Composition containing anoectochilus formosanus extract and three factors and having moisturizing and soothing effects and application of composition

Abstract

The invention discloses an anoectochilus formosanus three-cause composition with moisturizing and soothing effects and application thereof, and relates to the technical field of cosmetic formulas. The composition is formed by compounding an anoectochilus formosanus extract, ectoine, ergothioneine and vitamine according to the mass ratio of (150-500): 100: 1: 29, and through the synergistic interaction among the compositions, the genes of caspase-14 (Caspase-14) and aquaporin 3 (AQP3) can be effectively improved, the release of inflammatory factors (IL-1alpha) by macrophages in an inflammatory reaction is remarkably inhibited, the water content of skin is improved, and the skin care effect is improved. The skin a * value is reduced. Compared with the single use of the four components and the compound use of the ectoine, the ergothioneine and the glassine, the moisturizing and relieving composition has better moisturizing and relieving activity, and has a very good application prospect in moisturizing and relieving cosmetics.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetic formulas, and in particular to a composition containing a roxburghii extract and three factors with moisturizing and soothing effects and an application thereof. Background Art

[0002] Anoectochilus roxburghii (Wall.) Lindl., also known as golden thread orchid and golden silk grass, is a rare perennial Chinese herbal medicine belonging to the genus Anoectochilus of the Orchidaceae family. Anoectochilus roxburghii has high medicinal value, and the whole plant can be used as medicine. It has the effects of clearing heat and cooling blood, detoxifying and reducing swelling, moistening the lungs and relieving cough, lowering high blood pressure, lowering blood lipids, and lowering high uric acid. In addition, Anoectochilus roxburghii has skin care effects such as anti-oxidation, dehumidification, antibacterial, and anti-inflammatory, and can be widely used in the field of cosmetics.

[0003] Ectoine, ergothioneine and botoxine are currently widely used in cosmetics. They can not only be used alone, but also combined with other ingredients to achieve better skin care effects. As consumers become more and more aware of cosmetic ingredients, more and more consumers begin to pay attention to the overall efficacy and formula of the product.

[0004] The use of active ingredients from natural plants as cosmetic raw materials is a hot area in the current cosmetics research and development. About 82.64% of cosmetics in my country add plant extracts, and 80.17% of cosmetics are compounded with two or more plant extracts. The compounding of several raw materials with similar effects may produce a synergistic effect of enhancing each other's activity, or it may produce an antagonistic effect of weakening each other's activity. However, many formulators only empirically compound each plant extract in a certain proportion when designing cosmetics, ignoring the possible synergistic or antagonistic interactions between ingredients with similar activities.

[0005] Therefore, Anoectochilus roxburghii is used in combination with ectoine, ergothioneine and botoxine to complement each other and improve the overall efficacy and stability of cosmetics. At the same time, plant extracts are generally considered to be safer and gentler, and compounding technology can create products with personalized effects through different combinations to meet the specific needs of different consumers. Summary of the invention

[0006] The purpose of the present invention is to provide a composition containing aroxithium roxburghii extract and three factors with moisturizing and soothing effects and application thereof.

[0007] To achieve the above object, the present invention adopts the following technical scheme: A composition containing aroximate extract and three factors with moisturizing and soothing effects. The composition is prepared by compounding the aroximate extract and three factors; the three factors are ectoine, ergothioneine and bosine.

[0008] Furthermore, the golden thread lotus extract, ectoine, ergothioneine and bosine are compounded in a mass ratio of (150-500):100:1:29.

[0009] Furthermore, 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 of a mass ratio of freeze-dried powder to pure water of 1:10 and a temperature of 10-40°C, each extraction time being 1-2 hours, and concentrated under reduced pressure and low temperature to a sugar content of 65%-75%, and filtered with 300-mesh gauze to obtain the roxburghii extract, wherein the main active ingredient is total polysaccharides with a content of 10wt%-20wt%.

[0010] Furthermore, the temperature during the low-temperature concentration under reduced pressure is 45°C-50°C, and the pressure range is 10 -2 -10 -5 mbar.

[0011] The above-mentioned composition is used in the preparation of cosmetics with moisturizing and soothing effects, and the application concentrations of the roxburghii extract, ectoine, ergothioneine and bosoneine in the cosmetics are (0.15%-0.5%): 0.1%: 0.001%: 0.029%.

