbent paper, fill each well with washing buffer, let stand for 1 min, discard the washing buffer, pat dry on absorbent paper, and repeat this washing process 5 times. Add 50 μL each of substrate A and B to each well, and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm within 15 min.

[0097] Figure 3 This indicates the effect of Sanguisorba officinalis ferment on soothing and repair. Figure 3 Figure A in the figure shows its effect on hyaluronidase. The results show that the fermented product of Sanguisorba officinalis inhibits the activity of hyaluronidase in a concentration-dependent manner, and 10 mg / mL of fermented product of Sanguisorba officinalis reaches the half-maximal inhibitory concentration. Figure 3 Figure B in the figure shows the effect of different concentrations of Sanguisorba officinalis ferment on cell viability. The results showed that the Sanguisorba officinalis ferment at concentrations of 0.1-6 mg / mL had no cytotoxicity to Hacat cells, 8 mg / mL of Sanguisorba officinalis ferment produced slight cytotoxicity, while 1 mg / mL of Sanguisorba officinalis ferment significantly promoted cell viability. Therefore, a concentration of 1 mg / mL of Sanguisorba officinalis ferment was selected for subsequent studies. Figure 3Figure C in the figure represents the effect of Sanguisorba officinalis ferment on cell repair ability. The results show that, compared with the control group (CTR), the 1 mg / mL Sanguisorba officinalis ferment treatment group significantly promoted cell wound healing after 24 h, and had a good repair effect. Figure 3 The D diagram in the middle is Figure 3 Statistical analysis of the results in Figure C; Figure 3 Figure E in the figure shows the effect of Sanguisorba officinalis fermentation product on the inflammatory factor TNF-α. Figure 3 Figure F in the figure shows the effect of Sanguisorba officinalis fermentation product on IL-1. Figure 3 Figure G in the figure shows the effect of Sanguisorba officinalis ferment on IL-6. The results show that, compared with the blank control group (CTR), the addition of LPS significantly promoted the release of inflammatory factors TNF-α, IL-1 and IL-6. The addition of Sanguisorba officinalis ferment (1 mg / mL) could reverse the upregulated expression of inflammatory factors TNF-α, IL-1 and IL-6 stimulated by LPS, indicating that Sanguisorba officinalis ferment can significantly inhibit the inflammatory response induced by LPS and has a significant soothing effect.

[0098] Test Example 4: Evaluation of Oil Control and Whitening Efficacy 1. The role of 5-alpha reductase in acne development is primarily manifested in promoting sebum secretion, a crucial process. 5-alpha reductase (especially type I) converts testosterone into the more potent DHT in sebaceous glands. DHT binds to androgen receptors, strongly stimulating sebaceous gland cell proliferation and sebum secretion. Multiple studies have found that DHT levels in acne patients (both male and female) are significantly higher than in those without acne, suggesting that enhanced 5-alpha reductase activity may be one of the intrinsic driving factors of acne development. The oil-controlling effect of the raw material was evaluated by assessing its impact on 5-alpha reductase activity in Hacat cells.

[0099] The conventional method in this art is employed, comprising: selecting Hacat cells in good growth condition and in the logarithmic growth phase; digesting them with trypsin and centrifuging them, discarding the supernatant; gently pipetting the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension; thoroughly mixing the cell suspension; taking 10 μL for cell counting using a hemocytometer; and diluting the cell suspension to a concentration of 4 × 10⁻⁶. 5Cells / mL; mix the cell suspension thoroughly, then seed it into six-well plates, setting up three replicates for each concentration gradient. Note that after adding several wells, the cells should be resuspended by pipetting. The next day, discard the old culture medium, replace the CTR group with fresh culture medium, and add different concentrations of prepared Sanguisorba officinalis ferment broth to the sample group. Continue culturing in a cell culture incubator for 24 hours. After 24 hours of treatment, discard the original culture medium, wash several times with PBS, extract the cells to obtain the precipitate, and use sonication to disrupt the cells and obtain the cell supernatant. After incubation, perform the experiment using an ELISA kit. Remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a self-sealing bag and return them to 4℃. Set up standard wells and sample wells. Add 50 μL of different concentrations of standard to each standard well. Add 10 μL of the sample to be tested to each sample well, followed by 40 μL of sample diluent. Do not add any to the blank wells. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each standard and sample well. Add 50 μL of washing buffer to each well, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 60 min. Discard the liquid, pat dry on absorbent paper, add washing buffer to each well, let stand for 5 min, discard the washing buffer, pat dry on absorbent paper, and repeat this washing process 5 times (or a plate washer can be used). Add 50 μL each of substrate A and B to each well, and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well, and within 15 min, measure the OD value of each well at a wavelength of 450 nm.

