Composition containing anoectochilus formosanus extract, collagen I and collagen III and having repairing effect and application of composition

Abstract

The invention discloses a composition containing an anoectochilus formosanus extract, I-type collagen and III-type collagen and having a repairing effect and application of the composition. The composition is prepared by compounding the anoectochilus formosanus extract, the I-type collagen and the III-type collagen according to the mass ratio of (60-200): 1: 3. The invention provides a new recorded raw material, namely the anoectochilus formosanus extract, and the composition prepared by compounding the anoectochilus formosanus extract and the two collagens can be applied to repair cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of cosmetic raw materials, and in particular to a composition prepared by compounding a roxburghii roxburghii extract with type I collagen and type III collagen in a specific ratio and an application thereof. Background Art

[0002] The core function of cosmetics is only to protect the epidermis. The more superficial the effect is, the more obvious it is, and the deeper the effect is, the smaller the effect is. The limit of the effect of cosmetics is the basal layer of the skin's epidermis.

[0003] The skin can be roughly divided into the epidermis, dermis and subcutaneous tissue. The epidermis can be further divided into the stratum corneum (mainly dead cells), stratum granulosum (mainly containing keratin, etc.), stratum spinosum (mainly containing tight junction proteins such as Claudin-5), and stratum basale (mainly containing laminin, which is closely related to the fibroblasts in the dermis, etc.) from top to bottom.

[0004] Anoectochilus roxburghii is a perennial herbaceous plant of the genus Orchidaceae. It is recorded in monographs such as the Chinese Medicine Dictionary, the National Collection of Chinese Herbal Medicines, the Chinese Materia Medica, and the Fujian Pharmacopoeia. It is rich in polysaccharides, steroid compounds, organic acids, terpenoids, alkaloids, volatile oils, flavonoids, and glycosides, and has anti-inflammatory, anti-tumor, liver protection, and antioxidant biological activities. The present invention found that the extract of Anoectochilus roxburghii can not only inhibit the 9 major stimulatory factors (TRPV1, Ca 2+ Ions, NF-κB, IL-1α, IL-1β, IL-8, TNF-α, COX-2, PCE2), and can also promote the production of four major barrier proteins in the epidermis (FLG, LOR, IVL, TGM1), but cannot play a role in the barrier repair in the dermis.

[0005] Recombinant collagen raw materials include type I collagen and type III collagen. Due to their high processing difficulty and high price, they are more often used in anti-wrinkle and firming cosmetics, and rarely in repair cosmetics. Type I collagen is the most abundant type of collagen in the skin, accounting for more than 80% of human collagen. It can promote the proliferation of epidermal cells and is beneficial to epidermal repair. When the skin already has micro-injuries or micro-damages that are visible or invisible to the naked eye, the skin barrier has been opened and macromolecules can also enter; and type I collagen can promote wound healing and tissue reconstruction, which is beneficial to improve cell microcirculation, promote skin metabolism, and accelerate tissue regeneration. Type III collagen is mainly distributed around type I collagen at the junction of the epidermis and dermis, and its shape is loose and small. Type III collagen is higher in infancy and is called "baby collagen". It has a good nutritional recovery and repair and healing effect. Type I and type III collagen can promote the barrier repair effect at the junction of the true epidermis, but the repair effect on the epidermis is weak.

[0006] Therefore, the present invention compounds the extract of Anoectochilus roxburghii with type I collagen and type III collagen, which has a strong repairing effect on the junction between the epidermis and the true epidermis. Summary of the invention

[0007] The purpose of the present invention is to provide a compound of aroximate extract and type I collagen and type III collagen and its application, wherein the compound can play a strong repairing effect from the junction of the skin epidermis to the true epidermis.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] A composition with repairing effect containing aroximate extract, type I collagen and type III collagen. The composition is prepared by compounding the aroximate extract, type I collagen and type III collagen in a mass ratio of (60-200):1:3.

[0010] Preferably, the preparation method of the extract of Anoectochilus roxburghii comprises the following steps: freeze-drying and crushing the fresh Anoectochilus roxburghii, extracting 1-2 times with water at a mass ratio of freeze-dried powder to pure water of 1:10 and a temperature of 10-40°C, each extraction time being 1-2h, and concentrating under reduced pressure and low temperature (temperature of 45°C-55°C and pressure range of 10 -2 -10 -5 mbar) to a sugar content of 65%-75%, and then filtered with a 300-mesh gauze to obtain a roxburghii roxburghii extract, the main active ingredient of which is total polysaccharide with a content of 10wt%-20wt%.

