Composition of plant extracts and application thereof

Abstract

The invention discloses a plant extract composition and application thereof. The composition comprises a scutellaria baicalensis extract, a willow herb extract and a lavender extract in a mass ratio of 1: 1: 1. The composition disclosed by the invention can be applied to preparation of products for reducing the irritation influence of the surfactant on the skin.

Description

TECHNICAL FIELD

[0001] The present application relates to a composition, in particular a composition of plant extracts and its application. BACKGROUND

[0002] The occurrence of sensitive skin is related to the use of daily care products, and the high frequency use of hair care products such as hair conditioner tends to increase the risk of scalp irritation, and the cationic surfactant contained therein is considered to be the main irritant source. For example, the representative cationic surfactant ingredient cetyltrimethylammonium chloride (CTAC) has multiple dimensional irritation effects on the scalp. Therefore, there is an urgent need for a solution to the skin sensitivity problem caused by the irritation of cationic surfactant ingredients. SUMMARY

[0003] The purpose of the present application is to provide a composition of plant extracts and its application. The composition of the present application can effectively reduce the irritation of surfactants to the skin.

[0004] The technical solution of the present application is as follows: A composition of plant extracts, characterized in that it comprises Scutellaria baicalensis Georgi extract, Spiraea salicifolia L. extract and Lavandula angustifolia Mill. extract in a mass ratio of 1:1:1.

[0005] The use of the aforementioned composition of plant extracts in the preparation of a product having reduced irritation of surfactants to the skin. The skin comprises the scalp.

[0006] In the aforementioned use, the irritation includes skin irritation, skin inflammation, skin barrier effect, skin oxidative stress and sebaceous gland oil secretion.

[0007] In the aforementioned use, as a preferred embodiment, the product is a care product or a care product.

[0008] In the aforementioned use, as a preferred embodiment, the surfactant is a cationic surfactant.

[0009] In the aforementioned use, as the most preferred embodiment, the cationic surfactant is cetyltrimethylammonium chloride.

[0010] Compared with the prior art, the present application has the following advantages: The present application can achieve a synergistic effect by compounding three natural plant extracts, Scutellaria baicalensis Georgi extract, Spiraea salicifolia L. extract and Lavandula angustifolia Mill. extract, in an equal ratio of 1:1:1. When used, the composition obtained by compounding is added to a care product or a care product, which can reduce the irritation of surfactants to the scalp, thereby inhibiting the skin sensitivity problem caused by surfactants. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is the result of cytotoxicity experiment in experimental example 1; the vertical coordinate in the figure is the cell survival rate; Figure 2 is the graph of IL-6 inflammatory factor expression in experimental example 1; Figure 3 is the graph of IL-8 inflammatory factor expression in experimental example 1; Figure 4 is the ROS fluorescence graph of human immortalized keratinocytes in experimental example 2; Figure 5 is the graph of ROS expression change of human immortalized keratinocytes in experimental example 2; Figure 6 is the histomorphology graph of epidermis model under CTAC stimulation in experimental example 3; Figure 7 is the graph of epidermis cell thickness change of epidermis model under CTAC stimulation in experimental example 3; Figure 8 is the immunofluorescence graph of barrier-related protein FLG of epidermis model under CTAC stimulation in experimental example 3; Figure 9 is the graph of barrier-related protein FLG expression change of epidermis model under CTAC stimulation in experimental example 3; Figure 10 is the immunofluorescence graph of NRF2 of epidermis model under CTAC stimulation in experimental example 3; Figure 11 is the graph of NRF2 expression change of epidermis model under CTAC stimulation in experimental example 3; Figure 12 is the graph of IL-8 inflammatory factor expression of epidermis model under CTAC stimulation in experimental example 3; Figure 13 is the graph of IL-1α inflammatory factor expression of epidermis model under CTAC stimulation in experimental example 3; Figure 14 is the graph of PGE2 inflammatory factor expression of epidermis model under CTAC stimulation in experimental example 3; Figure 15 is the Nile red staining graph of sebaceous gland cells stimulated by CTAC in experimental example 4; Figure 16 is the IOD change graph of Nile red staining of sebaceous gland cells stimulated by CTAC in experimental example 4; IOD represents the total pixel optical density value in the selected area in the fluorescence picture divided by the total number of pixels in the selected area, reflecting the average brightness of the fluorescence in the area, and then indicating the expression of the protein; Figure 17This is an immunofluorescence staining image of PPARγ in sebaceous gland cells stimulated by CTAC in Experiment Example 5; in the image, DAPI represents the cell nucleus (blue fluorescence), and merge is the image after combining the protein and cell nucleus; Figure 18 This is a diagram showing the changes in IOD of sebaceous gland cells after PPARγ immunofluorescence staining stimulated by CTAC in Experiment Example 5. Figure 19 This is an immunofluorescence staining image of SREBP1 in sebaceous gland cells stimulated by CTAC in Experiment Example 5; Figure 20 This is a diagram showing the changes in IOD of SREBP1 immunofluorescence staining of sebaceous gland cells stimulated by CTAC in Experiment Example 5. Detailed Implementation

