A soothing and repairing gentiana scabra extract composition, and a preparation method and application thereof

Abstract

This invention discloses a soothing and repairing Gentiana macrophylla extract composition, its preparation method, and its application, specifically belonging to the technical field of daily necessities raw material preparation. The Gentiana macrophylla extract composition is obtained by solvent extraction after solid-state fermentation of Gentiana macrophylla by Aspergillus oryzae, Leuconostoc mesenteroides, and Saccharomyces cerevisiae. The Gentiana macrophylla extract composition provided by this invention has a high oleanolic acid content, proving that Aspergillus oryzae, Leuconostoc mesenteroides, and Saccharomyces cerevisiae can effectively enrich the active ingredients in Gentiana macrophylla, and at the same time, they exhibit a certain synergistic effect on the enrichment of active ingredients during the fermentation process. The Gentiana macrophylla extract composition provided by this invention can effectively inhibit the release of inflammatory factors and promote skin repair.

Description

Technical Field

[0001] This invention belongs to the technical field of raw material preparation for daily necessities, specifically relating to a soothing and repairing Gentiana macrophylla extract composition, its preparation method, and its application. Background Technology

[0002] Gentiana macrophylla, a traditional Chinese medicinal herb, is rich in various active ingredients, among which oleanolic acid is one of the key substances that exerts its anti-inflammatory, antioxidant, and immunomodulatory effects. Studies have shown that oleanolic acid can effectively alleviate skin inflammation and promote the repair of skin barrier function by inhibiting the release of inflammatory factors (such as TNF-α and IL-6) and regulating the NF-κB signaling pathway. However, the active ingredients such as oleanolic acid in Gentiana macrophylla exist mostly in bound or large molecular complex forms. Traditional water or alcohol extraction processes are difficult to fully release their free forms, resulting in low extraction efficiency and limited bioavailability, which seriously restricts its application potential in functional skin care products.

[0003] In recent years, microbial fermentation technology has attracted much attention due to its ability to transform plant substrates through enzymatic hydrolysis and improve the bioavailability of active ingredients. Existing research shows that fermentation with a single strain (such as Aspergillus oryzae or Lactobacillus) can partially enhance the efficacy of plant extracts, but it suffers from drawbacks such as the single metabolite and insufficient synergistic effect. Especially in solid-state fermentation systems, the metabolic synergistic mechanism of complex microbial communities has not been fully explored, and how to optimize the fermentation process through multi-strain compatibility to enhance the release efficiency of active ingredients remains a technical challenge. In addition, the lack of systematic optimization of key parameters such as temperature and humidity control and nutrient substrate ratio in traditional fermentation processes leads to poor stability of fermentation products and a high loss rate of active ingredients.

[0004] In the field of skincare product development, while chemically synthesized soothing ingredients offer rapid results, they pose a risk of allergic reactions. Meanwhile, existing natural plant extracts generally suffer from low transdermal absorption efficiency and insufficient duration of efficacy. Therefore, developing a novel Gentiana macrophylla extraction process based on multi-strain synergistic fermentation, which can increase the content of active ingredients while enhancing their skin compatibility, has become an important direction for the development of raw materials for functional skincare products. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a fermented extract of Gentiana macrophylla based on multi-strain synergistic fermentation. By optimizing the strain compatibility and fermentation process, the extraction yield of active ingredients such as oleanolic acid is improved, thereby enhancing the soothing, anti-inflammatory, and repairing effects of Gentiana macrophylla.

[0006] To achieve the above objectives, the present invention discloses the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a soothing and repairing Gentiana macrophylla extract composition, the preparation method comprising the following steps:

[0008] Step 1. Grind the dried Gentiana macrophylla into powder and sieve it to obtain Gentiana macrophylla powder;

[0009] Step 2. Mix Aspergillus oryzae liquid, Leuconostoc mesenteroides liquid and Saccharomyces cerevisiae liquid at a volume ratio of (2-2.5):1:(1-1.5) to obtain a compound bacterial solution;

[0010] Step 3. Take sterile water to moisten the Gentiana macrophylla powder obtained in Step 1, control the moisture content to 45-55%, mix evenly, and sterilize at 121℃ for 30 minutes to obtain solid fermentation powder for later use.

[0011] Step 4. Spray the compound bacterial solution obtained in Step 2 into the solid fermentation powder at an inoculation rate of 8-10 wt%, and ferment it for 4-5 days at a temperature of 30-37℃, a humidity of 70-75%, and an aeration rate of 0.2-0.4 L / min·kg to obtain the fermentation product of Gentiana macrophylla.

