Relieving and repairing gentiana macrophylla extract composition as well as preparation method and application thereof

Abstract

The invention discloses a soothing and repairing gentiana macrophylla extract composition as well as a preparation method and application thereof, and particularly belongs to the technical field of preparation of raw materials of daily necessities. The gentiana macrophylla extract composition is obtained by performing compound solid-state fermentation on gentiana macrophylla through aspergillus oryzae, leuconostoc citreum and saccharomyces cerevisiae and then performing solvent extraction. The gentiana macrophylla extract composition provided by the invention has relatively high oleanolic acid content, which proves that the aspergillus oryzae, the leuconostoc citreum and the saccharomyces cerevisiae can effectively enrich active ingredients in gentiana macrophylla, and meanwhile, the aspergillus oryzae, the leuconostoc citreum and the saccharomyces cerevisiae have a certain synergistic effect on the enrichment of the active ingredients in the fermentation process; the gentiana macrophylla extract composition provided by the invention can effectively inhibit release of inflammatory factors and promote skin repair.

Description

Technical Field

[0001] The invention belongs to the technical field of daily necessities raw material preparation, and particularly relates to a soothing and repairing gentiana macrophylla extract composition, a preparation method and an application thereof. Background Art

[0002] As a traditional Chinese medicinal material, Gentiana macrophylla is rich in a variety of active ingredients, among which oleanolic acid is one of the key substances that exert its anti-inflammatory, antioxidant and immunomodulatory effects. Studies have shown that oleanolic acid can effectively relieve skin inflammatory responses 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 are mostly present in the form of bound or macromolecular complexes. Traditional water extraction 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 matrices through enzymatic hydrolysis and improve the bioaccessibility of active ingredients. Existing studies have shown that fermentation with a single bacterial species (such as Aspergillus oryzae or lactic acid bacteria) can partially enhance the efficacy of plant extracts, but there are defects such as a single metabolite and insufficient synergistic effect. Especially in solid-state fermentation systems, the metabolic synergistic mechanism of complex bacterial communities has not been fully explored. How to optimize the fermentation process by combining multiple bacterial species to enhance the release efficiency of active ingredients remains a technical difficulty. In addition, the lack of systematic optimization of key parameters such as temperature and humidity control and nutrient matrix ratio in traditional fermentation processes has led to poor stability of fermentation products and a high loss rate of effective ingredients.

[0004] In the field of skincare product development, chemically synthesized soothing ingredients, while effective quickly, carry the risk of allergens. Existing natural plant extracts generally suffer from issues like low transdermal absorption efficiency and insufficient long-lasting efficacy. Therefore, developing a novel Gentiana macrophylla extraction process based on multi-strain synergistic fermentation, which increases the active ingredient content while enhancing skin compatibility, has become a key focus in the development of ingredients for functional skincare products. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a Qinjiao fermentation extract product based on multi-strain collaborative fermentation, by optimizing the strain compatibility and fermentation process, to improve the extraction yield of active ingredients such as oleanolic acid, and to enhance the soothing, anti-inflammatory and repairing effects of Qinjiao.

[0006] In order to achieve the above object, 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 and sieve it to obtain Gentiana macrophylla powder;

[0009] Step 2. Mixing the Aspergillus oryzae solution, the Leuconostoc citraceus solution, and the Saccharomyces cerevisiae solution in a volume ratio of (2-2.5):1:(1-1.5) to obtain a composite bacterial solution;

[0010] Step 3. Moisten the gentiana powder obtained in step 1 with sterile water to control the moisture content to 45-55%, mix well, and sterilize at 121° C. for 30 min to obtain a solid-state fermentation powder for later use;

[0011] Step 4. Spraying the composite bacterial solution obtained in step 2 into the solid fermentation powder at an inoculum amount of 8-10 wt%, and fermenting and culturing at a temperature of 30-37° C., a humidity of 70-75%, and an aeration rate of 0.2-0.4 L / min·kg for 4-5 days to obtain a Gentiana macrophylla fermentation product;

[0012] Step 5. extracting the fermentation product of Gentiana macrophylla by soaking it in an ethanol-water solution, and concentrating the extract under reduced pressure and drying it to obtain the fermentation extract of Gentiana macrophylla;

[0013] Step 6. The fermented extract of Gentiana macrophylla and the solvent are mixed and stirred at a mass ratio of 1:(5-50) at 300-500 r / min and room temperature 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, camellia oil, jojoba seed oil, avocado oil, 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 gentiana macrophylla is crushed and then passed through a 60-mesh sieve.