[0012] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0013] Example: A composition of plant extracts, hereinafter referred to as SSC, comprising Scutellaria baicalensis extract (an aqueous solution containing >90 mg / 100 g of baicalin), willowherb extract (an aqueous solution containing 0.12–0.36% by mass of evening primrose B) and lavender extract (an aqueous solution containing 100–300 mg / kg of total flavonoids and 400–900 mg / kg of polyphenols) in a mass ratio of 1:1:1.

[0014] The composition of this plant extract can be used in the preparation of products that reduce the irritant effects of surfactants on the skin (especially the scalp), including skin irritation, skin inflammation, skin barrier dysfunction, skin oxidative stress, and sebum production. The products are repair or conditioning products. Adding SSC to the product not only directly reduces the skin irritation caused by surfactants in the product itself during use, but also reduces the skin irritation caused by surfactants in other products used subsequently.

[0015] Experiment 1: Experiment on the protective effect of SSC on CTAC-stimulated keratinocytes.

[0016] I. Principle Sensitive scalp is associated with improper daily use of conditioner, and cetyltrimethylammonium chloride (CTAC), a commonly used cationic surfactant in conditioners, is considered a major irritant. This study used keratinocytes as the research subject and evaluated the protective efficacy of the ingredients against CTAC-induced keratinocyte irritation by measuring cell viability and inflammatory factors using the CCK8 assay.

[0017] II. Instruments and Equipment Microbalance, clean bench, CO2 incubator, ELISA reader, cell counter, inverted microscope, low-speed centrifuge.

[0018] III. Reagents and Materials Cell material: Human immortalized keratinocytes HaCaT.

[0019] Reagents: trypsin, phosphate-buffered saline (PBS), DMEM medium (containing 10% newborn calf serum), DMEM high glucose medium (pyruvate-free, containing 10% newborn calf serum), CCK-8 solution, Human IL-6 Elisa Kit, Human IL-8 Elisa Kit, Human PGE2 Elisa Kit.

[0020] IV. Operating Procedures 4.1 CTAC Stimulation Method for Keratinocytes 1) The cells were loaded at a rate of 1×10 4 Cells / well were seeded in 96-well plates, with 6 replicates per sample, 100 μL per well, and 100 μL of sterile PBS solution was added to the edge of the plate. The experiment included a zeroing group (DMEM complete medium without cells), a control group (cell group), a stimulation group, a positive control group (100 μg / mL dexamethasone), and a sample group, with 6 replicates per group. The sample loading information is shown in Table 1.

[0021] 2) Drug administration: Prepare working solutions of different concentrations of the test substance according to the experimental groups. After the cells are seeded in 96-well plates for 24 h, the drugs are administered in groups, 100 μL per well, with 6 replicates. After drug administration, the 96-well plates are placed in an incubator (37℃, 5% CO2) for 4 h.

[0022] Table 1 Sample Addition Information Table 3) Preparation of CTAC working solution: Dissolve an appropriate amount of CTAC in PBS to prepare a 1 mg / mL stock solution. After filtration at 0.22 μm, dilute the stock solution with PBS to obtain the CTAC working solution (2 μg / mL) and vortex to mix.

[0023] 4) CTAC stimulation: After the sample incubation is completed, the culture medium is discarded and CTAC is administered to stimulate the sample. 100 μL is administered to each well, with 6 replicates. After administration, the 96-well plate is placed in an incubator (37℃, 5% CO2) for 20 h.

