Application of salicin in preparing and / or serving as exfoliating product

Abstract

The invention relates to the technical field of cosmetics, in particular to application of salicin in preparing and / or serving as an exfoliating product. Studies find that salicin can improve the level of KLK7 and significantly reduce the thickness of skin cuticle, has no obvious irritation, and also can significantly reduce the content of SPINK5, which indicates that salicin can be used for preparing and / or serving as an exfoliating product. The invention provides a new raw material salicin for the development of exfoliating products, and can realize the effects of no skin irritation and mild exfoliating.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics, and in particular to application of salicin in preparation and / or as an exfoliating product. Background Art

[0002] When the skin metabolism becomes irregular due to aging or other reasons, the stratum corneum cells cannot carry out normal metabolism. The excess keratin cells "semi-adhere" to the surface, making it difficult for the skin to absorb daily skin care products. Secondly, it will also cause the skin to look less smooth, affecting the appearance. At this time, exfoliation is needed to accelerate the renewal of skin cells and let the excess keratin on the skin surface fall off. This can not only restore the skin to a smooth and vibrant state, but also have the effect of removing wrinkles and anti-aging.

[0003] Exfoliation can be achieved by enzymatically hydrolyzing the desmosome adhesion proteins (desmosome core proteins, desmocollins) between keratinocytes. The main enzymes for enzymatic hydrolysis of desmosome adhesion proteins are: steroid sulfatase and serine protease. Among them, the key protease is the kallikrein (KLK), a kallikrein-related peptidase expressed by keratinocytes in the stratum granulosum and stratum corneum. KLK belongs to the serine protease family, including KLK7 and KLK5. Under physiological conditions, serine protease inhibitors (LEKTI) inhibit the activity of KLK7 and KLK5, making them inactive. When the epidermis migrates to the stratum granulosum, LEKTI is gradually released into the intercellular matrix, and the inhibition of KLK7 and KLK5 is weakened or disappears. KLK7 hydrolyzes desmocollin 1, and KLK5 hydrolyzes desmocollin 1, so that keratinocytes reach the stratum corneum of the epidermis and fall off.

[0004] Among the exfoliating ingredients in existing products, salicylic acid is a commonly used ingredient, but it has certain skin irritation: its use can cause dryness, redness, swelling, stinging and other discomfort. Therefore, it is urgent to find new, milder and non-irritating exfoliating active ingredients. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the use of salicin in the preparation and / or as an exfoliating product.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides the use of salicin in the preparation and / or as an exfoliating product.

[0008] The present invention has found that salicin can increase the level of KLK7, significantly reduce the thickness of the stratum corneum of the skin, has no obvious irritation, and can also significantly reduce the content of SPINK5, which indicates that salicin can be used to prepare and / or be used as an exfoliating product. The present invention provides a new raw material for the development of exfoliating products, namely salicin, which can achieve the effect of mild exfoliation without skin irritation.

[0009] Preferably, the effective concentration of salicin is 0.5%-10%.

[0010] Preferably, the salicin achieves the exfoliating effect by acting on KLK5, KLK7, LEKTI and STS.

[0011] In the docking analysis of salicin molecules and protein molecules, the present invention found that salicin molecules had good binding with KLK5, KLK7, LEKTI, STS protein molecules and salicin molecules.

[0012] Preferably, the salicin increases the content of KLK7 and decreases the content of SPINK5 protein, thereby promoting the shedding of keratinocytes and reducing the thickness of the stratum corneum, thereby achieving the effect of exfoliation.

[0013] The present invention is based on 3D epidermal skin model The exfoliation effect of salicylic acid was investigated by detecting the changes in tissue morphology, kallikrein 7 (KLK7), serine peptidase inhibitor Kazal type 5 (SPINK5) recombinant protein and total protein content. The results showed that salicin can achieve exfoliation effect by acting on KLK7 and SPINK5.

[0014] In a second aspect, the present invention provides a cosmetic, wherein the active ingredient of the cosmetic includes salicin.

[0015] Preferably, the cosmetic is an exfoliating product.

[0016] Preferably, the cosmetics include leave-on cosmetics and rinse-off cosmetics.

[0017] The beneficial effects of the present invention are:

[0018] The present invention has found that salicin can increase the level of KLK7, significantly reduce the thickness of the stratum corneum of the skin, has no obvious irritation, and can also significantly reduce the content of SPINK5, which indicates that salicin can be used to prepare and / or be used as an exfoliating product. The present invention provides a new raw material for the development of exfoliating products, namely salicin, which can achieve the effect of mild exfoliation without skin irritation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is the result of total protein in exfoliated stratum corneum cells.

[0020] Figure 2 This is a test diagram of salicin 3D skin tissue morphology.

[0021] Figure 3 This is the result of salicin 3D skin KLK7 immunofluorescence.

[0022] Figure 4 This is the result of SPINK5 immunofluorescence on salicin 3D skin.