[0011] Application: The composition is used in repair cosmetics. The concentrations of roxburghii extract, type I collagen and type III collagen in cosmetics are 0.15wt%-0.5wt%, 0.0025wt% and 0.0075wt%, respectively.

[0012] The advantages of the present invention are:

[0013] The combination of golden thread lotus extract and a small amount of type I collagen and type III collagen significantly enhances the repair effect of the junction between the epidermis and the dermis, and plays a synergistic role in the repair effect of the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the histological morphology of the group of Anoectochilus roxburghii extract: (Type I: Type III) = 30: (1:3);

[0015] Figure 2 This is the histological morphology of the group of Anoectochilus roxburghii extract: (Type I: Type III) = 60: (1:3);

[0016] Figure 3This is the histological morphology of the group of Anoectochilus roxburghii extract: (Type I: Type III) = 120: (1:3);

[0017] Figure 4 This is the histological morphology of the group of Anoectochilus roxburghii extract: (Type I: Type III) = 200: (1:3);

[0018] Figure 5 This is the tissue morphology diagram of the group of Anoectochilus roxburghii extract: (Type I: Type III) = 300: (1:3). DETAILED DESCRIPTION

[0019] In order to make the above features and advantages of the present invention more clearly understood, the following embodiments are given for detailed description. The methods of the present invention are all conventional methods in the art unless otherwise specified.

[0020] The present invention was tested by the following experiments and standards:

[0021] 1. Based on human keratinocytes, test the cell activity of different concentrations of golden thread lotus extract, type I collagen, and type III collagen to determine their addition amount. When the cell activity is ≥80%, it is judged to be non-cytotoxic and belongs to a safe concentration.

[0022] 2. This experiment is based on the SDS-induced human keratinocyte (HaCaT) model, and enzyme-linked immunosorbent assay was used to detect the changes in the contents of keratin, claudin-5, and laminin to evaluate the repair efficacy of golden thread lotus extract, type I collagen, type III collagen and their combination.

[0023] 3. This experiment is based on the suckling pig skin-Franz cell system. By measuring the concentrations of the active ingredients of Anoectochilus roxburghii extract, type I collagen, type III collagen and their combination in the receiving fluid at different time points, their penetration behavior into the skin was evaluated.

[0024] 4. This experiment is based on a 3D skin model and evaluates the repair efficacy of different proportions of roxburghii extract and type I and type III collagen by detecting changes in tissue morphology.

[0025] Example 1

[0026] The preparation method of the extract of Anoectochilus roxburghii comprises the following steps: freeze-drying and crushing the fresh Anoectochilus roxburghii, extracting twice with water at a mass ratio of freeze-dried powder to pure water of 1:10 and a temperature of 35°C, with each extraction time of 1.5 hours, and concentrating under reduced pressure and low temperature (temperature of 50°C and pressure range of 10 -3 mbar) until the sugar content reached 70% and then filtered through 300-mesh gauze to obtain a roxburghii roxburghii extract, the main active ingredient of which was total polysaccharide with a content of 10wt%.

[0027] Solution preparation:

[0028] 1. Preparation of type I collagen solution: weigh 0.02 g of type I collagen and add 1.98 mL of deionized water to prepare a 1% stock solution. When using, dilute with the corresponding solvent to the corresponding concentration.

[0029] 2. Preparation of type III collagen solution: Weigh 0.02 g of type III collagen and add 1.98 mL of deionized water to prepare a 1% stock solution. When using, dilute with the corresponding solvent to the corresponding concentration.

[0030] Example 2

[0031] Cell activity test (MTT method)

[0032] 1. Plate: Take the cultured cells, digest and centrifuge, count, and configure the cell concentration to 3×10 5 / mL, pipette evenly, spread into 96-well plate, 100μL / well, and incubate in the incubator for 24h±2h.

[0033] 2. Administration: After 24h±2h, add the prepared sample solution (concentration is twice the test concentration), 100μL per well, set up 3 replicates, add the same volume of culture medium (with cells) to the blank group, and only add culture medium (without cells) to the solvent group, and continue incubation for 24h±2h.

[0034] 3. Test: After 24 h ± 2 h, discard the supernatant, add 200 μL of 0.5 mg / mL MTT solution to each well, and place in a CO2 incubator for 4 h.