[0024] 5) Detection: After CTAC stimulation, cells were incubated in an incubator (37℃, 5% CO2) for 20 h. The supernatant was discarded, and CCK8 working solution was added. Cells were incubated at 37℃ in the dark for 1 h. After incubation, the OD value was read at 490 nm.

[0025] 6) Calculation of relative cell viability: Calculated according to the formula. Cell viability (%) = (sample well OD - zeroing well OD) / (blank well OD - zeroing well OD) × 100%.

[0026] 4.2 Method for assessing inflammatory factors and stimulating keratinocytes 1) The cells were loaded at a rate of 1×10 5 One cell / well was seeded in a 24-well plate, with three replicates for each sample, 1 mL per well; the experimental setup, blank group (cell group), stimulation group, positive control group (100 μg / mL dexamethasone), and sample group, and the sample loading information are shown in Table 2.

[0027] 2) Drug administration: Prepare working solutions of different concentrations of the test substance according to the experimental groups. After the cells are seeded in 24-well plates for 24 h, administer the drug to each group, 1 mL per well, with 3 replicates. After drug administration, place the 24-well plates in an incubator (37℃, 5% CO2) for 4 h.

[0028] Table 2 Sampling Information Table 3) CTAC stimulation: After the sample incubation is completed, discard the culture medium and administer CTAC to stimulate the sample. 1 mL per well. After administration, place the 24-well plate in an incubator (37℃, 5% CO2) for 20 h.

[0029] 4) Collect the supernatant: After CTAC stimulation, the cells were incubated in an incubator (37℃, 5% CO2) for 20 h and the supernatant was collected.

[0030] 5) Centrifugation: Centrifuge at 12000 rpm for 5 min, discard the precipitate, and store at -20℃.

[0031] 6) Kit testing: Follow the instructions in the ELISA kit for subsequent operations.

[0032] V. Judgment Basis Compared with the negative control group, the release of inflammatory factors from human immortalized keratinocytes was significantly reduced in the sample group, indicating that the test substance has an inhibitory effect on the release of inflammatory factors from human immortalized keratinocytes and has the effect of reducing irritation.

[0033] VI. Test Results Results of cytotoxicity and inflammatory factor assays are shown in Figures 1-3As shown, the cell viability of the SSC group was significantly increased compared to the CTAC group, while the levels of IL-6 and IL-8 were significantly decreased. Furthermore, the SSC group exhibited a significant inhibitory effect on IL-8 secretion in the CTAC stimulation model, showing a synergistic effect. Where # indicates a significant difference from the BC group; * indicates a significant difference from the CTAC group; * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001; **** indicates P-value < 0.0001.

[0034] VII. Experimental Conclusions SSC can significantly reduce the inflammatory response caused by CTAC stimulation, thus reducing irritation.

[0035] Experiment Example 2: Immunofluorescence staining to assess ROS expression in keratinocytes.

[0036] I. Principle The intracellular redox state is one of the key factors in maintaining normal cellular physiological functions. Reactive oxygen species (ROS), as important participants in intracellular redox reactions, directly reflect the redox balance of cells through changes in their levels. By detecting ROS levels, we can understand whether cells are in a state of oxidative stress or a relatively stable reducing state, thus assessing the overall health of the cells. For example, under normal physiological conditions, the production and clearance of ROS within cells are in dynamic equilibrium. When cells are exposed to external stimuli such as ultraviolet radiation or chemical toxins, ROS production increases. If this exceeds the cell's clearance capacity, it disrupts the redox balance, leading to oxidative stress and potentially causing cell damage or even death. The specific binding of fluorescent dyes to ROS, followed by observation of the fluorescence signal intensity using a fluorescence microscope, allows for the assessment of ROS levels in keratinocytes. Commonly used fluorescent dyes, such as DCFH-DA (dichlorofluorescein diacetate), are non-fluorescent themselves. After entering the cell, they are hydrolyzed by intracellular esterases to generate DCFH. DCFH can be oxidized by ROS to form fluorescent DCF (dichlorofluorescein). The fluorescence intensity is directly proportional to the intracellular ROS content.

[0037] II. Instruments and Equipment Microbalance, clean bench, CO2 incubator, fluorescence microscope, centrifuge, cell counting chamber.

[0038] III. Reagents and Materials Cell material: human immortalized keratinocytes.

[0039] Reagents: trypsin / EDTA solution, phosphate-buffered saline (PBS), DMEM medium (containing 10% newborn calf serum), DCFH-DA.