[0023] Figure 5 This is the result of total protein of stratum corneum cells exfoliated from the skin by salicin 3D. DETAILED DESCRIPTION

[0024] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0025] Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0026] Unless otherwise specified, all raw materials involved in the following examples and comparative examples are commercially available general materials.

[0027] The salicin raw material used in the present invention is commercially available salicin with a concentration of 70%, which was diluted with sterile water in the experiment.

[0028] Example 1: In vitro pig skin experiment to determine the exfoliating effect of salicin

[0029] 1. After thawing and cleaning the pig skin, fix the skin between the supply chamber and the receiving chamber of the Franz cell diffusion tank, with the stratum corneum facing the supply chamber and the dermis facing the receiving chamber; add receiving solution (PBS) to the receiving chamber, tighten and fix the pig skin, add a small amount of receiving solution to the receiving chamber through the sampler, exhaust the air, and make the dermis layer of the skin in close contact with the receiving solution.

[0030] 2. Sample application: Add 50 μL of sample (0.5% and 1% salicin solution) to the surface of pig skin (PBS buffer was added to the surface of the control group as a blank control), and spread the sample evenly from the center of the skin to the edge in a radial pattern. Repeat 3 times for each sample in parallel, keep the water bath at 32°C, and ensure that there are no bubbles in the water bath.

[0031] 3. Sample collection: After incubation for 24 hours, put on a finger cot and rub the sample application area. After rubbing for two minutes, add 0.5 mL of cleaning solution (0.1% Triton X-100) to the supply chamber, blow and clean the keratinocytes shed from the skin surface, put the collected cleaning solution into a high-speed centrifuge, and collect the keratinocytes by centrifugation.

[0032] 4. Lysis: After resuspending the above keratinocytes in deionized water, add 25 μL of 12M NaOH solution to the liquid, boil in a water bath for 30 minutes to lyse the keratinocytes, and finally add 25 μL of 12M HCl solution to neutralize the above lysate.

[0033] 5. Determination of total protein by BCA method: Determine total protein according to the operating instructions of the BCA test kit.

[0034] Salicin exfoliation test of pig skin, the results of total protein of detached stratum corneum cells are as follows Figure 1 As shown in the figure, the total protein content of the sample salicin group was significantly increased compared with the normal control group, revealing that the sample salicin has the effect of exfoliating.

[0035] Example 2: Docking analysis of salicin molecule and protein molecule

[0036] The present invention uses a CADD system to perform docking analysis between salicin molecules and protein molecules. First, the protein 3D structure is downloaded from the PDB database. The protein structure is checked using PyMOL 2.5.4 software for docking. The salicin molecule (Pubchem CID: 439503) is downloaded from the PubChem database, and the small molecule structure is optimized using the MMFF94 force field of OpenBabel3.1.1 software, and the optimal molecular structure with the lowest energy state is finally obtained. Then, the protein is hydrogenated using AutoDockTools1.5.6 software, and the small molecule is hydrogenated and the torsion bond is determined. The molecular docking range parameters are set using the Grid panel. The docking mode is set to semi-flexible docking, the docking precision exhaustiveness is 25, and the docking algorithm is the Lamarckian genetic algorithm. Finally, the Auto Dock Vina 1.2.0 software is run to perform molecular docking, and the docking binding free energy and docking results are obtained.

[0037] After research and analysis, it was found that the docking of tissue kallikrein 5 (KLK5), tissue kallikrein 7 (KLK7), serine protease inhibitor (LEKTI), steroid sulfatase (STS) protein molecules and salicin molecules all had good binding. That is, in the model analysis, salicin can achieve the exfoliating effect by acting on KLK5, KLK7, LEKTI, and STS.

[0038] Example 3: 3D skin in vitro exfoliation efficacy experiment

[0039] 1. The groups are shown in Table 1.

[0040] Table 1 Test groups

[0041]

[0042] 2. Drug administration: According to the experimental grouping, the model was transferred to a 6-well plate (0.9 mL EpiGrowth culture medium was added in advance), and the experimental group number was marked on the 6-well plate. The BC group did not receive any treatment, the PC group added the corresponding concentration of working solution, and the sample group evenly distributed the sample working solution on the surface of the model, placed in a CO2 incubator (37°C, 5% CO2) for 24 hours, rubbed and applied for 2 minutes, and washed twice with cleaning solution (0.1% TritonX-100), 250 μL each time, and the cleaning solution was collected for later use.

[0043] 3. Tissue morphology test: Take the model for detection, fix it with 4% paraformaldehyde, and after fixation for 24 hours, perform H&E staining, observe under a microscope, collect pictures and analyze them.

[0044] 4. Immunofluorescence test: Take the model to be tested, fix it with 4% paraformaldehyde, and after fixation for 24 hours, perform immunofluorescence test, take pictures under a microscope, collect pictures and analyze them.

[0045] 5. Protein content test: Detect the total protein content according to the instructions of the BCA protein assay kit.

[0046] 6. Calculate the enhancement rate and inhibition rate according to formula (1) and (2).