[0012] The beneficial effects of the present invention are: The combination of Anoectochilus roxburghii extract and ectoine, ergothioneine and botoxine has synergistic effects, significantly enhancing the moisturizing and soothing effects. It has better effects than using the four ingredients alone or using the three synthetic raw materials of ectoine, ergothioneine and botoxine in combination, and can be better used in cosmetic formulas. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Comparison of VISIA®CR facial images after 28 days of using a 0.15% carbomer hydrogel system product containing a complex of roxburghii extract (roxburghii extract: ectoine: ergothioneine: bosoneine = 150:100:1:29) (left: before use; right: after use). DETAILED DESCRIPTION

[0014] 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.

[0015] This experiment was tested by the following experiments and standards: 1. Based on the human keratinocyte (HaCaT) model, the moisturizing effect of the sample to be tested was evaluated by detecting the changes in the gene expression of caspase-14 (Caspase-14) and aquaporin 3 (AQP3) after the composition acted on human keratinocytes.

[0016] 2. Based on the bacterial lipopolysaccharide (LPS)-induced mouse macrophage RAW264.7 model, the ELISA method was used to determine the IL-1α concentration in the cell culture supernatant, the inhibition rate of the composition on the release of IL-1α by mouse macrophages was detected, and the soothing effect of the test substance was evaluated.

[0017] 3. Based on 30 Asian adult subjects (age: 35.64±0.52) using a 0.15% carbomer hydrogel system product containing the composition for 28 consecutive days under normal conditions, the water content of the stratum corneum was measured by a Corneometer instrument to evaluate the moisturizing efficacy of the composition.

[0018] 4. Based on 30 Asian adult subjects (age: 35.64±0.52) using a 0.15% carbomer hydrogel system product containing the composition for 28 consecutive days under normal conditions, the a* value was measured by the VISIA® CR instrument to evaluate the soothing efficacy of the composition.

[0019] Example 1 Preparation of Anoectochilus roxburghii extract 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) to a sugar content of 70% and then filtered through 300-mesh gauze to obtain a roxburghii roxburghii extract, the main active ingredient of which was total polysaccharides with a content of 10wt%.

[0020] Example 2 Cell activity test 1. Preparation of mother solutions of each component 1) Preparation of the roxburghii extract solution: Weigh 0.2 g of the roxburghii extract obtained in Example 1, add 1.8 mL of deionized water to prepare a 10% mother solution, and dilute it with DMEM complete medium to a corresponding concentration when used.

[0021] 2) Preparation of ectoine solution: Weigh 0.2 g ectoine and add 1.8 mL deionized water to prepare a 10% stock solution. When using, dilute it with DMEM complete medium to the corresponding concentration.

[0022] 3) Preparation of thioneine solution: Weigh 0.2 g of thioneine, add 1.8 mL of deionized water to prepare a 10% stock solution, and dilute to the corresponding concentration with DMEM complete medium when using.

[0023] 4) Preparation of bosense solution: weigh 0.2 g bosense and add 1.8 mL deionized water to prepare a 10% stock solution. Dilute with DMEM complete medium to the corresponding concentration when using.

[0024] 2. Cell viability test (MTT) 1) Plate: Take the cultured cells, digest and centrifuge, count, and set 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.

[0025] 2) Administration: 24h±2h later, 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.

[0026] The final concentrations of the sample solutions were as follows: Anoectochilus roxburghii extract (the final tested concentrations were 1%, 0.5%, 0.1%, 0.05%, 0.01%, and 0.005%, respectively), Ecdoine (the final tested concentrations were 2%, 1%, 0.5%, 0.1%, 0.05%, and 0.01%, respectively), Ergothioneine (the final tested concentrations were 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, and 0.0005%, respectively), and Bosoneine (the final tested concentrations were 2%, 1%, 0.5%, 0.1%, 0.05%, and 0.01%, respectively).

[0027] 3) Test: After incubation for 24 hours, the supernatant was discarded, and 200 μL of 0.5 mg / mL MTT solution was added to each well and placed in a CO2 incubator for 4 hours.

[0028] 4) Test: After 4 hours, the supernatant was discarded, 150 μL DMSO was added to each well, and the cells were shaken at room temperature in the dark for 10 minutes. The absorbance was measured at a wavelength of 490 nm. The MTT test results are shown in Tables 1 and 2.