[0040] IV. Operating Procedures 4.1 Test substance pretreatment and test grouping 4.1.1 Cell Plating After cell counting, dilute to the desired concentration and add cell suspension to the cell culture plate to achieve a culture volume of 1 mL / well and a viable cell count of 5 × 10⁶ cells / well. 4 cell / well.

[0041] 4.1.2 Sample Addition Processing After 24 hours of plating, the cell fusion rate was 40-60%. Different concentrations of the test substance were added to achieve the test concentration, and three parallel test wells were set up. The positive control group and the sample group were incubated with the test substance for 24 hours, followed by stimulation with the stimulant for 6 hours in the negative control group, positive control group, sample group, and test group. The sample addition information for each group is shown in Table 3.

[0042] Table 3 Sampling Information Table 4.1.3 Immunofluorescence staining After 6 hours of CTAC stimulation, the culture medium was aspirated and the cells were washed three times with PBS.

[0043] DCFH-DA was diluted with PBS at a dilution ratio of 1:500.

[0044] Add DCFH-DA working solution and incubate at 37°C for 30 minutes. Aspirate the liquid and wash three times with washing solution, 3-5 minutes each time.

[0045] Observed under a fluorescence microscope. ROS staining shows green fluorescence.

[0046] V. Judgment Basis If the expression level of ROS in human immortalized keratinocytes is significantly lower than that in the negative control group under the action of the sample group, it is determined that the sample can effectively downregulate the expression of ROS in human immortalized keratinocytes.

[0047] VI. Test Results Experimental results are as follows Figure 4 and 5As shown in the figure, green fluorescence represents ROS. The green fluorescence intensity of the SSC group was significantly lower than that of the CTAC group, indicating that ROS expression in the SC group was significantly lower than that in the CTAC group. Where # indicates a significant difference from the BC group; * indicates a significant difference from the CTAC group; * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001; **** indicates P-value < 0.0001.

[0048] VII. Experimental Conclusions The test substance SSC reduced the expression of ROS in human immortalized keratinocytes at the tested concentration.

[0049] Experiment Example 3: CTAC stimulation of the epidermal model to detect changes in tissue morphology, barrier proteins and inflammatory factors.

[0050] 1. Experimental Objective Skin irritation refers to reversible damage to the skin after contact with a substance. A reconstructed human epidermal model is a tissue model with a similar structure and function to normal skin, obtained by using human skin-derived cells and tissue engineering techniques for three-dimensional structural reconstruction. It can be used to replace live animals in experiments. This test uses 3D skin as the research object and evaluates the protective efficacy of the raw material on the CTAC-stimulated epidermal model by testing changes in 3D skin tissue morphology, barrier-related proteins, and inflammatory factors under CTAC stimulation.

[0051] 2. Scope of Application It is suitable for determining the skin irritation of cosmetics and chemicals.

[0052] 3. Test materials 3.1 Experimental Model Epskin-3D skin model.

[0053] 3.2 Main Equipment Biosafety cabinet, constant temperature water bath, carbon dioxide incubator, multi-functional microplate reader, 6-well plate, 24-well plate, sterile forceps, absorbent paper, sterile cotton swabs, sealing film.

[0054] 3.3 Experimental Reagents PBS, Epi growth medium, Epi maintenance medium, Human IL-1α Elisa Kit, Human IL-8 Elisa Kit, Human PGE2 Elisa Kit.

[0055] 4. Experimental Design 4.1 Acceptance and Verification of Skin Models Upon receiving the skin model, open the outer packaging. Visually inspect the interface between the model and the solid culture medium for air bubbles. If obvious air bubbles are found at the interface, the model should be discarded.

[0056] 4.2 Model Recovery 1) Prepare sufficient sterile 6-well plates (one 6-well plate for every 3 models). After removing the outer packaging of the 6-well plates, place them in a laminar flow hood. Add 0.9 mL of growth medium to each well of the 6-well plate, and store the remaining growth medium in a 4°C refrigerator for later use.

[0057] 2) Open the inner packaging bag of the model and observe whether there is any liquid seepage or shrinkage on the surface and around it. If there is a lot of water on the surface, it should be discarded.

[0058] 3) Use sterile tweezers to remove the model along the upper edge of the plastic wall, and gently wipe away the agar or other liquid from the bottom of the model on the non-woven fabric provided in the model packaging plate.