[0012] Step 5. Extract the fermentation product of Gentiana macrophylla by soaking in an ethanol-water solution. The extract is then concentrated under reduced pressure and dried to obtain the fermentation extract of Gentiana macrophylla.

[0013] Step 6. Mix the fermented extract of Gentiana macrophylla with the solvent at a mass ratio of 1:(5-50) at 300-500 r / min and at room temperature until homogeneous to obtain the Gentiana macrophylla extract composition;

[0014] The solvent is selected from at least one of vegetable oils, synthetic oils, and polyols.

[0015] Preferably, the vegetable oil is selected from at least one of olive oil, tea seed oil, jojoba seed oil, shea butter, and meadowfoam seed oil.

[0016] Preferably, the polyol is selected from at least one of butanediol, 1,3-propanediol, propylene glycol, glycerol, and 1,2-hexanediol.

[0017] Preferably, in step 1, the gentian root is pulverized and then passed through a 60-mesh sieve.

[0018] Preferably, in step 2, the Aspergillus oryzae culture is prepared by inoculating a spore suspension into a liquid medium containing 0.1% peptone PDA and culturing in a shake flask at 28°C and 120 r / min for 48-60 h to obtain the Aspergillus oryzae culture, wherein the spore concentration of the Aspergillus oryzae culture is 1×10⁻⁶. 8 -5×10 8 CFU / mL.

[0019] Preferably, in step 2, the preparation of *Leuconostoc mesenteroides* bacterial suspension involves: inoculating frozen bacterial strains into MRS liquid medium containing 0.5% maltose, and incubating at 37°C for 24-36 hours to obtain *Leuconostoc mesenteroides* bacterial suspension, wherein the viable count of *Leuconostoc mesenteroides* is 1×10⁻⁶. 8 -2×10 8 CFU / mL.

[0020] Preferably, in step 2, the preparation of the Saccharomyces cerevisiae culture involves: inoculating frozen inoculum into YPD liquid medium and culturing in a shake flask at 30°C and 150 r / min for 18-24 h to obtain the Saccharomyces cerevisiae culture, wherein the viable count of the Saccharomyces cerevisiae culture is 1×10⁻⁶. 8 -2×10 8 CFU / mL.

[0021] Preferably, in step 5, the fermented product of Gentiana macrophylla is mixed with a 60-70 v / v% ethanol aqueous solution at a ratio of 1:(8-10) g / mL, and ultrasonically assisted extraction is performed 2-3 times at 60-70℃ for 30-40 min each time. The combined extracts are filtered through a 120-200 mesh sieve, concentrated under reduced pressure at 50-60℃ to a paste state, and dried until the water content is ≤5% to obtain the fermented extract of Gentiana macrophylla.

[0022] In a second aspect, the present invention provides a soothing and repairing Gentiana macrophylla extract composition, wherein the Gentiana macrophylla extract composition is prepared by the preparation method described in the first aspect.

[0023] Thirdly, the present invention provides the application of the soothing and repairing Gentiana macrophylla extract composition described in the second aspect in the preparation of skin care products with repairing and soothing effects.

[0024] The beneficial effects of this invention are:

[0025] 1. The product obtained by extracting Gentiana macrophylla in this invention through a combination of Aspergillus oryzae, Leuconostoc mesenteroides and Saccharomyces cerevisiae has a high content of oleanolic acid, which proves that Aspergillus oryzae, Leuconostoc mesenteroides and Saccharomyces cerevisiae can effectively enrich the active ingredients in Gentiana macrophylla. At the same time, they have a certain synergistic effect on the enrichment of active ingredients during the fermentation process.

[0026] 2. The Gentiana macrophylla extract composition provided by this invention can effectively inhibit the release of inflammatory factors. Experimental verification shows that, compared with the negative control group, the 1%, 0.5%, and 0.1% concentrations of Gentiana macrophylla extract composition inhibited the release of TNF-α inflammatory factors in macrophages by 25.797%, 20.470%, and 12.923%, respectively.

[0027] 3. The Gentiana macrophylla extract composition provided by the present invention has a significant effect on promoting skin repair. Compared with the blank control group, the keratinocyte migration area of ​​the 1% Gentiana macrophylla extract composition increased significantly, with a migration rate of 18.909%, and the keratinocyte migration area of ​​the 0.5% Gentiana macrophylla extract composition increased significantly, with a migration rate of 11.576%. Attached Figure Description

[0028] Figure 1 This is a standard curve diagram of oleanolic acid.