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

[0019] Preferably, in step 2, the preparation of the Leuconostoc citrifolia liquid is as follows: frozen strains are inoculated into a MRS liquid medium containing 0.5% maltose, and the culture is statically cultured at 37° C. for 24-36 hours to obtain a Leuconostoc citrifolia liquid, wherein the number of viable Leuconostoc citrifolia cells is 1×10 8 -2×10 8 CFU / mL.

[0020] Preferably, in step 2, the preparation of the saccharomyces cerevisiae liquid is as follows: frozen strains are inoculated into YPD liquid medium, and cultured in a shake flask at 30°C and 150 r / min for 18-24 h to obtain a saccharomyces cerevisiae liquid, wherein the number of viable cells in the saccharomyces cerevisiae liquid is 1×10 8 -2×10 8 CFU / mL.

[0021] Preferably, in step 5, the gentiana fermentation product is mixed with a 60-70 v / v% ethanol aqueous solution at a ratio of 1: (8-10) g / mL, and ultrasonic-assisted extraction is performed at 60-70° C. for 2-3 times, each time for 30-40 minutes. The combined extract is filtered through a 120-200 mesh sieve, concentrated under reduced pressure at 50-60° C. to an extract, and dried to a water content of ≤5% to obtain the gentiana fermentation extract.

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

[0023] In a third aspect, the present invention provides the use of the soothing and repairing Gentiana macrophylla extract composition described in the second aspect in the preparation of a skin care product with repairing and soothing effects.

[0024] Beneficial effects of the present invention:

[0025] 1. The product obtained by extracting the gentiana macrophylla by organic solvent after composite fermentation with Aspergillus oryzae, Leuconostoc citrifolia, and Saccharomyces cerevisiae has a high oleanolic acid content, demonstrating that Aspergillus oryzae, Leuconostoc citrifolia, and Saccharomyces cerevisiae can effectively enrich the active ingredients in the gentiana macrophylla and exhibit a certain synergistic effect on the enrichment of the active ingredients during the fermentation process.

[0026] 2. The gentiana extract composition provided by the present invention can effectively inhibit the release of inflammatory factors. Experimental verification shows that compared with the negative control group, the gentiana extract composition at concentrations of 1%, 0.5%, and 0.1% has an inhibition rate of 25.797%, 20.470%, and 12.923% on the inflammatory factor TNF-α of macrophages, respectively;

[0027] 3. The Qinjiao extract composition provided by the present invention has the effect of significantly promoting skin repair. Compared with the blank control group, the keratinocyte migration area of the Qinjiao extract composition with a concentration of 1% has a significant increase, and the migration rate is 18.909%. The keratinocyte migration area of the Qinjiao extract composition with a concentration of 0.5% has a significant increase, and the migration rate is 11.576%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the standard curve of oleanolic acid;

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

[0030] Figure 3 is the HPLC chart of Gentiana macrophylla extract composition 2;

[0031] Figure 4 This is a graph showing the effects of different samples on the content of TNF-α inflammatory factors in macrophages;

[0032] Figure 5 This is a graph showing the effects of samples at different concentrations on keratinocyte migration;

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

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

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

[0036] Figure 9 Cell migration images of sample group 3 at 0 h (left) and 24 h (right); DETAILED DESCRIPTION

[0037] The present invention will be further described 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. In addition, the embodiments of the present invention referred to in the following description are generally only part of the embodiments of the present invention, rather than all of the embodiments.

[0038] Therefore, based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without inventive effort shall fall within the scope of protection of the present invention. Unless otherwise specified, the raw materials used in the embodiments of the present invention are commercially available or available to persons skilled in the art; unless otherwise specified, the methods used in the embodiments of the present invention are methods known to those skilled in the art.