[0035] 4. Test: After 4 hours, discard the supernatant, add 150 μL DMSO to each well, shake at room temperature in the dark for 10 minutes, and measure the absorbance at a wavelength of 490 nm.

[0036] Table 1 MTT test

[0037]

[0038] Therefore, 0.5% of the roxburghii extract, 0.0025% of the type I collagen, and 0.0075% of the type III collagen were selected as the maximum addition amount.

[0039] Example 3

[0040] This experiment was based on the SDS-induced human keratinocyte (HaCaT) model and used enzyme-linked immunosorbent assay to detect changes in the contents of keratin, Claudin-5, and laminin to evaluate the repair efficacy of roxburghii extract, type I collagen, type III collagen, a combination of type I collagen and type III collagen, and a combination of roxburghii extract and type I and type III collagen.

[0041] 1. Plate: digest and centrifuge the cultured cells, count them, and set the cell concentration to 3×10 5 pcs / mL, pipette evenly, add 1 mL to each well of a 12-well plate, and incubate in an incubator for 24±2 h.

[0042] 2. After 24±2h, the drug was administered.

[0043] 3. Test: Collect the cell supernatant and test the content of keratin, Claudin-5 and laminin using ELISA kit.

[0044] (1) Take out the keratin, claudin-5, and laminin kits from the refrigerator 1 hour in advance and equilibrate to room temperature. Take out the collected supernatant from the -80°C refrigerator in advance, thaw it, and centrifuge it at 10,000 rpm for 20 minutes.

[0045] (2) Take 2 mL of 20× washing buffer and prepare washing solution with 38 mL of distilled water. Take out the strip from the aluminum foil bag and set up standard wells and sample wells. Add 100 μL of each standard of 8 concentrations to the standard wells, and add 100 μL of each collected cell supernatant to the sample wells. Cover with a sealing film and incubate at 37°C for 60 min.

[0046] (3) After 60 min, remove the ELISA plate, discard the liquid, add 100 μL of biotinylated antibody working solution to each well, cover with sealing film, and incubate in a 37°C incubator for 60 min.

[0047] (4) After 60 minutes, remove the ELISA plate, discard the liquid, add 300 μL of the prepared washing solution to each well, let it stand for 1 minute, discard the washing solution, pat dry on absorbent paper, and repeat the washing three times. Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film, and incubate at 37°C incubator for 30 minutes.

[0048] (5) After 30 minutes, remove the ELISA plate, discard the liquid, add 300 μL of the prepared washing solution to each well, let it stand for 1 minute, discard the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times. Add 90 μL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37°C for 15 minutes.

[0049] (6) After 15 min, remove the ELISA plate, add 50 μL of stop solution to each well, immediately measure the OD value of each well at a wavelength of 450 nm, and calculate the upregulation rate of each protein content.

[0050] This experiment is based on the suckling pig skin-Franz cell system. The concentrations of the active ingredients of Anoectochilus roxburghii extract and type I and type III collagen in the receiving solution at different time points were measured to evaluate their penetration behavior into the skin.

[0051] 1. Preparation of ex vivo suckling pig skin: Thaw the back skin of one-month-old suckling pig stored at -20°C with deionized water at room temperature and rinse repeatedly with PBS buffer.

[0052] 2. In vitro penetration test of active substances: Fix the skin between the supply chamber and the receiving chamber of the Franz cell diffusion pool, with the stratum corneum facing the supply chamber and the dermis facing the receiving chamber; add receiving solution to the receiving chamber, exhaust the air, and make the dermis layer of the skin in close contact with the receiving solution; add samples to the skin surface in the supply chamber, and record the effective penetration area S. Take sample solutions at 2h, 4h, 6h, 8h, 10h, and 12h respectively, and use a sampler to extract the receiving solution through the sampling tube. In addition, add an equal amount of receiving solution after each sampling.

[0053] 3. Test of the effective ingredient transdermal content: After 12 hours of penetration, the recovered liquids of different experimental groups and the blank group were tested for the content of the effective ingredient. The specific processing methods are as follows:

[0054] (1) Test of the content of type I soluble protein, the effective component of type I collagen: The qT-PCR method was used to determine the content of type I collagen in the recovered solution of type I collagen at different time points, the recovered solution of type I collagen and type III collagen in different proportions, and the recovered solution of Anoectochilus roxburghii extract and type I collagen and type III collagen in different proportions;

[0055] (2) Test of the content of type III soluble protein, the effective component of type III collagen: The qT-PCR method was used to determine the content of type III collagen in the recovered solution of type III collagen at different time points, the recovered solution of different proportions of type I collagen and type III collagen, and the recovered solution of different proportions of the extract of Anoectochilus roxburghii and type I collagen and type III collagen in a fixed ratio;

[0056] (3) Calculate the upregulation rate of polysaccharide content and type I and III collagen, that is, the permeability upregulation rate.