[0100] 2. Post-inflammatory hyperpigmentation is also a skin care concern. In order to investigate whether Sanguisorba officinalis ferment can inhibit post-inflammatory hyperpigmentation, the effects of Sanguisorba officinalis ferment on tyrosinase and melanin production were tested.

[0101] The method for testing the effect of Sanguisorba officinalis fermentation product on tyrosinase includes the following steps: A1 control group: 800 μL of L-tyrosine solution and 1500 μL of PBS (pH=6.8) buffer were added; A0 blank group: 800 μL of L-tyrosine solution and 2500 μL of PBS (pH=6.8) buffer were added; B1 sample group: 800 μL of L-tyrosine solution, 800 μL of sample, and 700 μL of PBS (pH=6.8) buffer were added; B0 blank group: 800 μL of L-tyrosine solution, 800 μL of sample, and 1700 μL of PBS were added. (pH=6.8) buffer solution was used. After mixing all systems, the mixture was heated in a 37°C water bath for 10 min. At the same time, the tyrosinase was preheated in a 37°C water bath for 10 min. After the water bath heating was completed, 1000 μL of tyrosinase solution was added to each of the A1 control group and the B1 sample group. After mixing, the mixture was heated in a 37°C water bath for 40 min. After the reaction was completed, each experimental group was quickly transferred to a 96-well plate, and the absorbance (OD) was immediately measured at 475 nm using a microplate reader. The tyrosinase activity inhibition rate was calculated.

[0102] Formula for tyrosinase activity inhibition rate: Inhibition rate = ((A1 control group - A0 blank group) - (B1 sample group - B0 sample blank group)) / (A1 control group - A0 blank group).

[0103] 3. The CCK-8 assay method for detecting the effect of Sanguisorba officinalis fermentation on melanoma cell viability includes the following steps: selecting B16 cells in good growth condition and logarithmic growth phase, digesting them with trypsin, centrifuging and discarding the supernatant, gently pipetting the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension; diluting the cell suspension to a concentration of 4 × 10⁻⁶. 4 Cells were seeded at a density of 1 / mL into 96-well plates, with sterile PBS used in the edge wells. Three replicates were set up. After the cells had fully adhered to the plate and entered the logarithmic growth phase, the control group was replaced with fresh culture medium, while the experimental groups were added with 200 μL of culture medium containing different concentrations of Sanguisorba officinalis fermentation material, and cultured for another 24 h. After the culture was completed, 10% CCK-8 solution was added to each well for another 2.5 ± 0.5 h. The absorbance (OD value) of each well was measured using 490 nm as the detection wavelength, and the cell viability was calculated.

[0104] Cell viability (%) = (OD experimental group - OD zero-adjustment group) / (OD blank control group - OD zero-adjustment group) ×100%.

[0105] 4. Test method for the inhibition of melanin production by Sanguisorba officinalis fermentation product: The steps include: selecting B16 cells in good growth condition and in the logarithmic growth phase, digesting them with trypsin, centrifuging and discarding the supernatant, gently pipetting the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension; diluting the cell suspension to a concentration of 4 × 10⁻⁶.4 Cells were seeded at a density of 100 cells / mL into 96-well plates, with sterile PBS used in the edge wells. Three replicates were set up. After cell adhesion, the control group was replaced with fresh culture medium, while the experimental groups were cultured with medium containing 1, 5, and 10 mg / mL of Sanguisorba officinalis ferment filtrate, and cultured for 22 ± 2 h. After treatment, the cells were washed twice with PBS, digested with trypsin, and centrifuged at 4000 rpm for 5 min to obtain cell pellet. 200 μL of melanin extraction solution was added to each tube, shaken well, and heated in an 80°C water bath for 1 h. After cooling, the droplets on the tube wall were centrifuged moderately, and the mixture was blown around to mix. 150 μL of the solution from each centrifuge tube was transferred to a 96-well plate, and the absorbance at 405 nm was measured using a microplate reader. The melanin synthesis inhibition rate was calculated.