[0039] In the present invention:

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

[0041] Leuconostoc citrina: CGMCC accession number: 1.2461, purchased from China General Microorganism Collection Center;

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

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

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

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

[0046] Gentiana macrophylla extract composition

[0047] Step 1-1. Take dried Gentiana macrophylla and grind it in a universal grinder, pass it through a 60-mesh sieve, and the sieve material is ≤0.1% to obtain Gentiana macrophylla powder for later use;

[0048] Step 1-2. The Aspergillus oryzae solution, the Leuconostoc citrifolia solution and the Saccharomyces cerevisiae solution were compounded and mixed in a volume ratio of (2-2.5): 1: (1-1.5) to obtain a composite bacterial solution;

[0049] Preparation of Aspergillus oryzae liquid: Take spore suspension and inoculate it into liquid medium containing 0.1% peptone PDA, shake flask culture at 28 ° C and 120 r / min for 48-60 h to obtain Aspergillus oryzae liquid, wherein the spore concentration of Aspergillus oryzae liquid is 1×10 8-5×10 8 CFU / mL;

[0050] Preparation of Leuconostoc lemon bacterial solution: frozen bacterial strains were inoculated into MRS liquid medium containing 0.5% maltose and cultured at 37°C for 24-36 hours to obtain Leuconostoc lemon bacterial solution, in which the number of viable Leuconostoc lemon cells was 1×10 8 -2×10 8 CFU / mL;

[0051] Preparation of Saccharomyces cerevisiae liquid: frozen strains were inoculated into YPD liquid medium and cultured in a shake flask at 30°C and 150 rpm for 18-24 h to obtain a Saccharomyces cerevisiae liquid with a viable cell count of 1 × 10 8 -2×10 8 CFU / mL;

[0052] Step 1-3 solid-state fermentation powder preparation: take sterile water to moisten the Gentiana macrophylla powder, control the moisture content to 45-55%, 121 ℃ high pressure steam sterilization for 30min to obtain a solid-state fermentation powder;

[0053] Step 1-4. The solid-state fermentation powder was transferred to a solid fermentation tank, and the composite bacterial solution was evenly sprayed on the solid-state fermentation powder at an inoculum size of 8-10wt%. The fermentation was started while stirring the fermentation tank. The stirring paddle was stirred and sprayed. After the spraying was completed, the fermentation was cultured at 30-37 ° C, 70-75% humidity, and 0.2-0.4 L / min kg ventilation for 4-5 days. During the fermentation process, intermittent stirring was performed and the mixture was stirred every 2h. After the fermentation was completed, the fermentation product of Gentiana macrophylla was obtained.

[0054] Step 1-5. The fermentation product of Gentiana macrophylla was mixed with 60-70 v / v% ethanol aqueous solution at a ratio of 1: (8-10) g / mL, and ultrasonic-assisted extraction was performed at 60-70 ° C for 2-3 times, each time for 30-40 min. The combined extract was filtered through a 120-200 mesh sieve, concentrated under reduced pressure at 50-60 ° C to an extract, and dried to a water content of ≤5% to obtain a fermentation extract of Gentiana macrophylla;

[0055] Step 1-6. The fermented extract of Gentiana macrophylla and olive oil are mixed and stirred uniformly at a mass ratio of 1:(9-19) at 300-500 r / min and room temperature to obtain a Gentiana macrophylla extract composition 1-3.

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

[0057] Table 1 Fermentation process parameters

[0058]

[0059]

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

[0061] Composition 4: In step 1-2, the Aspergillus oryzae solution is missing, and the volume ratio of the remaining bacterial solution remains unchanged. The rest is consistent with the preparation method of Gentiana macrophylla extract composition 2;

[0062] Composition 5: In step 1-2, the Leuconostoc citrifolia bacterial solution is missing, and the volume ratio of the remaining bacterial solution remains unchanged. The rest of the preparation method is consistent with that of Gentiana macrophylla extract composition 2;

[0063] Composition 6: In step 1-2, the Saccharomyces cerevisiae bacterial solution is missing, and the volume ratio of the remaining bacterial solution remains unchanged. The rest of the preparation method is consistent with that of Gentiana macrophylla extract composition 2;

[0064] Composition 7: The lemon Leuconostoc liquid in step 1-2 was replaced with Lactobacillus plantarum liquid, and the volume ratio of the liquid remained unchanged. The rest of the preparation method was consistent with that of Gentiana macrophylla extract composition 2. The Lactobacillus plantarum was purchased from the China Center for Type Culture Collection, with the deposit number CCTCC AB 2022276. The strain was activated by conventional activation means to a viable cell count of 1×10 8 CFU / mL;

[0065] Composition 8: Take the dried root of Gentiana macrophylla and place it in a universal grinder for crushing, pass it through a 60-mesh sieve to obtain Gentiana macrophylla powder, mix the Gentiana macrophylla powder with a 65v / v% ethanol aqueous solution at a material-liquid ratio of 1:9g / mL, and perform ultrasonic-assisted extraction at 65°C for 3 times, each time for 30 minutes. After the combined extract is filtered through a 150-mesh sieve, it is concentrated under reduced pressure at 55°C to an extract state, and dried to a water content of ≤5% to obtain an extract. The extract is mixed with olive oil at a mass ratio of 1:14 at 400r / min and room temperature, and stirred evenly to obtain Composition 8.