[0029] Table 1 Results of MTT assay for HaCaT cell activity Table 2 Results of MTT assay for RAW264.7 cell activity As shown in Tables 1 and 2, the maximum addition amount of Anoectoine is 1%, the maximum addition amount of Ecdoin is 0.5%, the maximum addition amount of ergothioneine is 0.001%, and the maximum addition amount of Bosoneine solution is 1%.

[0030] Example 3 Determination of Moisturizing and Soothing Efficacy 1. Single-component moisturizing and soothing efficacy test Based on the human keratinocyte (HaCaT) model, the moisturizing effect of the test sample was evaluated by detecting the changes in the expression of Caspase-14 and AQP3 genes after the test sample acted on human keratinocytes. The experimental steps are as follows: 1) Plating: 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.

[0031] 2) Administration: 24±2h later, place the sample in an incubator and incubate for 24±2h. The final test concentration of each sample is as follows: Anoectochilus roxburghii extract (final test concentration 0.15%, 0.3%, 0.5%), Ecdoine (final test concentration 0.5%), Ergothioneine (final test concentration 0.001%), and Bosoneine (final test concentration 1%).

[0032] 3) Test: After 24 hours of culture, the supernatant was discarded and total RNA was extracted using an RNA extraction kit. The total RNA concentration and purity were measured using an ultra-micro UV-visible spectrophotometer, and reverse transcription was performed using a reverse transcription kit in a PCR amplifier to synthesize cDNA. After using fluorescent dyes to spot the plate, the expression of Caspase-14 and AQP3 genes was detected using a fluorescent quantitative PCR instrument. The results are shown in Table 3.

[0033] Based on the bacterial lipopolysaccharide (LPS)-induced mouse macrophage RAW264.7 model, the ELISA method was used to determine the IL-1α concentration in the cell culture supernatant, and the inhibition rate of the test sample on the release of IL-1α by mouse macrophages was detected to evaluate its soothing effect. The experimental steps are as follows: 1) Plating: 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.

[0034] 2) After 24±2h, administer the drug.

[0035] 3) Test: Collect the cell supernatant and test the IL-1α content using an ELISA kit. The steps are as follows. The results are shown in Table 3.

[0036] S1: Take the IL-1α kit out of the refrigerator 1 hour in advance and equilibrate to room temperature. After thawing, centrifuge the collected supernatant at 10,000 rpm for 20 min.

[0037] S2: 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 the 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 minutes.

[0038] S3: After 60 min, take out 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.

[0039] S4: After 60 min, remove the ELISA plate, discard the liquid, add 300 μL of the prepared washing solution to each well, let it stand for 1 min, 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 min.

[0040] S5: After 30 min, remove the ELISA plate, discard the liquid, add 300 μL of the prepared washing solution to each well, let it stand for 1 min, discard the washing solution, pat dry on absorbent paper, and repeat the washing 5 times. Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C incubator for 15 min.

[0041] S6: After 15 min, the ELISA plate was taken out, 50 μL of stop solution was added to each well, the OD value of each well was immediately measured at a wavelength of 450 nm, and the inhibition rate of IL-1α content was calculated.

[0042] Table 3 Test results of single-component moisturizing and soothing efficacy As shown in Table 3, Anoectochilus roxburghii extract and botoxine can effectively inhibit the expression of IL-1α to a certain extent, and have a soothing effect; Ecdoine can promote the expression of Caspase-14 gene to a certain extent, and ergothioneine can promote the expression of AQP3 to a certain extent, and has a moisturizing effect.

[0043] 2. The moisturizing and soothing effect of the three-factor combination (1) Test ratio of three-factor combination 1) Ectoine: ergothioneine: bosine = 20:1:5 (i.e. 0.02%: 0.001%: 0.005%); 2) Ectoine: ergothioneine: bosine = 50:1:20 (i.e. 0.05%: 0.001%: 0.02%); 3) Ectoine: ergothioneine: bosine = 100:1:29 (i.e. 0.1%: 0.001%: 0.029%); 4) Ectoine: ergothioneine: bosine = 150:1:50 (i.e. 0.1%: 0.000667%: 0.0333%); 5) Ectoine: ergothioneine: bosine = 200:1:70 (i.e. 0.1%: 0.0005%: 0.035%); (2) The experimental process was the same as step 1 of Example 3. The expression of Caspase-14 and AQP3 genes and the inhibition rate of IL-1α were measured respectively. The results are shown in Table 4.