[0029] Figure 2 HPLC chromatograms of oleanolic acid standards at concentrations of 200 μg / mL, 240 μg / mL, 300 μg / mL, 360 μg / mL, and 400 μg / mL;

[0030] Figure 3 The HPLC chromatogram of Gentiana macrophylla extract composition 2 is shown below.

[0031] Figure 4 Figure showing the effect of different samples on the content of TNF-α inflammatory factor in macrophages;

[0032] Figure 5 This figure shows the effect of different concentrations of samples on keratinocyte migration.

[0033] Figure 6 Cell migration diagrams of the blank control group at 0h (left) and 24h (right);

[0034] Figure 7 Cell migration diagrams for sample group 1 at 0h (left) and 24h (right);

[0035] Figure 8 Cell migration diagrams for sample group 2 at 0h (left) and 24h (right);

[0036] Figure 9 Cell migration diagrams for sample groups 0h (left) and 24h (right); Detailed Implementation

[0037] The present invention will be further described clearly and in detail below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, and not all, of the embodiments of the present invention.

[0038] Therefore, based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort should fall within the scope of protection of this invention. Unless otherwise specified, the raw materials used in the embodiments of this invention are commercially available or obtainable by those skilled in the art; unless otherwise specified, the methods used in the embodiments of this invention are methods mastered by those skilled in the art.

[0039] In this invention:

[0040] Aspergillus oryzae: accession number CGMCC: 3.13905, purchased from China General Microbiological Culture Collection Center;

[0041] Leuconostoc mesenteroides: accession number CGMCC: 1.2461, purchased from China General Microbiological Culture Collection Center;

[0042] Saccharomyces cerevisiae: CGMCC accession number: 2.1364, purchased from China General Microbiological Culture Collection Center;

[0043] PDA liquid culture medium: Take 200g of peeled potatoes, cut them into small pieces, add 1000ml of water and boil for 30 minutes. Filter out the potato pieces, add the filtrate to 1000ml, and add 20g of glucose.

[0044] MRS liquid culture medium: peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, Tween-80 1.0 mL / L, dipotassium hydrogen phosphate 2.0 g / L, sodium acetate 5.0 g / L, triammonium citrate 2.0 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·4H2O 0.05 g / L;

[0045] YPD liquid culture medium: peptone 20 g / L, glucose 20 g / L, yeast extract 10 g / L

[0046] Gentiana macrophylla extract composition

[0047] Step 1-1. Place the dried Gentiana macrophylla in a universal pulverizer and pulverize it. Pass it through a 60-mesh sieve. The amount of material on the sieve should be ≤0.1% to obtain Gentiana macrophylla powder for later use.

[0048] Step 1-2. Mix Aspergillus oryzae liquid, Leuconostoc mesenteroides liquid and Saccharomyces cerevisiae liquid in a volume ratio of (2-2.5):1:(1-1.5) to obtain a compound bacterial solution;

[0049] Preparation of Aspergillus oryzae suspension: Spore suspension was inoculated into liquid medium containing 0.1% peptone PDA and cultured in shake flasks at 28℃ and 120 r / min for 48-60 h to obtain Aspergillus oryzae suspension. The spore concentration of the Aspergillus oryzae suspension was 1×10⁻⁶. 8-5×10 8 CFU / mL;

[0050] Preparation of Leuconostoc mesenteroides suspension: Frozen bacterial culture was inoculated into MRS liquid medium containing 0.5% maltose and incubated at 37℃ for 24-36 hours to obtain Leuconostoc mesenteroides suspension, in which the viable count of Leuconostoc mesenteroides was 1×10⁻⁶. 8 -2×10 8 CFU / mL;

[0051] Preparation of Saccharomyces cerevisiae culture: Frozen inoculum was inoculated into YPD liquid medium and cultured in shake flasks at 30℃ and 150 r / min for 18-24 h to obtain Saccharomyces cerevisiae culture, wherein the viable count of the Saccharomyces cerevisiae culture was 1×10⁻⁶. 8 -2×10 8 CFU / mL;

[0052] Steps 1-3. Preparation of solid fermentation powder: Take sterile water to wet the Gentiana macrophylla powder, control the moisture content to 45-55%, and sterilize it with high pressure steam at 121℃ for 30 minutes to obtain solid fermentation powder;

[0053] Steps 1-4. Transfer the solid fermentation powder to a solid fermentation tank. Take the compound bacterial solution and spray it evenly onto the solid fermentation powder at an inoculation rate of 8-10 wt%. Start the fermentation tank agitator while spraying. After spraying, ferment and culture for 4-5 days at 30-37℃, 70-75% humidity, and 0.2-0.4 L / min·kg aeration rate. During the fermentation process, stir intermittently and turn the mixture over every 2 hours. After fermentation, the fermented product of Gentiana macrophylla is obtained.