[0047] Improvement rate (%) = (sample group - BC) / BC × 100% - Formula (1)

[0048] Inhibition rate (%) = (BC-sample group) / BC×100% - Formula (2)

[0049] Salicin 3D skin in vitro exfoliation efficacy experiment, tissue morphology test results are as follows Figure 2 As shown in Table 2; Kallikrein 7 (KLK7) test results are as follows Figure 3 As shown in Table 3; The test results of serine peptidase inhibitor Kazal type 5 (SPINK5) recombinant protein are as follows Figure 4 As shown in Table 4; The results of total protein of detached stratum corneum cells are as follows Figure 5 And as shown in Table 5.

[0050] Based on 3D epidermal skin model Compared with the control group, the thickness of the stratum corneum decreased significantly at a concentration of 3% (m / v) in the sample salicin, with an inhibition rate of 43.28%; the content of kallikrein 7 (KLK7) and the total protein content of the detached stratum corneum cells increased significantly, with an increase rate of 71.00% and 71.43% respectively; the content of the recombinant protein of the serine peptidase inhibitor Kazal type 5 (SPINK5) decreased significantly, with an inhibition rate of 32.00%. This indicates that salicin can promote the shedding of keratinocytes, reduce the thickness of the stratum corneum, and achieve the effect of exfoliation by increasing the content of kallikrein 7 (KLK7) and reducing the content of the recombinant protein of the serine peptidase inhibitor Kazal type 5 (SPINK5), which means that salicin has an exfoliating effect.

[0051] Table 2 Summary of stratum corneum thickness results

[0052]

[0053] Table 3 Summary of KLK7 immunofluorescence analysis results

[0054]

[0055]

[0056] Table 4 Summary of SPINK5 immunofluorescence analysis results

[0057]

[0058] Table 5 Summary of the results of total protein of detached stratum corneum cells

[0059]

[0060] Example 4: Chicken chorioallantoic membrane test for cosmetic eye irritation / corrosion

[0061] 1. Experimental process:

[0062] CAM preparation: 9-day-old chick embryos were candled and the eggshell of the air chamber was peeled off with dental serrated curved forceps to expose the white egg membrane, and the integrity of the egg membrane was carefully not damaged. A drop of 0.9% sodium chloride (NaCl) solution was dripped with a pipette to moisten the egg membrane, and the inner membrane was carefully removed with forceps to ensure that the vascular membrane was not damaged.

[0063] Reaction time method: Take 0.3 mL of the test substance and apply it to the CAM, ensuring that at least 50% of the CAM surface is covered by the test substance. Observe the CAM reaction and record the time of occurrence of each toxic effect within 5 minutes of action.

[0064] The test samples were: salicylic acid with a concentration of 1.25%, and salicin with a concentration of 2%. Six chicken embryos were set for each sample, and one chicken embryo each for the negative control (olive oil) and the positive control (1% SDS).

[0065] Result evaluation: Stimulus score method, the test was conducted using the reaction time method, and the stimulation score (IS) was calculated according to formula (3), and the result was rounded to two decimal places:

[0066] IS=(301-secH)*5 / 300+(301-secL)*7 / 300+(301-secC)*9 / 300——Formula (3)

[0067] In the above formula: secH (bleeding time) - the average time when bleeding begins to be observed on the CAM membrane, in seconds (s);

[0068] secL (vascular lysis time) - the average time in seconds (s) when vascular lysis begins to be observed on the CAM membrane;

[0069] secC (clotting time) - the average time in seconds (s) for the onset of coagulation to be observed on the CAM membrane.

[0070] Based on the calculated IS values, classify the eye irritation of the test substances according to Table 6.

[0071] Table 6 Evaluation of stimulation scoring method results

[0072]

[0073] 2. Result evaluation

[0074] The results of the eye irritation test on salicylic acid and salicin are shown in Table 7.

[0075] Table 7 Eye irritation scoring results

[0076]

[0077]

[0078] As shown in Table 7, salicin at a concentration of 2% is non-irritating, while salicylic acid at 1.25% is moderately irritating at a concentration lower than the tested concentration of salicin, indicating that compared with salicylic acid, salicin is mild and non-irritating in the chicken embryo eye irritation test.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of salicin in the preparation and / or as an exfoliating product.

2. The use according to claim 1, characterized in that The effective concentration of salicin is 0.5%-10%.

3. The use according to claim 1, characterized in that The salicin achieves the exfoliating effect by acting on KLK5, KLK7, LEKTI and STS.

4. The use according to claim 1, characterized in that The salicin increases the content of KLK7 and decreases the content of SPINK5 protein, thereby promoting the shedding of keratinocytes and reducing the thickness of the stratum corneum, thereby achieving the effect of exfoliation.

5. A cosmetic, characterized in that: The active ingredient of the cosmetic includes salicin.

6. The cosmetic according to claim 5, characterized in that The cosmetic is an exfoliating product.

7. The cosmetic according to claim 5, characterized in that The cosmetics include leave-on cosmetics and rinse-off cosmetics.

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