[0044] Table 4 Test results of moisturizing and soothing effects of the three-factor composition As shown in Table 4, the three-factor combination can promote the expression of Caspase-14 and AQP3 genes to a certain extent, and inhibit the expression of IL-1α, and has moisturizing and soothing effects. Among them, ectoine: ergothioneine: bosine = 100:1:29 (i.e. 0.1%: 0.001%: 0.029%) has the best effect.

[0045] 3. Moisturizing and soothing effects of the combination of roxburghii extract and three factors Set up the following experimental groups: 1) Anoectochilus roxburghii extract: ectoine: ergothioneine: bosoepine = 150:100:1:29 (i.e. 0.15%:0.1%:0.001%:0.029%); 2) Anoectochilus roxburghii extract: ectoine: ergothioneine: bosoepine = 300:100:1:29 (i.e. 0.3%:0.1%:0.001%:0.029%); 3) Anoectochilus roxburghii extract: ectoine: ergothioneine: bosoepine = 500:100:1:29 (i.e. 0.5%:0.1%:0.001%:0.029%); The steps for determining the expression of Caspase-14 and AQP3 genes and the inhibition rate of IL-1α are the same as those in step 1 of Example 3. The experimental results are shown in Table 5.

[0046] Table 5 Moisturizing and soothing test results of the combination of Anoectochilus roxburghii extract and three factors According to Tables 3, 4 and 5, the best ratio of the combination of Anoectochilus roxburghii extract and three factors in terms of moisturizing and soothing effect is Anoectochilus roxburghii extract: Ectoin: Ergothioneine: Bosoein = (150-500): 100: 1: 29. The combination of Anoectochilus roxburghii extract and Ectoin, Ergothioneine and Bosoein in a specific ratio significantly enhances its moisturizing and soothing effect. Compared with the use of the four ingredients alone and the combination of Ectoin, Ergothioneine and Bosoein, the combination of the four ingredients produces a synergistic effect.

[0047] 4. Moisturizing and soothing efficacy test of roxburghii extract and three-factor combination on human body The best ratio of the combination of roxburghii extract and three factors in moisturizing and soothing effect is roxburghii extract: ectoine: ergothioneine: bosoneine = (150-500): 100:1:29. The lowest concentration ratio of roxburghii extract, i.e., roxburghii extract: ectoine: ergothioneine: bosoneine = 150:100:1:29, was selected for moisturizing and soothing human efficacy test.

[0048] According to QB / T 4256-2011 Guidelines for Evaluation of Cosmetic Moisturizing Efficacy, 30 Asian adult subjects (age: 35.64±0.52) used a 0.15% carbomer hydrogel system product containing a combination of roxburghii extract (roxburghii extract: ectoine: ergothioneine: bosoneine = 150:100:1:29) for 28 consecutive days under normal conditions. The moisture content of the stratum corneum was measured by a Corneometer instrument to evaluate the moisturizing efficacy of the composition.

[0049] According to the T / GDCDC 021-2022 Cosmetic Soothing Efficacy Test Method, 30 Asian adult subjects (age: 35.64±0.52) used a 0.15% carbomer hydrogel system product containing a combination of roxburghii extract (roxburghii extract: ectoine: ergothioneine: bosacein = 150:100:1:29) for 28 consecutive days under normal conditions. The a* value was measured by the VISIA® CR instrument to evaluate the soothing efficacy of the composition.

[0050] Compared with before use, after 28 days of using a 0.15% carbomer hydrogel system product containing a compound of Anoectochilus roxburghii extract (Anoectochilus roxburghii extract: Ectoin: Ergothioneine: Bosine = 150:100:1:29), the water content of the stratum corneum increased significantly, with an increase rate of 4.02%; the a* value of the red zone decreased significantly, with a decrease rate of 5.59% ( Figure 1 ) 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 containing aroxithium roxburghii extract and three factors with moisturizing and soothing effects, characterized in that The composition is composed of a compound of a roxburghii extract and three factors; the three factors are ectoine, ergothioneine, and bosoneine; the roxburghii extract, ectoine, ergothioneine, and bosoneine are compounded in a mass ratio of (150-500):100:1:

29.

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 concentrated under reduced pressure and low temperature until the sugar content is 65%-75%, 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 any one of claims 1 or 2 in the preparation of cosmetics with moisturizing and soothing effects, characterized in that The application concentrations of the golden thread vine extract, ectoine, ergothioneine and bosine in cosmetics are (0.15%-0.5%): 0.1%: 0.001%: 0.029%.

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