[0054] Steps 1-5. Mix the fermented product of Gentiana macrophylla with 60-70 v / v% ethanol aqueous solution at a ratio of 1: (8-10) g / mL, and extract with ultrasonic assistance at 60-70℃ for 2-3 times, each time for 30-40 min. Combine the extracts and filter them through a 120-200 mesh sieve. Concentrate under reduced pressure at 50-60℃ to a paste-like consistency, and dry until the water content is ≤5% to obtain the fermented extract of Gentiana macrophylla.

[0055] Steps 1-6. Mix the fermented extract of Gentiana macrophylla with olive oil at a mass ratio of 1:(9-19) at 300-500 r / min and room temperature to obtain Gentiana macrophylla extract composition 1-3.

[0056] The specific preparation process parameters are shown in Table 1.

[0057] Table 1 Fermentation process parameters

[0058]

[0059]

[0060] To verify the difference between the Gentiana macrophylla extract composition prepared by the above method and compositions obtained by other methods, the following operations were performed:

[0061] Composition 4: The Aspergillus oryzae liquid is missing in steps 1-2, the volume ratio of the remaining liquid remains unchanged, and the rest is the same as the preparation method of Gentiana macrophylla extract composition 2;

[0062] Composition 5: The Leuconostoc mesenteroides bacterial solution is missing in steps 1-2, the volume ratio of the remaining bacterial solution remains unchanged, and the rest is the same as the preparation method of Gentiana macrophylla extract composition 2;

[0063] Composition 6: The brewer's yeast liquid is missing in steps 1-2, the volume ratio of the remaining liquid remains unchanged, and the rest is the same as the preparation method of Gentiana macrophylla extract composition 2;

[0064] Composition 7: The Leuconostoc mesenteroides culture in steps 1-2 is replaced with Lactobacillus plantarum culture, with the volume ratio of the culture remaining unchanged. The rest of the preparation method is the same as that of Gentiana macrophylla extract composition 2. The Lactobacillus plantarum was purchased from the China Center for Type Culture Collection, accession number CCTCC AB 2022276, and the strain was activated to a viable count of 1×10⁻⁶ using conventional activation methods. 8 CFU / mL;

[0065] Composition 8: Take dried Gentiana macrophylla root and place it in a universal pulverizer for pulverization. Pass it through a 60-mesh sieve to obtain Gentiana macrophylla powder. Mix the Gentiana macrophylla powder with a 65% v / v% ethanol aqueous solution at a material-liquid ratio of 1:9 g / mL. Extract the mixture three times at 65℃ with ultrasonic assistance, each time for 30 min. Combine the extracts and filter them through a 150-mesh sieve. Concentrate the extract under reduced pressure at 55℃ to a paste state. Dry the extract until the water content is ≤5% to obtain the extract. Mix the extract with olive oil at a mass ratio of 1:14 at 400 r / min and room temperature until homogeneous to obtain Composition 8.

[0066] Performance testing

[0067] 1. Oleanolic acid content detection

[0068] 1.1 Principle

[0069] The samples were analyzed by high performance liquid chromatography, with retention time used for qualitative analysis and external standard method for quantitative analysis.

[0070] 1.2 Main Instruments and Equipment

[0071] High-performance liquid chromatograph (HPLC) Thermo Fisher U3000, UV detector or diode array detector; ultrasonic cleaner; analytical balance; vortex mixer.

[0072] 1.3 Reagents

[0073] Unless otherwise specified, all reagents used are of analytical grade. Water is Grade I water as specified in GB / T 6682. Acetonitrile (chromatographic grade); methanol (chromatographic grade); oleanolic acid standard (HPLC): content not less than 98%.

[0074] 1.4 Measurement Procedure

[0075] 1.4.1 Preparation of stock solution for oleanolic acid standard curve

[0076] Accurately weigh 20 mg of oleanolic acid standard into a 50 mL brown volumetric flask, dissolve it in 40 mL of methanol, and then dilute to the mark with methanol to prepare a 400 μg / mL standard stock solution.

[0077] 1.4.2 Preparation of Oleanolic Acid Standard Working Solution

[0078] Pipette 5.0 mL, 6.0 mL, 7.5 mL, and 9.0 mL of the 400 μg / mL standard stock solution into 10 mL brown volumetric flasks, respectively, and dilute to the mark with methanol to form a series of standard solutions of 200 μg / mL, 240 μg / mL, 300 μg / mL, 360 μg / mL, and 400 μg / mL, together with the standard stock solution.

