Synergistic anti-wrinkle composition and application thereof

Abstract

The invention discloses a synergistic anti-wrinkle composition and application thereof, and relates to the technical field of skin care products. The synergistic anti-wrinkle composition provided by the invention is prepared from 5-35 parts by mass of gynura procumbens extract, 5-30 parts by mass of VC ethyl ether, 5-30 parts by mass of hydrolyzed collagen and 5-20 parts by mass of ginseng essential oil, the four components are combined to have a remarkable synergistic effect on inhibition of elastase activity, the combination index CI among the four components is less than 1, and the synergistic anti-wrinkle composition has the advantages that the synergistic anti-wrinkle effect is obvious; the inhibition rate of the composition on the activity of elastinase is up to 15.43% or above, the composition has a remarkable anti-wrinkle effect, the collapsed tail fin of the zebra fish can be recovered to a normal state, and the recovery rate is up to 59.69%. The composition provided by the invention also has a DPPH clearance rate up to 92.32% or above, can realize oxidation resistance by scavenging free radicals, and has a wide prospect in the aspect of aging resistance in combination with inhibition on the activity of elastinase.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care products, in particular to a synergistic anti-wrinkle composition and application thereof. Background Art

[0002] Skin is the largest organ in the human body and is composed of multiple layers of tissue, including the epidermis, dermis, and subcutaneous tissue. Among them, the most important cell type in the dermis is fibroblasts, which secrete collagen and elastin to form collagen fibers and elastic fibers, which together with matrix components and fibroblasts constitute the main components of the dermis.

[0003] The elastic fibers in the dermis are the key to maintaining the elasticity and firmness of human skin, but fibroblasts not only secrete elastin, but also secrete elastase that can degrade elastin. Elastase degrades elastin, which can cause skin aging. With the increase of age, the human body's ability to inhibit elastase is getting weaker and weaker, and the activity of elastase is getting stronger and stronger. It can not only degrade most of the extracellular matrix proteins including elastin, but also activate other matrix metalloproteinases, accelerate the degradation of the extracellular matrix of the dermis, thereby causing the normal structure and function of the dermis of human skin to be seriously damaged, causing the skin to produce obvious wrinkles and lose elasticity. Based on this, it is currently in urgent need of developing a skin care product that can inhibit the activity of elastase to achieve anti-wrinkle. Summary of the invention

[0004] In order to address the deficiencies of the prior art, the present invention provides a synergistic anti-wrinkle composition, which combines notoginseng extract, VC ethyl ether, hydrolyzed collagen and ginseng essential oil. Through the synergistic enhancement of the four components, the inhibition rate of elastase activity is greatly improved, and the composition has a significant anti-wrinkle effect.

[0005] Another object of the present invention is to provide the use of the above synergistic anti-wrinkle composition in the preparation of products for inhibiting the activity of elastase.

[0006] Another object of the present invention is to provide an anti-wrinkle skin care product, comprising the above synergistic anti-wrinkle composition.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A synergistic anti-wrinkle composition, comprising the following components calculated by weight:

[0009] 5-35 parts of Chrysanthemum procumbentum notoginseng extract, 5-30 parts of VC ethyl ether, 5-30 parts of hydrolyzed collagen, and 5-20 parts of ginseng essential oil.

[0010] In the synergistic anti-wrinkle composition provided by the present invention, the extract of Panax notoginseng and ginseng essential oil are both plant extracts, which have the effect of inhibiting the activity of elastase, and can improve skin elasticity and achieve anti-wrinkle effect when used in skin care products. VC ethyl ether (3-o-ethyl ascorbic acid) can enter the dermis, directly participate in the synthesis of collagen to increase collagen, repair skin cells, improve its activity, so that the skin becomes full and elastic, delicate and smooth. Hydrolyzed collagen is a small molecule collagen that is hydrolyzed, which can be better absorbed by the human body and is rich in biologically active substances such as various polypeptides necessary for human metabolism. Hydrolyzed collagen can enter the deep layer of the skin to increase hydration, improve the elasticity and firmness of the skin, reduce wrinkles, and promote skin regeneration. There is a synergistic effect between the above four components, so that the composition provided by the present invention has an excellent inhibition rate of elastase activity, and can be applied to skin care products to play a significant anti-wrinkle effect. In addition, the composition provided by the present invention also has an excellent antioxidant effect, and can remove the DPPH free radicals in the skin. Therefore, the composition provided by the present invention has a broad application prospect in the field of anti-aging.

[0011] It should be noted that in the composition provided by the present invention, the mass proportions of Panax notoginseng extract, VC ethyl ether, hydrolyzed collagen and ginseng essential oil need to be controlled to 5-35 parts, 5-30 parts, 5-30 parts and 5-20 parts respectively, so that the obtained composition can fully synergize and have a significantly improved elastase activity inhibition rate.