[0059] 4) Place three molds in the first row of a 6-well plate containing growth medium. When placing the molds, tilt the bottom of the molds slightly to ensure full contact between the molds and the growth medium and prevent air bubbles from forming.

[0060] 5) Transfer all the 6-well plates containing the model to a CO2 incubator (37±1℃, 5±1%CO2, 95% relative humidity) and incubate for 60±5 min.

[0061] 6) After the culture is completed, use sterile forceps to transfer the model from the first row of the 6-well plate to the second row of wells, and then put the 6-well plate back into the CO2 incubator (37±1℃, 5±1%CO2, 95% relative humidity) for overnight incubation (18±3h).

[0062] 4.3 Test Procedure 1) CTAC stimulation: Prepare a 30 μg / mL CTAC solution and stimulate the epidermal models of the NC / PC / sample groups. Apply 80 μL of the drug to the skin and stimulate for 0.5 h. Place at room temperature. Repeat the process for 3 epidermal models in each group. For the BC group, the drug administration operation is not carried out, only the model is cleaned and followed up.

[0063] 2) Cleaning: After administration of the test substance, the cleaning procedure begins. Clean the surface of the model with a wash bottle containing sterile PBS solution to remove any remaining test substance, ensuring a constant and uninterrupted flow of PBS. Repeat the cleaning 15 times. Then, immerse the inner and outer surfaces of the model in a beaker containing sterile PBS solution, and gently wipe away any remaining liquid with a sterile cotton swab. The total cleaning time for each model should be controlled between 1 and 2 minutes to ensure consistent administration times for this model and subsequent models in the experiment.

[0064] 3) Sample protection: Prepare working solutions of different concentrations of test substance according to experimental groups. After 3D skin cleansing, administer the drug to the groups. The PC group is administered the drug submerged, while the sample group is administered the drug topically. The dosage is 25 μl. After administration, place the 6-well plate in an incubator (37℃, 5% CO2) for 24 h.

[0065] Table 4 Sample Addition Information Table 4) Collect the supernatant: Collect the subcutaneous solution after the sample has been protected for 24 hours.

[0066] 5) Centrifugation: Centrifuge at 12000 rpm for 5 min, discard the precipitate, and store at -20℃.

[0067] 6) Kit testing: Follow the instructions in the ELISA kit for subsequent operations.

[0068] 7) Skin model collection: After cleaning, gently wipe away any residual liquid inside and outside the model with a sterile cotton swab. Circumferentially section the model, fix it in 4% paraformaldehyde, embed it in paraffin, section it, and perform HE staining and immunofluorescence staining for detection.

[0069] V. Test Results Experimental results are as follows Figures 6-14 As shown. Figures 6-7 In the study, the thickness of viable epidermal cells in the SSC group was significantly increased compared to the CTAC group. Figures 8-9 In the diagram, green fluorescence represents FLG expression. The green fluorescence in the SSC group was significantly increased compared to the CTAC group, indicating that FLG expression was enhanced. Figures 10-11 In the diagram, green fluorescence represents NRF2 expression. The green fluorescence in the SSC group was significantly increased compared to the CTAC group, indicating that NRF2 expression was enhanced. Figures 12-14 In the study, the levels of IL-6, IL-8, and PGE2 in the SSC group were significantly lower than those in the CTAC group. The values ​​are indicated by: # indicating a significant difference from the BC group; * indicating a significant difference from the CTAC group; * indicating P-value < 0.05; ** indicating P-value < 0.01; *** indicating P-value < 0.001; **** indicating P-value < 0.0001.

[0070] VI. Experimental Conclusions Compared with the CTAC group, SSC reduced the production of IL-8, IL-1α and PGE2 at the tested concentration, increased endogenous NRF2, and increased filaggrin FLG expression, thus exhibiting a repairing effect.

[0071] Experiment Example 4: Nile Red staining experiment to evaluate the oil control effect of samples in sebaceous gland cells.