[0066] Performance Testing

[0067] 1. Oleanolic acid content detection

[0068] 1.1 Principle

[0069] The samples were determined by high performance liquid chromatography (HPLC), with qualitative determination based on retention time and quantitative determination based on external standard method.

[0070] 1.2 Main instruments and tools

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

[0072] 1.3 Reagents

[0073] Unless otherwise specified, all reagents used were of analytical grade. Water was grade 1 water as specified in GB / T6682. Acetonitrile (chromatographic grade); methanol (chromatographic grade); oleanolic acid standard (HPLC): content not less than 98%.

[0074] 1.4 Determination procedure

[0075] 1.4.1 Preparation of Oleanolic Acid Standard Curve Stock Solution

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

[0077] 1.4.2 Preparation of oleanolic acid standard working solution

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

[0079] 1.5 Chromatographic conditions

[0080] The reference conditions for HPLC determination are as follows:

[0081] Chromatographic column: Hypersil GOLD C18 column (250 mm × Ø4.6 mm, 5 μm), or equivalent;

[0082] Mobile phase: acetonitrile: water = 94:6;

[0083] Detection wavelength: 210nm;

[0084] Column temperature: 30°C;

[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 processing

[0089] Accurately weigh 1 mL of composition 2, composition 4, composition 5, composition 6, composition 7, and composition 8 into stoppered colorimetric tubes, then add methanol to each colorimetric tube to dilute the sample 5-fold, vortex and shake for 3 minutes, let it stand for 10 minutes, extract the upper layer solution, filter it through a 0.45 μm filter membrane, and use the filtrate as the sample solution to be tested.

[0090] 1.8 Determination

[0091] Under the chromatographic conditions in 1.5, take the standard series solution 1.4.2 and inject them separately. Record the chromatogram. Draw the oleanolic acid standard curve with the concentration of the standard series solution as the horizontal axis and the peak area as the vertical axis. Figure 1 HPLC charts of oleanolic acid standards (200 μg / mL, 240 μg / mL, 300 μg / mL, 360 μg / mL, 400 μg / mL) are shown in Figure 2 .

[0092] Take the prepared sample test solution 1.7 and inject it, record the chromatogram, measure the peak area, obtain the content of oleanolic acid in the sample test solution according to the standard curve, and then calculate the content of oleanolic acid in the composition according to 1.9. The results are shown in Table 2. The HPLC diagram of composition 2 is shown in Figure 3 .

[0093] 1.9 Calculation formula

[0094] The oleanolic acid content in the sample was calculated according to the following formula:

[0095] X=5×Q

[0096] Where: Q: Oleanolic acid content of 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 area and linear regression equation of each concentration standard are shown in the following table:

[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] Result 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 followed by extraction with an organic solvent has a high content of oleanolic acid, which proves that Aspergillus oryzae, Leuconostoc citreum and Saccharomyces cerevisiae can effectively enrich the active ingredients in Gentiana macrophylla; in Compositions 4-6, Aspergillus oryzae, Leuconostoc citreum and Saccharomyces cerevisiae are missing respectively in the preparation process, resulting in a significant decrease in the oleanolic acid content of Compositions 4-6. At the same time, in the preparation process of Composition 7, Leuconostoc citreum is replaced by Lactobacillus plantarum, and the result shows that the content of oleanolic acid 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 Medical Experimental Animal Center (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 Medical Experimental Animal Center, 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 h 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 h later.

[0113] 2.6 Test results:

[0114] Table 3 Results of irritation experiment

[0115]

[0116]

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

[0118] 3 Soothing effect test

[0119] 3.1 Test basis

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

[0121] 3.2 Purpose of the test

[0122] By testing the product's ability to inhibit macrophage inflammatory factors, the soothing effect of the product is explored.