[0012] Preferably, the notoginseng procumbens extract is prepared by extracting notoginseng procumbens through water and / or organic solvent.

[0013] In a specific embodiment of the present invention, the notoginseng procumbent extract is obtained by water extraction, centrifugation, purification, concentration, sterilization and drying of notoginseng procumbent leaves / stems with a length of 1-3 cm.

[0014] Preferably, the content of ginsenoside in the ginseng essential oil is 0.1-1 mg / g.

[0015] The main component of ginseng essential oil that inhibits the activity of elastase is ginsenoside. Ginseng essential oil with a ginsenoside content of 0.1-1 mg / g has a more significant effect on the elastase inhibition rate and anti-wrinkle effect of the composition.

[0016] In a specific embodiment of the present invention, the preparation method of ginseng essential oil comprises the following steps: drying and crushing ginseng, performing microwave extraction, and then centrifuging, distilling, condensing, and separating oil from water to obtain ginseng essential oil.

[0017] Preferably, the synergistic anti-wrinkle composition comprises the following components calculated by weight:

[0018] 25-35 parts of Chrysanthemum procumbentum extract, 10-30 parts of VC ethyl ether, 5-30 parts of hydrolyzed collagen and 5-20 parts of ginseng essential oil.

[0019] When the mass fractions of the components satisfy the above relationship, the obtained composition has a more excellent elastase activity inhibition rate.

[0020] More preferably, the mass ratio of the notoginseng procumbent extract, VC ethyl ether, hydrolyzed collagen and ginseng essential oil is 1: (0.3-4): (0.167-6): (0.143-2).

[0021] When the mass ratio of each component is within the above preferred range, the obtained composition has a more excellent elastase activity inhibition rate.

[0022] Preferably, the synergistic anti-wrinkle composition has an elastase activity inhibition rate of ≥15.43%.

[0023] More preferably, the synergistic anti-wrinkle composition has an elastase activity inhibition rate of ≥48.72%.

[0024] Preferably, the synergistic anti-wrinkle composition has a combination index <1.

[0025] Preferably, the DPPH scavenging rate of the synergistic anti-wrinkle composition is ≥ 92.32%.

[0026] The present invention also protects the use of the synergistic anti-wrinkle composition in preparing products for inhibiting the activity of elastase.

[0027] The present invention also protects an anti-wrinkle skin care product, comprising the above synergistic anti-wrinkle composition.

[0028] Preferably, the concentration of the synergistic anti-wrinkle composition in the anti-wrinkle skin care product provided by the present invention is ≥20 mg / mL.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The synergistic anti-wrinkle composition provided by the present invention has a synergistic effect among the four components including the extract of Panax notoginseng, and the combination index CI is less than 1. The inhibition rate of the composition on the activity of elastase is as high as 15.43% or more, and it has a significant anti-wrinkle effect, and can restore the wrinkled tail fin of zebrafish to a normal state, with a recovery rate of up to 59.69%. In addition, the composition provided by the present invention also has a DPPH clearance rate of more than 92.32%, and has an excellent antioxidant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1It is the activity inhibition rate of elastase when each component in the synergistic anti-wrinkle composition of the present invention is used alone.

[0032] Figure 2 The anti-wrinkle effects of Example 1 and Comparative Examples 1-4 on zebrafish. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents. Among them, the raw material information used in each embodiment and comparative example is as follows:

[0034] Hydrolyzed collagen: purchased from Anhui Shengminuo Biotechnology Co., Ltd.

[0035] VC ethyl ether: purchased from Yantai Dongfang Chemical Co., Ltd.

[0036] Notoginseng procumbent extract: obtained by water extraction, centrifugation, purification, concentration, sterilization and drying of the dried leaves of Notoginseng procumbent with a length of 1-3 cm.

[0037] Ginseng essential oil: Ginseng is dried, crushed, and then subjected to microwave extraction, followed by centrifugation, distillation, condensation, and oil-water separation to obtain ginseng essential oil. According to testing, the content of ginsenosides in ginseng essential oil is 0.1-1 mg / g.

[0038] Examples 1 to 11 and Comparative Examples 1 to 12

[0039] This embodiment and comparative example provide a series of synergistic anti-wrinkle compositions with different mass fractions of components. The specific mass fractions of the components are shown in Table 1 below:

[0040] Table 1. Mass fractions of each component in Examples 1 to 11 and Comparative Examples 1 to 12

[0041]

[0042]

[0043] Comparative Example 13

[0044] This comparative example provides a synergistic anti-wrinkle composition, which differs from Example 1 only in that:

[0045] Replace the ginseng essential oil with an equal weight of Dendrobium officinale.

[0046] Comparative Example 14

[0047] This comparative example provides a synergistic anti-wrinkle composition, which differs from Example 1 only in that:

[0048] Replace hydrolyzed collagen with an equal weight of recombinant collagen.