[0106] Inhibition rate (%) = (CT) / C × 100%; C - blank control group, T - sample group; Figure 4 The image shown illustrates the oil-controlling and skin-whitening effects of fermented Sanguisorba officinalis extract. Figure 4 The results in Figure A show that the fermented product of Sanguisorba officinalis inhibits the activity of 5α-reductase in a concentration-dependent manner, and has a significant inhibitory effect on 5α-reductase at concentrations of 1, 3 and 5 mg / mL, indicating that the fermented product of Sanguisorba officinalis has a certain oil-controlling effect. Figure 4 Figure B shows that the fermented product of Sanguisorba officinalis inhibited the activity of tyrosinase in a concentration-dependent manner. The inhibition rate of tyrosinase by 30 mg / mL of fermented product of Sanguisorba officinalis was approximately ±50%, and the inhibition rate of tyrosinase by 50 mg / mL of fermented product of Sanguisorba officinalis was approximately ±80%. This indicates that the fermented product of Sanguisorba officinalis can inhibit the production of melanin by inhibiting the enzyme activity of tyrosine. Figure 4 The results in Figure C showed that 1-10 mg / mL of Sanguisorba officinalis ferment had no significant effect on B16 cell viability. Further investigation was conducted on the effects of 1, 5, and 10 mg / mL Sanguisorba officinalis ferment on melanin production, and the results are as follows... Figure 4 Figure D shows that Sanguisorba officinalis ferment precipitate inhibits melanin production in a concentration-dependent manner, indicating that Sanguisorba officinalis ferment precipitate has a certain whitening effect. Sanguisorba officinalis ferment precipitate can alleviate post-inflammatory pigmentation by inhibiting melanin deposition.

[0107] Test Example 5: Evaluation of antioxidant efficacy at biochemical and cellular levels Extrinsic skin aging is primarily caused by photoaging, which leads to the excessive accumulation of free radicals within cells, inducing oxidative stress and ultimately resulting in skin cell aging. ROS (reactive oxygen species) is a collective term for the "reactive oxygen species family," which includes both true free radicals (such as O2) and... 2- ·, ·OH and RO2·), and also some non-free radical but highly reactive oxygen-containing molecules (such as H2O2, O 2-ONOO - Prolonged skin exposure to ultraviolet radiation (especially UVA / UVB) and pollution can induce significant ROS / free radical generation. This test case biochemically analyzed the effects of this raw material on DPPH and O2. 2- The ability of the raw material to scavenge ·OH free radicals was then assessed, and its ability to scavenge ROS was further tested at the cellular level.

[0108] 1. The DPPH scavenging rate was tested using conventional methods in the field, including: preparing 0.1 mM DPPH solutions and 1, 5, 10, 20, 50, and 100 mg / mL solutions of Sanguisorba officinalis ferment; setting up sample groups, control groups, and blank groups; the sample groups consisted of 180 µL and 90 µL of Sanguisorba officinalis ferment in solution of different concentrations, resulting in concentrations of 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, or 100 mg / mL, respectively; the control group consisted of 180 µL of DMSO and 90 µL of DPPH; and the blank group consisted of 180 µL and 90 µL of Sanguisorba officinalis ferment in solution of different concentrations in anhydrous ethanol; the samples were shaken in the dark for 10 min, and the atmosphere was zeroed with anhydrous ethanol; the absorbance (A) was measured at 520 nm, and the data were recorded and analyzed.

[0109] DPPH clearance rate % = A control group - (A sample group - A blank group) / A control group × 100%.

[0110] 2. The scavenging rate of •OH was tested using conventional methods in the field. The method included: preparing solutions of 9 mmol / L ethanol-salicylic acid, 9 mmol / L ferrous sulfate, 8.8 mmol / L H2O2, and one of the following concentrations of Sanguisorba officinalis fermentation product: 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, and 100 mg / mL; the reaction system was carried out in a 1.5 mL colorimetric tube, and the order of sample addition was shown in Table 1; the mixture was shaken well, incubated in a water bath at 37°C for 15 min, and the absorbance A was measured; the data were recorded and analyzed.