[0123] 3.3 Test principle

[0124] In daily life, there are three main types of stimuli that cause skin irritation: physical stimuli such as ultraviolet radiation, microbial infections, and chemical stimuli such as hair dyes. Inflammation is a defensive response to exogenous or endogenous (harmful) stimuli, mainly involving the innate immune system. The skin manifests itself in the form of redness, swelling, itching, and other phenomena. Bacterial lipopolysaccharide (LPS) binds to antigen recognition receptors on the surface of macrophages, inducing macrophages to secrete a variety of cytokines such as tumor necrosis factor-α (TNF-α). TNF-α can activate three signaling pathways, namely Caspase, JNK, and NF-κB, to achieve its biological functions such as apoptosis and cellular inflammatory response, and is one of the important inflammatory factors. LPS-induced RAW264.7 macrophages is a classic cell model for studying inflammatory factors. By comparing the difference in TNF-α secretion levels by RAW264.7 after administration of the negative control and the test substance, the ability of the test substance to inhibit the secretion of cellular inflammatory factors is evaluated, and the soothing effect of the product is explored.

[0125] 3.4 Test cells

[0126] The mouse monocytic macrophage leukemia cell line RAW264.7 was used. The cell line passed the cell line quality control test and the results were qualified. The number of generations in this experiment was P20.

[0127] 3.5 Main instruments and reagents

[0128] 3.5.1 Main test instruments: CO2 incubator, multifunctional enzyme-linked immunosorbent assay (ELISA), biological safety cabinet, biological microscope, and 8-channel pipette.

[0129] 3.5.2 Main experimental 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 were sample solutions of the Gentiana macrophylla extract composition 2 prepared above and diluted with cell culture fluid to corresponding test concentrations; sample group 1: 1% sample group, sample group 2: 0.5% sample group, and sample group 3: 0.1% sample group.

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

[0135] 3.6.2 Operation steps

[0136] 1) Cell preparation: After stable passage twice from cryopreserved cell culture, cells were seeded into 96-well plates, and the 96-well plates were placed in a 5 v / v% CO 2 incubator and cultured overnight.

[0137] 2) Dosing: Discard the culture medium in the 96-well plate and proceed with dosing. The blank control group receives complete culture medium, the negative control group receives 0.5 μg / mL LPS, and the remaining groups receive 0.5 μg / mL LPS plus the corresponding concentration of the test substance. After dosing, place the 96-well plate in a CO2 incubator for the desired period of time.

[0138] 3) Inflammatory factor detection: After the incubation, the TNF-a enzyme-linked immunosorbent assay kit was used for detection according to the instruction manual, and the absorbance of each sample well was measured at 450 nm using a microplate reader.

[0139] 3.7 Judgment Basis

[0140]

[0141] 3.8 Experimental Results

[0142] Under LPS stimulation culture conditions, the content and inhibition rate of Gentiana macrophylla extract composition 2 on the secretion of TNF-α inflammatory factor by macrophages are shown in Table 4. Figure 4As shown. Compared with the negative control group, the 1% sample group had a significant reduction in the content of TNF-α inflammatory factors in macrophages, with an inhibition rate of 25.797%; the 0.5% sample group had a significant reduction in the content of TNF-α inflammatory factors in macrophages, with an inhibition rate of 20.470%; and the 0.1% sample group had a significant reduction in the content of TNF-α inflammatory factors in macrophages, 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 factors. Based on the experimental basis and the mechanism of skin irritation, this result can be used as one of the evidences to support the soothing effect claim of the Gentiana macrophylla extract composition.

[0143] Table 4 TNF-α inflammatory factor content and inhibition rate in macrophages

[0144]

[0145] Note: “*” indicates significant difference compared with the negative control group, “**”: p < 0.01, “***”: p < 0.001.

[0146] 4 Soothing effect test

[0147] 4.1 Test basis

[0148] This experiment was designed and tested according to the literature "Standards for the Evaluation of 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 Test purpose and principle

[0150] 4.2.1 Test Purpose

[0151] By testing the product's ability to promote keratinocyte migration, the product's repair effect is explored.

[0152] 4.2.2 Test principle

[0153] The stratum corneum is the outermost layer of the epidermis. Due to its unique "brick-wall" structure, it plays a vital role as a barrier against mechanical forces and invasion by pathogenic microorganisms, preventing the loss of water and nutrients. This physical barrier function is achieved through the tight connections between the stratum corneum and keratinocytes. Keratinocytes are the primary cells in the epidermis, and their behavior significantly influences the timely recovery of damaged skin. By comparing changes in keratinocyte migration between the blank control and test substance-treated groups, we can evaluate whether the test sample promotes the restoration of barrier function in the presence of damaged skin.