[0079] 1.5 Chromatographic conditions

[0080] The reference conditions for high performance liquid chromatography (HPLC) are as follows:

[0081] Chromatographic column: Hypersil GOLD C18 column (250mm × Ф4.6mm, 5um), or equivalent column;

[0082] Mobile phase: Acetonitrile: Water = 94:6;

[0083] Detection wavelength: 210nm;

[0084] Column temperature: 30℃;

[0085] Injection volume: 20 μL.

[0086] 1.6 System Adaptability

[0087] The theoretical plate number, calculated based on the oleanolic acid peak, should be no less than 5000.

[0088] 1.7 Sample Preparation

[0089] Accurately weigh 1 mL of each of the following compositions into stoppered colorimetric tubes: composition 2, composition 4, composition 5, composition 6, composition 7, and composition 8. Dilute the sample 5 times with methanol in each tube, vortex for 3 min, let stand for 10 min, extract the supernatant, filter through a 0.45 μm filter membrane, and use the filtrate as the sample solution to be tested.

[0090] 1.8 Measurement

[0091] Under the chromatographic conditions described in 1.5, standard solutions were injected separately according to section 1.4.2, and chromatograms were recorded. An oleanolic acid standard curve was plotted with the concentration of the standard solutions on the x-axis and the peak area on the y-axis. (See...) Figure 1 HPLC chromatograms of oleanolic acid standards (200 μg / mL, 240 μg / mL, 300 μg / mL, 360 μg / mL, 400 μg / mL) are shown below. Figure 2 .

[0092] The prepared sample solution was injected into 1.7, the chromatogram was recorded, and the peak area was measured. The content of oleanolic acid in the sample solution was obtained according to the standard curve. Then, the content of oleanolic acid in the composition was calculated according to 1.9. The results are shown in Table 2. The HPLC chromatogram of composition 2 is shown in Table 2. Figure 3 .

[0093] 1.9 Calculation Formula

[0094] The oleanolic acid content in the sample is calculated using the following formula:

[0095] X = 5 × Q

[0096] In the formula: Q: Oleanolic acid content in the sample solution, μg·mL -1 ;

[0097] 5: Dilution factor;

[0098] X: Oleanolic acid content of the corresponding composition, μg·mL -1 .

[0099] 1.10 Experimental Results

[0100] The peak areas and linear regression equations for each concentration of the standard are shown in the table below:

[0101]

[0102] Oleanolic acid content in the composition

[0103] Table 2. Oleanolic acid content in each group

[0104] Group <![CDATA[Oleanolic acid / μg·mL -1 > Composition 2 656.87 Composition 4 330.97 Composition 5 284.44 Composition 6 305.47 Composition 7 448.84 Composition 8 275.27

[0105] Results analysis:

[0106] According to the test results, it can be seen that the product obtained by compound fermentation of Gentiana macrophylla in the present invention with Aspergillus oryzae, Leuconostoc citreum and Saccharomyces cerevisiae and then extracted with an organic solvent has a high oleanolic acid content, which proves that Aspergillus oryzae, Leuconostoc citreum and Saccharomyces cerevisiae can effectively enrich the active ingredients in Gentiana macrophylla; Aspergillus oryzae, Leuconostoc citreum and Saccharomyces cerevisiae are missing in the preparation process of Compositions 4-6 respectively, resulting in a significant decrease in the oleanolic acid content of Compositions 4-6. At the same time, Lactobacillus plantarum was used to replace Leuconostoc citreum in the preparation process of Composition 7, and the result shows that the oleanolic acid content in Composition 7 is not higher than that in Composition 2, indicating that the selected strains Aspergillus oryzae, Leuconostoc citreum and Saccharomyces cerevisiae in the present invention play a key role in the fermentation process of Gentiana macrophylla, and they show a certain synergistic effect on the enrichment of active ingredients during the fermentation process.

[0107] 2 Animal irritation experiment

[0108] 2.1 Test animals: 4 ordinary New Zealand rabbits (female, 2290.95 - 2341.27 g), provided by the Guangdong Provincial Center for Medical Laboratory Animals (Sanshui Base), license number: SCXK(Yue)2019-0035, quality certificate number: 44411600011537.

[0109] 2.2 Feeding environment: Ordinary environment, temperature 20°C - 26°C, relative humidity 40% - 70%, license number: SYXK(Yue)2022-0194.

[0110] 2.3 Feed source: Guangdong Provincial Center for Medical Laboratory Animals, production license number: Yue Sizheng (2019)05073, production date: 2023-03-06.