[0111] ·OH removal rate % = [A0 - (A X -A X0 )] / A0×100%;A X The absorbance of the sample was measured, with A0 serving as a blank control. X0 Background for the sample.

[0112] Table 1. Sample addition sequence for determining hydroxyl radical scavenging ability using the ethanol-salicylic acid method.

[0113] 3. For O 2-The scavenging rate was tested using conventional methods in the art, which included: preparing solutions of 5 mmol / L pyrogallol, 0.1 mol / L Tris-HCl, and one of the following concentrations of Sanguisorba officinalis fermentation product: 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, and 100 mg / mL; the reaction was carried out in a cuvette, and the order of sample addition was shown in Table 2; after rapid mixing and shaking, the absorbance of the solution was measured at 325 nm as the first absorbance value, and measurements were taken every 1 minute for 4 minutes. The increase in absorbance per minute within the linear range was calculated, and the data were recorded and analyzed.

[0114] O 2- Scavenging rate (%) = (△A0 - △A) / △A0 × 100%; △A0 is the auto-oxidation rate of pyrogallol; △A is the auto-oxidation rate of pyrogallol after the sample solution is added.

[0115] Table 2. Sampling order for the determination of superoxide anion scavenging using the pyrogallol method.

[0116] 4. The effect on ROS scavenging ability was tested using conventional methods in the field. The methods included: Hacat cells were seeded at an appropriate density in six-well plates, and after cell adhesion, they were treated; the experimental group was treated with 200µM H2O2 for 2.5±0.5h, followed by washing three times with PBS; the control group was treated with fresh culture medium, while the experimental group was treated with 1mg / ml Sanguisorba officinalis fermentation solution for 24h, followed by ROS detection; the DCFH-DA probe was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10µM; the original culture medium was discarded, and an appropriate volume of the diluted DCFH-DA probe was added, and the cells were incubated at 37℃ for 20min; the cells were washed three times with serum-free cell culture medium to thoroughly remove any DCFH-DA probe that had not entered the cells; and the cells were observed under a fluorescence microscope.

[0117] The results are as follows Figure 5 As shown, Figure 5 In this context, A indicates that the fermentation product of Sanguisorba officinalis inhibits DPPH in a dose-dependent manner. Figure 5 In this context, B indicates that the fermentation product of Sanguisorba officinalis inhibits O in a dose-dependent manner. 2- The generation, Figure 5 The C in the figure indicates that the Sanguisorba officinalis ferment broth inhibits the production of •OH free radicals in a dose-dependent manner. Specifically, the Sanguisorba officinalis ferment broth achieves its effect on DPPH and O2 free radicals at a concentration of 10 mg / mL. 2- The half-maximal inhibitory concentration (WMC) of *Sanguisorba officinalis* ferment at 50 mg / mL for its effect on DPPH and O2. 2- The scavenging rates of OH radicals reached 85%, 80%, and 78%, respectively. Figure 5 The D value in the figure (white bar size is 100 μm) shows that, compared with the control group, H2O2 treatment of Hacat cells significantly promoted ROS accumulation, while the addition of Sanguisorba officinalis ferment significantly inhibited ROS production. These results indicate that Sanguisorba officinalis ferment has a good antioxidant effect.

[0118] The fermented products of Sanguisorba officinalis prepared in Example 3 (referred to as Y17), Example 4 (referred to as JM2-16), and Example 5 (referred to as JM4-20) were tested according to the methods of Test Examples 2 to 5. The test results are as follows: Fermentation products Y17, JM2-16, and JM4-20 all inhibited the growth of Staphylococcus aureus, Malassezia, and Propionibacterium acnes in a concentration-dependent manner. At 50 mg / mL, the inhibition rates of Y17, JM2-16, and JM4-20 fermentation products against Staphylococcus aureus were 30%, 25%, and 45%, respectively; at 50 mg / mL, the inhibition rates against Malassezia were 34%, 30%, and 48%, respectively; and at 20 mg / mL, the inhibition rates against Propionibacterium acnes were approximately 38%, 40%, and 42%, respectively, indicating that they all have good antibacterial effects.