[0154] 4.3 Test materials

[0155] 4.3.1 Test cells

[0156] HaCaT human keratinocytes were used. The cell line passed cell line quality control testing and STR genotyping. The generation number for this experiment was P15.

[0157] 4.3.2 Main instruments and reagents

[0158] 4.3.2.1 Main test instruments: CO2 incubator, multifunctional enzyme-linked immunosorbent assay (ELISA), biological safety cabinet, biological microscope, and 8-channel pipette.

[0159] 4.3.2.2 Main experimental 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 a sample solution of the Gentiana macrophylla extract composition 2 prepared above and 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 Operation steps

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

[0166] (2) Cell migration assay: Remove the cell culture insert from the cultured monolayer of cells and continue culturing the cells in serum-free medium containing the sample. Observe the area of cells that have migrated to the scratched area after 24 hours, and calculate the change in the area of cells that have migrated to the scratched area.

[0167] 4.4.3 Judgment basis

[0168]

[0169] 4.5 Test results

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

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

[0172] Table 5 Keratinocyte migration rate

[0173]

[0174] Note: “*” indicates significant difference compared with 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 aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a soothing and repairing Gentiana macrophylla extract composition, characterized in that: The preparation method comprises the following steps: Step 1. Grind the dried Gentiana macrophylla and sieve it to obtain Gentiana macrophylla powder; Step 2. Mixing the Aspergillus oryzae solution, the Leuconostoc citraceus solution, and the Saccharomyces cerevisiae solution in a volume ratio of (2-2.5):1:(1-1.5) to obtain a composite bacterial solution; Step 3. Moisten the gentiana powder obtained in step 1 with sterile water to control the moisture content to 45-55%, mix well, and sterilize at 121° C. for 30 min to obtain a solid-state fermentation powder for later use; Step 4. Spraying the composite bacterial solution obtained in step 2 into the solid fermentation powder at an inoculum amount of 8-10 wt%, and fermenting and culturing at a temperature of 30-37° C., a humidity of 70-75%, and an aeration rate of 0.2-0.4 L / min·kg for 4-5 days to obtain a Gentiana macrophylla fermentation product; Step 5. extracting the fermentation product of Gentiana macrophylla by soaking it in an ethanol-water solution, and concentrating the extract under reduced pressure and drying it to obtain the fermentation extract of Gentiana macrophylla; Step 6. The fermented extract of Gentiana macrophylla and the solvent are mixed and stirred at a mass ratio of 1:(5-50) at 300-500 r / min and room temperature to obtain the Gentiana macrophylla extract composition; The solvent is selected from at least one of vegetable oils, synthetic oils, and polyols.

2. The preparation method according to claim 1, characterized in that In the 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 Preparation of Aspergillus oryzae liquid in step 2: taking spore suspension and inoculating it into liquid culture medium containing 0.1% peptone PDA, shaking flask culture at 28°C and 120r / min for 48-60h to obtain Aspergillus oryzae liquid, wherein the spore concentration of Aspergillus oryzae liquid is 1×10 8 -5×10 8 CFU / mL.

4. The preparation method according to claim 1, characterized in that In step 2, the preparation of the Leuconostoc citrifolia liquid is as follows: frozen strains are inoculated into a MRS liquid medium containing 0.5% maltose, and the culture is statically cultured at 37°C for 24-36 hours to obtain a Leuconostoc citrifolia liquid, wherein the number of viable Leuconostoc citrifolia cells is 1×10 8 -2×10 8 CFU / mL.

5. The preparation method according to claim 1, characterized in that Preparation of the brewer's yeast liquid in step 2: frozen strains were inoculated into YPD liquid medium, and cultured in a shake flask at 30°C and 150 rpm for 18-24 hours to obtain a brewer's yeast liquid, wherein the number of viable cells in the brewer's yeast liquid was 1×10 8 -2×10 8 CFU / mL.

6. The preparation method according to claim 1, characterized in that In step 5, the gentiana fermentation product is mixed with a 60-70 v / v% ethanol aqueous solution at a ratio of 1: (8-10) g / mL, and ultrasonic-assisted extraction is performed at 60-70° C. for 2-3 times, each time for 30-40 minutes. The combined extract is filtered through a 120-200 mesh sieve, concentrated under reduced pressure at 50-60° C. to an extract, and dried to a water content of ≤5% to obtain the gentiana fermentation extract.

7. 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 to 6.

8. Use of the soothing and repairing Gentiana macrophylla extract composition according to claim 7 in the preparation of skin care products with repairing and soothing effects.

Images