[0072] I. Principle Nile Red staining is a fluorescent staining technique commonly used to assess oil content, and it plays an important role in evaluating the oil-controlling effects of cosmetics (such as skincare products). Utilizing the specific binding characteristics of Nile Red to oils, changes in oil content can be reflected by the intensity of the fluorescence signal, thus determining the effectiveness of oil-controlling products. Nile Red is a lipophilic fluorescent dye with a highly lipid-soluble molecular structure. In oils, Nile Red emits strong fluorescence (maximum excitation wavelength approximately 550 nm, emission wavelength approximately 630 nm, exhibiting red fluorescence); its fluorescence intensity decreases significantly (almost no fluorescence) in water or polar solvents. Nile Red embeds itself into the hydrophobic structure of oils (such as triglycerides, fatty acids, sebum, etc.) through hydrophobic interactions, forming a stable bond, thus exhibiting a clear fluorescence signal under a fluorescence microscope or fluorescence spectrophotometer.

[0073] II. Instruments and Equipment Clean bench, CO2 incubator, fluorescence microscope, centrifuge, cell counting chamber, cell culture dish.

[0074] III. Reagents and Materials Cell material: Human sebaceous gland cells SZ95.

[0075] Reagents: DMEM high glucose culture medium (pyruvate-free), fetal bovine serum, penicillin-dextrin antibiotics, PBS, trypsin, isotretinoin, Nile red staining solution.

[0076] IV. Operating Procedures 4.1 Test substance pretreatment and test grouping 4.1.1 Cell Plating After cell counting, dilute to the desired concentration and add cell suspension to the cell culture plate to achieve a culture volume of 1 mL / well and a viable cell count of 5 × 10⁶ cells / well. 4 cell / well.

[0077] 4.1.2 Sample Addition Processing After 24 hours of plating, the cell fusion rate was 40-60%. Different concentrations of the test substance were added to achieve the test concentration, and the test wells were set up in triplicate. The cells were cultured for another 24 hours. The sample addition information for the blank group, negative control group, positive control group, and sample group is shown in Table 5.

[0078] Table 5. Nile Red Staining Sample Application Information Table 4.1.3 Nile Red Staining Rinse: Discard the culture medium and wash 3 times with PBS; Fixation: Add 4% paraformaldehyde (300 µL) for 30 min; Staining: Prepare Nile Red staining solution (working concentration is 10 μg / mL), discard the fixative, rinse the cells 3 times with PBS, add Nile Red staining solution (300 µL) and stain for 15 min; Photography: Observe the staining of cells in each group under an inverted fluorescence microscope and take photos.

[0079] Results analysis: The fluorescence intensity was quantitatively analyzed using Image Pro Plus software.

[0080] V. Judgment Basis The fluorescence intensity emitted by Nile Red after binding with lipids is positively correlated with the lipid content in the sample. The presence of a distinct red area within cells indicates the presence of lipids, and a deeper color generally indicates a relatively high lipid content.

[0081] VI. Test Results Experimental results are as follows Figures 15-16 As shown in the figure, the red fluorescence of the SSC group was significantly lower than that of the CTAC group, indicating a reduction in lipid droplet secretion; where # indicates a significant difference between the LA and BC groups; * indicates a significant difference between the sample group and the BC group; * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001; **** indicates P-value < 0.0001.

[0082] VII. Experimental Conclusions Compared with the control group, the test substance reduced the secretion of sebum from sebaceous gland cells at the tested concentration.

[0083] Experiment Example 5: Immunofluorescence staining to assess the expression of PPARγ and SREBP1 in sebaceous gland cells.

[0084] I. Principle PPARγ (peroxisome proliferator-activated receptor γ) and SREBP1 (sterol regulatory element-binding protein 1) are core transcription factors in cellular lipid metabolism, energy homeostasis, and inflammation regulation. They play crucial roles in adipocyte differentiation, lipid synthesis, and glucose metabolism, and exhibit close synergistic and cross-regulatory functions. Abnormal expression of these proteins is closely associated with metabolic diseases such as obesity, type 2 diabetes, and fatty liver. Downregulating the expression of PPARγ and SREBP1 proteins in cells can help control lipid production.

[0085] II. Instruments and Equipment Microbalance, clean bench, carbon dioxide incubator, fluorescence microscope.

[0086] III. Reagents and Materials Cell material: SZ95 sebaceous gland cells Reagents: rabbit PPARγ antibody, rabbit SREBP1 antibody, DAPI staining solution, paraformaldehyde fixative, PBS, immunostaining blocking solution, immunostaining permeabilization solution.