[0119] Y17 fermentation product inhibits hyaluronidase activity in a concentration-dependent manner, with an inhibition rate of approximately 65% ​​at 50 mg / mL. 1 mg / mL of Y17 fermentation product reversed the increase in TNF-α, IL-1, and IL-6 levels induced by LPS stimulation with inhibition rates of 30%, 25%, and 20%, respectively, indicating that Y17 fermentation product also has a soothing effect.

[0120] JM2-16 ferment broth inhibits tyrosinase activity in a concentration-dependent manner. At 50 mg / mL, the inhibition rate of JM2-16 ferment broth against tyrosinase is approximately 60%. At 10 mg / mL, the ferment broth can significantly inhibit melanin production, with an inhibition rate of approximately 35%, indicating that it has good whitening effects.

[0121] JM4-20 fermentation product inhibits DPPH and O in a concentration-dependent manner. 2- The formation of OH- and •OH, at 100 mg / mL, JM4-20 fermentation product has an effect on DPPH and O2. 2- The inhibition rates of α, β, and OH were 84%, 75%, and 70%, respectively, indicating that the JM4-20 ferment has good antioxidant effects.

[0122] This invention demonstrates that the fermented product of Sanguisorba officinalis prepared by Saccharomyces cerevisiae (Y17) has antibacterial and soothing effects; the fermented product of Sanguisorba officinalis prepared by Schizosaccharomyces cerevisiae JM-16 has antibacterial and whitening effects; and the fermented product of Sanguisorba officinalis prepared by Pichia pastoris JM4-20 has antibacterial and antioxidant effects.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a fermented product of Sanguisorba officinalis that controls oil, removes acne, inhibits bacteria, and repairs the skin barrier, characterized in that, Includes the following steps: The material derived from Sanguisorba officinalis was mixed with yeast extract peptone glucose medium at a mass ratio of (10~15):(7~10) to prepare the culture medium. Inoculate yeast onto the culture medium at a total inoculum amount of 1-1.5% and ferment at 30-35°C for 44-52 hours. The fermentation product was centrifuged and the supernatant was collected to obtain the Sanguisorba officinalis fermentation product; The fermentation bacteria are Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, or Pichia pastoris. The *Schizophyllum commune* strain is *Schizophyllum commune* JM2-16 (… Schizosaccharomyces pombe JM2-16), with accession number M 20242810; The Pichia pastoris strain is Pichia pastoris JM4-20 ( Pichia kudriavzevii JM4-20 was deposited on December 13, 2024, at the China Center for Type Culture Collection, located in Wuhan, China, with accession number M 20242811.

2. The preparation method of the Sanguisorba officinalis ferment as described in claim 1, characterized in that, Materials derived from Sanguisorba officinalis are selected from dried Sanguisorba officinalis or Sanguisorba officinalis extract.

3. The method for preparing the Sanguisorba officinalis ferment as described in claim 1, characterized in that, The culture medium was prepared by mixing the material derived from Sanguisorba officinalis with yeast extract peptone glucose medium and adding 10-15 times the weight of water.

4. The preparation method of the Sanguisorba officinalis ferment as described in claim 1, characterized in that, The culture medium is sterilized by autoclaving.

5. The method for preparing the Sanguisorba officinalis ferment as described in claim 1, characterized in that, The fermentation process involves sealing the container with multiple layers of gauze and shaking it at 160-180 rpm.

6. The method for preparing the Sanguisorba officinalis ferment as described in claim 1, characterized in that, After being sterilized by high-pressure steam, the fermentation products were centrifuged at 10,000-12,000 rpm for 8-12 minutes.

7. The preparation method of the Sanguisorba officinalis ferment as described in claim 1, characterized in that, The supernatant was then filtered.

8. The method for preparing the Sanguisorba officinalis ferment as described in claim 1, characterized in that, The process also includes the following steps: decolorizing the Sanguisorba officinalis ferment with activated carbon, centrifuging the activated carbon to obtain a second supernatant, and filtering the second supernatant with a filter membrane.

9. The Sanguisorba officinalis fermented product prepared by a method for preparing the Sanguisorba officinalis fermented product with oil control, acne removal, antibacterial and barrier repair properties as described in any one of claims 1-8.

10. The use of the Sanguisorba officinalis ferment as described in claim 9 in the preparation of acne-reducing and anti-inflammatory drugs or cosmetics.

Images