[0057] Permeation upregulation rate = (permeation amount of drug administration group - permeation amount of blank group) / permeation amount of blank group * 100%

[0058] Table 2 Repair efficacy and permeability test of Anoectochilus roxburghii extract, type I collagen, and type III collagen

[0059]

[0060] The experimental results show that 0.15%, 0.3%, 0.5% of golden thread lotus extract, 0.0025% of type I collagen, and 0.0075% of type III collagen cannot increase the content and permeability of characteristic proteins in various layers of the skin epidermis.

[0061] Table 3 Repair efficacy and permeability test of type I collagen and type III collagen complex

[0062]

[0063] Experimental results show that a concentration ratio of 1:3 between type I collagen and type III collagen can increase the content and permeability of characteristic proteins in each layer of the skin's epidermis compared to other ratios, and has a repairing effect.

[0064] Table 4 Repair efficacy and permeability test of the compound of Anoectochilus roxburghii extract and type I collagen and type III collagen

[0065]

[0066]

[0067] The experimental results show that the concentration ratio of (60-200):1:3 of golden thread lotus extract and type I collagen and type III collagen can better increase the content and permeability of characteristic proteins in each layer of the skin epidermis compared with other ratios, and has a repairing effect.

[0068] Example 4

[0069] This experiment is based on the SDS-stimulated 3D epidermal skin model, and the repair effect of the test sample is evaluated by detecting the changes in barrier thickness.

[0070] 1. Take the corresponding number of 3D epidermal skin models for resuscitation operation. After the resuscitation operation, prepare a 6-well cell culture plate according to the total number of models, and add 0.9mL of model culture solution to each well. According to the test group, move the model to the corresponding well, mark the test group number on the 6-well plate, and place it in a 37℃, 5% CO2 incubator for incubation for 24±2h.

[0071] 2. After 24±2h, administer the drug. After administration, place the cells back in a 37°C, 5% CO2 incubator and continue incubating for 24±2h.

[0072] 3. After the incubation, clean the test substance remaining on the surface of the model with sterile PBS solution, and wipe off the residual liquid inside and outside the model with a sterile cotton swab.

[0073] 4. The model for tissue morphology detection was removed by ring cutting, fixed with 4% paraformaldehyde, and after fixation for 24 hours, H&E staining was performed, and photos were taken under a microscope for observation, collection and analysis.

[0074] The experimental results are as follows Figure 1-5 As shown, the combination of Anoectochilus roxburghii extract with type I collagen and type III collagen in a concentration ratio of (60-200):1:3 can thicken the stratum corneum (the area on the double-headed arrow), making it denser, and also thicken the living cell layer (at the double-headed arrow), showing a stronger repair effect compared with other ratios.

[0075] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A composition with repairing effect containing aroximate extract and type I collagen and type III collagen, characterized in that: The composition is prepared by compounding aroximate extract, type I collagen and type III collagen in a mass ratio of (60-200):1:

3.

2. The composition according to claim 1, characterized in that The preparation method of the roxburghii extract comprises the following steps: fresh roxburghii grass is freeze-dried and crushed, extracted 1-2 times under the conditions that the mass ratio of freeze-dried powder to pure water is 1:10 and the temperature is 10-40°C, the extraction time for each time is 1-2h, and the extraction is terminated when the sugar content is 65%-75% under reduced pressure and low temperature, and filtered with 300-mesh gauze to obtain the roxburghii extract, wherein the main active component is total polysaccharide with a content of 10wt%-20wt%.

3. The composition according to claim 2, characterized in that The temperature during low-temperature concentration is 45℃-55℃ and the pressure range is 10 -2 -10 -5 mbar.

4. Use of the composition according to claim 1 or 2 in repair cosmetics, characterized in that: The application concentrations of roxburghii roxburghii extract, type I collagen and type III collagen in cosmetics are 0.15wt%-0.5wt%, 0.0025wt% and 0.0075wt%, respectively.

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