[0087] IV. Operating Procedures 4.1 Test substance pretreatment and test grouping 4.1.1 Cell Plating After cell counting, dilute to the desired concentration and add cell suspension to cell culture dishes to achieve a culture volume of 1 mL / well and a viable cell count of 5 × 10⁶ cells / well. 4 cell / well.

[0088] 4.1.2 Sample Addition Processing After 24 hours of plating, the cell fusion rate was 40-60%. Different concentrations of the test substance were added to achieve the test concentration, and the test wells were set up in triplicate. The cells were cultured for another 24 hours. The sample addition information for the blank group, negative control group, positive control group, and sample group is shown in Table 5.

[0089] Table 6. Immunofluorescence staining sample loading information 4.1.3 Sample Addition Processing After 24 hours of sample addition, the culture medium was aspirated and the sample was washed three times with PBS.

[0090] Add fixative and fix at 37°C for 30 minutes. Remove the fixative and wash three times with washing buffer for 3-5 minutes each time.

[0091] Add immunostaining permeabilization buffer and permeabilize at room temperature for 10 minutes. Aspirate the permeabilization buffer and wash three times with washing buffer, 3-5 minutes each time.

[0092] Add immunostaining blocking solution and incubate at room temperature for 60 minutes. The amount of immunostaining blocking solution should be sufficient to cover the sample; for 24-well plates, 0.5 mL of immunostaining blocking solution is usually added.

[0093] Remove the immunostaining blocking solution, add rabbit PPARγ antibody and rabbit SREBP1 antibody respectively, and incubate overnight at 4°C in the dark. Aspirate the antibody and wash three times with washing buffer for 5-10 minutes each time.

[0094] Add secondary antibody and incubate at 37°C for 1 hour. Wash three times with washing buffer, 5-10 minutes each time.

[0095] Add DAPI, the nuclear staining solution, and stain at room temperature for 10 minutes. Aspirate the nuclear staining solution and wash three times with washing buffer, 3-5 minutes each time.

[0096] Observe under a fluorescence microscope.

[0097] V. Judgment Basis If the expression levels of PPARγ and SREBP1 in SZ95 cells under the action of the sample group are significantly lower than those in the CTAC group, then the sample can be judged to effectively downregulate the expression of PPARγ and SREBP1 in SZ95 cells and has an oil-controlling effect.

[0098] VI. Test Results Experimental results are as follows Figures 17-20 As shown, Figures 17-18 In the diagram, green fluorescence represents PPARγ expression. The green fluorescence in the SSC group was significantly lower than that in the CTAC group, indicating that PPARγ expression was reduced. Figures 19-20 In the diagram, green fluorescence represents SREBP1 expression. The green fluorescence in the SSC group was significantly lower than that in the CTAC group, indicating a reduction in SREBP1 expression. # indicates a significant difference between the CTAC and BC groups; * indicates a significant difference between the sample group and the CTAC group; * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001; **** indicates P-value < 0.0001.

[0099] VII. Experimental Conclusions Compared with the control group, the test substance at the tested concentration reduced the expression of PPARγ and SREBP1, and had the effect of reducing abnormal sebum secretion.

[0100] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Those skilled in the art can modify the technical solutions described in the above embodiments, or make equivalent substitutions for some of the technical features; and all such modifications and substitutions should fall within the protection scope of the appended claims of the present invention.

Claims

1. A composition of plant extracts, characterized in that: It includes Scutellaria baicalensis extract, Willowherb extract and Lavender extract in a mass ratio of 1:1:

1.

2. The use of the plant extract composition according to claim 1 in the preparation of products that reduce the skin irritation caused by surfactants.

3. The use of the plant extract composition according to claim 2 in the preparation of a product having the effect of reducing the skin irritation of surfactants, characterized in that: The stimulating effects include skin irritation, skin inflammation, skin barrier dysfunction, skin oxidative stress, and sebum production.

4. The use of the plant extract composition according to claim 2 in the preparation of a product having the effect of reducing the skin irritation of surfactants, characterized in that: The product in question is a repair product or a washing and care product.

5. The use of the plant extract composition according to claim 2 in the preparation of a product having the effect of reducing the skin irritation of surfactants, characterized in that: The surfactant is a cationic surfactant.

6. The use of the plant extract composition according to claim 5 in the preparation of a product having the effect of reducing the skin irritation of surfactants, characterized in that: The cationic surfactant is hexadecyltrimethylammonium chloride.

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