[0111] 2.4 Sample preparation: Use the Gentiana macrophylla extraction Composition 2 prepared above.

[0112] 2.5 Test method: Approximately 24 hours before the test, shave the hair on both sides of the back spine of the test animals without damaging the epidermis, and the hair removal area is about 3 cm × 3 cm on the left and right. Take 0.5 mL of the test substance and apply it to the shaved skin on the left side with an area of 2.5 cm × 2.5 cm, and the skin on the other side is used as a control. Apply it once a day for 14 consecutive days. Starting from the second day, shave the hair before each application, remove the residual test substance with warm water, and observe the local skin reaction and score it 1 hour later.

[0113] 2.6 Test results:

[0114] Table 3 Results of irritation experiment

[0115]

[0116]

[0117] Experimental conclusion: The sample concentrate had an average score of 0 per animal per day in multiple skin irritation tests on New Zealand rabbits. According to the skin irritation intensity classification of the "Cosmetic Safety Technical Specifications" (2015 edition), it is non-irritating.

[0118] 3. Soothing Efficacy Test

[0119] 3.1 Test Basis

[0120] This experiment was designed and conducted in accordance with the standard "T / SHRH034-2021 Test of Cosmetic Soothing Efficacy - In Vitro TNF-α Inflammatory Factor Content Determination and Lipopolysaccharide-Induced Macrophage Inflammatory Cell Model Test Method".

[0121] 3.2 Experimental Objective

[0122] The product's soothing effects were explored by testing its ability to inhibit macrophage inflammatory factors.

[0123] 3.3 Experimental Principle

[0124] In daily life, skin irritants mainly fall into three categories: physical stimuli such as ultraviolet radiation, microbial infections, and chemical irritants such as hair dyes. Inflammation is a defensive response to exogenous or endogenous (harmful) stimuli, primarily involving the innate immune system. Skin symptoms include redness, swelling, and itching. Bacterial lipopolysaccharide (LPS) binds to antigen recognition receptors on the surface of macrophages, inducing macrophages to secrete various cytokines, including tumor necrosis factor-α (TNF-α). TNF-α can activate three signaling pathways: caspase, JNK, and NF-κB, enabling its biological functions such as apoptosis and cellular inflammatory responses, making it one of the important inflammatory factors. LPS-induced RAW264.7 macrophages are a classic cell model for studying inflammatory factors. By comparing the difference in TNF-α secretion levels in RAW264.7 macrophages after administration of the test substance to the negative control and the test substance, the ability of the test substance to inhibit the secretion of cellular inflammatory factors was evaluated, and the soothing efficacy of the product was explored.

[0125] 3.4 Test Cells

[0126] The mouse mononuclear macrophage leukemia cell line RAW264.7 was selected. The cell line passed the cell line quality control test, and the results were satisfactory. The passage number for this experiment was P20.

[0127] 3.5 Main Instruments and Reagents

[0128] 3.5.1 Main experimental instruments: carbon dioxide incubator, multifunctional enzyme-linked immunosorbent assay (ELISA) analyzer, biosafety cabinet, biological microscope, 8-channel pipette.

[0129] 3.5.2 Main test reagents: fetal bovine serum, high glucose DMEM medium, penicillin-streptomycin solution, trypsin-EDTA solution, TNF-α detection kit, lipopolysaccharide, dexamethasone sodium phosphate.

[0130] 3.6 Test Methods

[0131] 3.6.1 Grouping and Control

[0132] Product dosage grouping:

[0133] The sample groups consist of the Gentiana macrophylla extract composition 2 prepared above diluted with cell culture medium to the corresponding experimental concentration of the sample solution; Sample group 1: 1% sample group, Sample group 2: 0.5% sample group, Sample group 3: 0.1% sample group.

[0134] Experimental control information: ① Blank control: complete culture medium; ② Negative control: stimulation with 0.5 μg / mL LPS; ③ Positive control: 0.5 μg / mL LPS + 100 μg / mL dexamethasone sodium phosphate (DSP).

[0135] 3.6.2 Operating Procedures

[0136] 1) Cell preparation: After stable passage of the cryopreserved cell culture twice, the cells were seeded into 96-well plates and incubated overnight in a 5V / V %CO2 incubator.

[0137] 2) Drug administration: Discard the culture medium in the 96-well plate and proceed with the drug administration procedure. The blank control group was given complete culture medium, the negative control group was given 0.5 μg / mL LPS, and the other groups were given 0.5 μg / mL LPS + the corresponding concentration of the test substance. After drug administration, the 96-well plate was placed in a CO2 incubator and incubated for a certain period of time.

[0138] 3) Detection of inflammatory factors: After incubation, the TNF-α enzyme-linked immunosorbent assay kit was tested according to the instructions for use. The absorbance of each sample well was measured at 450 nm using an ELISA reader.

[0139] 3.7 Judgment Criteria

[0140]

[0141] 3.8 Experimental Results

[0142] Under LPS-stimulated culture conditions, the effects of Gentiana macrophylla extract composition 2 on the content and inhibition rate of TNF-α inflammatory factor secreted by macrophages are shown in Table 4. Figure 4As shown in the figure, compared with the negative control group, the content of macrophage TNF-α inflammatory factor in the 1% sample group was significantly reduced, with an inhibition rate of 25.797%; the content of macrophage TNF-α inflammatory factor in the 0.5% sample group was significantly reduced, with an inhibition rate of 20.470%; and the content of macrophage TNF-α inflammatory factor in the 0.1% sample group was significantly reduced, with an inhibition rate of 12.923%. This indicates that the Gentiana macrophylla extract composition has the ability to inhibit the secretion of TNF-α inflammatory factor. Based on the experimental evidence and the mechanism of skin irritation, this result can serve as one of the pieces of evidence supporting the claim of soothing efficacy of the Gentiana macrophylla extract composition.

[0143] Table 4. Content and inhibition rate of TNF-α inflammatory factor in macrophages

[0144]

[0145] Note: "*" indicates a significant difference compared to the negative control group, "**": p < 0.01, "***": p < 0.001.

[0146] 4. Soothing Efficacy Test

[0147] 4.1 Test Basis

[0148] This experiment was designed and conducted in accordance with the literature "Evaluation Standards for Cosmetic Efficacy Claims", "Bioactive glass promotes the barrier fiunctional behaviors of keratinocytes and improves the Re-epithelialization in wound healing in diabetic rats", and "Expression profile of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins during skin barriet repair".

[0149] 4.2 Experimental Objectives and Principles

[0150] 4.2.1 Experimental Objective

[0151] By testing the product's ability to promote keratinocyte migration, we can explore the product's repair efficacy.

[0152] 4.2.2 Experimental Principle

[0153] The stratum corneum, located on the outermost layer of the epidermis, plays a crucial barrier role due to its unique "brick-and-wall" structure. It protects against mechanical forces and pathogenic microorganisms, and prevents the loss of moisture and nutrients. This physical barrier function is based on the tight junctions between the stratum corneum and keratinocytes. Keratinocytes are the most abundant cells in the epidermis, and their behavior significantly impacts the timely recovery of damaged skin. By comparing changes in keratinocyte migration in the control group and the test substance group, we can evaluate whether the experimental sample is beneficial for the reconstruction of skin barrier function in cases of skin barrier damage.

[0154] 4.3 Test Materials

[0155] 4.3.1 Test Cells

[0156] HaCaT human keratinocytes were selected. The cell line passed the cell line quality control test and STR genotyping identification was completed. Passage number of this experiment: P15.

[0157] 4.3.2 Main Instruments and Reagents

[0158] 4.3.2.1 Main experimental instruments: carbon dioxide incubator, multifunctional enzyme-linked immunosorbent assay (ELISA) analyzer, biosafety cabinet, biological microscope, 8-channel pipette.

[0159] 4.3.2.2 Main test reagents: fetal bovine serum, high glucose DMEM medium, penicillin-streptomycin solution, trypsin-EDTA solution.

[0160] 4.4 Test Methods

[0161] 4.4.1 Grouping and Control

[0162] (1) Product dosage grouping: The sample group is the sample solution of the Gentiana macrophylla extract composition 2 prepared above diluted with cell culture medium to the corresponding test concentration; Sample group 1: 1% sample group, Sample group 2: 0.5% sample group, Sample group 3: 0.1% sample group.

[0163] (2) Experimental control information: Blank control group: complete culture medium.

[0164] 4.4.2 Operating Procedures

[0165] (1) Cell preparation: After stable passage of the cryopreserved cell culture twice, the cells were seeded into 24-well plates and the plates were placed in a CO2 incubator overnight.

[0166] (2) Cell migration assay: The cell culture insert was removed from the cultured monolayer, and the cells were cultured again in serum-free medium containing the sample. The area of ​​cells that migrated to the scratch area was observed after 24 hours, and the change in the area of ​​cells that migrated to the scratch area was calculated.

[0167] 4.4.3 Judgment Criteria

[0168]

[0169] 4.5 Test Results

[0170] Under normal culture conditions, the migration of keratinocytes is shown in Table 5 and... Figure 5-8 As shown.

[0171] Compared with the blank control group, the keratinocyte migration area in the 1% sample group was significantly increased, with a migration rate of 18.909%; the keratinocyte migration area in the 0.5% sample group was significantly increased, with a migration rate of 11.576%; and the keratinocyte migration rate in the 0.1% sample group was 4.931%.

[0172] Table 5. Keratinocyte migration rate

[0173]

[0174] Note: "*" indicates a significant difference compared to the blank control group, "*": P < 0.05, "**": P < 0.01.

[0175] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a soothing and repairing Gentiana macrophylla extract composition, characterized in that the preparation method includes the following steps: Step 1. Grind the dried Gentiana macrophylla into powder and sieve it to obtain Gentiana macrophylla powder; Step 2. Mix Aspergillus oryzae liquid, Leuconostoc mesenteroides liquid and Saccharomyces cerevisiae liquid in a volume ratio of (2-2.5):1:(1-1.5) to obtain a compound bacterial solution; Step 3. Take sterile water to moisten the Gentiana macrophylla powder obtained in Step 1, control the moisture content to 45-55%, mix evenly, and sterilize at 121℃ for 30 minutes to obtain solid fermentation powder for later use. Step 4. Spray the compound bacterial solution obtained in Step 2 into the solid fermentation powder at an inoculation rate of 8-10 wt%, and ferment it for 4-5 days at a temperature of 30-37℃, a humidity of 70-75%, and an aeration rate of 0.2-0.4 L / min·kg to obtain the fermentation product of Gentiana macrophylla. Step 5. Extract the fermentation product of Gentiana macrophylla by soaking in an ethanol-water solution. The extract is then concentrated under reduced pressure and dried to obtain the fermentation extract of Gentiana macrophylla. Step 6. Mix the fermented extract of Gentiana macrophylla with the solvent at a mass ratio of 1:(5-50) at 300-500 r / min and at room temperature to obtain the Gentiana macrophylla extract composition. The solvent is selected from at least one of vegetable oils, synthetic oils, and polyols; In step 2, the Aspergillus oryzae culture is prepared by inoculating a spore suspension into a liquid medium containing 0.1% peptone PDA and culturing in a shake flask at 28°C and 120 rpm for 48-60 hours to obtain the Aspergillus oryzae culture, wherein the spore concentration of the Aspergillus oryzae culture is 1×10⁻⁶. 8 -5×10 8 CFU / mL; In step 2, the preparation of *Leuconostoc mesenteroides* bacterial suspension involves inoculating frozen bacterial strains into MRS liquid medium containing 0.5% maltose and incubating at 37°C for 24-36 hours to obtain *Leuconostoc mesenteroides* bacterial suspension, wherein the viable count of *Leuconostoc mesenteroides* is 1×10⁻⁶. 8 -2×10 8 CFU / mL; In step 2, the preparation of the Saccharomyces cerevisiae culture involves: inoculating frozen inoculum into YPD liquid medium and culturing in a shake flask at 30°C and 150 rpm for 18-24 hours to obtain the Saccharomyces cerevisiae culture, wherein the viable count of the Saccharomyces cerevisiae culture is 1×10⁻⁶. 8 -2×10 8 CFU / mL; The Aspergillus oryzae described was obtained from the China General Microbiological Culture Collection Center, with the accession number CGMCC: 3.13905. The preservation number of Leuconostoc mesenteroides is CGMCC: 1.2461, purchased from China General Microbiological Culture Collection Center; The brewing yeast, with accession number CGMCC: 2.1364, was purchased from the China General Microbiological Culture Collection Center.

2. The preparation method according to claim 1, characterized in that, In step 1, the Gentiana macrophylla is crushed and then passed through a 60-mesh sieve.

3. The preparation method according to claim 1, characterized in that, In step 5, the fermented product of Gentiana macrophylla is mixed with a 60-70 v / v% ethanol aqueous solution at a ratio of 1: (8-10) g / mL, and extracted with ultrasound at 60-70℃ for 2-3 times, each time for 30-40 min. The combined extracts are filtered through a 120-200 mesh sieve and concentrated under reduced pressure at 50-60℃ to a paste state. The paste is then dried until the water content is ≤5% to obtain the fermented extract of Gentiana macrophylla.

4. A soothing and repairing Gentiana macrophylla extract composition, characterized in that, The Gentiana macrophylla extract composition is prepared by the preparation method according to any one of claims 1-3.

5. The use of the Gentiana macrophylla extract composition of claim 4 in the preparation of skin care products with repairing and soothing effects.

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