[0049] Comparative Example 15

[0050] This comparative example provides a synergistic anti-wrinkle composition, which differs from Example 1 only in that:

[0051] VC ethyl ether was replaced with an equal weight of vitamin A liposomes.

[0052] Comparative Example 16

[0053] This comparative example provides a synergistic anti-wrinkle composition, which differs from Example 1 only in that:

[0054] The notoginseng procumbent extract was replaced with an equal weight of rhodiola rosea extract.

[0055] Performance Testing

[0056] Next, experiments were conducted using the synergistic anti-wrinkle compositions of Examples 1 to 11 and Comparative Examples 1 to 16 to explore their inhibitory effects on elastase activity and anti-wrinkle effects.

[0057] 1. Elastase activity inhibition test

[0058] Take the composition of the embodiment and the comparative example and dissolve it in PBS solution, and prepare the sample solution of corresponding concentration according to the mass fraction of Table 1. For example, the composition of Example 1 contains 25 mass fractions of the flat chrysanthemum Panax notoginseng extract, and the concentration of the flat chrysanthemum Panax notoginseng extract in the sample solution obtained in Example 1 is 25 mg / mL. 1mL of the sample solution and 1mL of the elastase solution are added to a centrifuge tube, and the mixture is reacted at 37°C for 5min after being fully mixed, and then 1mL of the substrate solution (N-succinyl-Ala-Ala-Ala-p-nitroaniline solution) is added, and the reaction is continued at 37°C for 30min. After the incubation is completed, centrifuge at 1000rpm for 5min, and 200μL of the supernatant is drawn into a 96-well plate, and the absorbance is detected at a wavelength of 405nm with an ELISA reader. At the same time, a blank substrate group (10% DMSO of equal volume replaces the substrate), a 100% enzyme activity control group and a blank control group are set. Three parallel experiments are performed in each group, with sivelestat sodium as a positive control. The calculation formula of elastase inhibition rate is as follows:

[0059] Elastase inhibition rate = [1-(Y1-Y2) / (Y3-Y4)] × 100%

[0060] Where:

[0061] Y1 is the absorbance value of the test sample + elastase solution + substrate solution (experimental group);

[0062] Y2 is the absorbance value of the test sample + elastase solution + 10% DMSO (blank substrate group);

[0063] Y3 is the absorbance value of PBS buffer solution + elastase solution + substrate solution (100% enzyme activity control group);

[0064] Y4 is the absorbance value of PBS buffer solution + elastase solution + 10% DMSO (blank control group).

[0065] 2. Determination of the joint index

[0066] Fa = elastase inhibition rate. According to the experimental results of elastase activity inhibition, the Fa of each raw material (Psoralea corylifolia extract, VC ethyl ether, hydrolyzed collagen, ginseng essential oil) in the experimental group and the composition Fa prepared from each embodiment and comparative example in the experimental group were statistically analyzed by isobologram (through analysis software CompuSyn) to obtain a combination index (CI). The combination index is used to determine whether the composition has a synergistic effect. If the CI of the combined effect of each raw material is less than 1, it indicates that the combination of each raw material has a synergistic enhancement effect.

[0067] The specific data of the above determination are shown in Table 2 and Figure 1 As shown:

[0068] Table 2. Elastase activity inhibition rate and combination index (CI) of the examples and comparative examples

[0069]

[0070]

[0071] As can be seen from Table 2, in Examples 1 to 11, when the notoginseng extract, ginseng essential oil, VC ethyl ether and hydrolyzed collagen are used together, a synergistic effect (CI < 1) is achieved; in Comparative Examples 1 to 4, any three component combinations and Comparative Examples 13 to 16, when components with similar efficacy are used to replace a component in the composition of the present invention, the CIs are all > 1, and their ability to inhibit elastase activity is also much lower than that of the sample in Example 1 of the present invention.

[0072] According to the data of Examples 1 to 6, it can be seen that when the composition of the synergistic anti-wrinkle composition is 25 to 35 parts of chrysanthemum notoginseng extract, 10 to 30 parts of VC ethyl ether, 5 to 30 parts of hydrolyzed collagen, and 5 to 20 parts of ginseng essential oil (Examples 1 to 2, 5 to 6), the inhibition rate of the composition on elastase activity is higher; when the mass ratio of chrysanthemum notoginseng extract, VC ethyl ether, hydrolyzed collagen and ginseng essential oil in the composition is 1: (0.83 to 1): (0.67 to 1.2): (0.27 to 0.6) (Examples 1 and 6), the resulting composition also has a further improved inhibition rate of elastase activity. According to the data of Examples 7 to 8 and 10, it can be seen that when the synergistic anti-wrinkle composition of the present invention is used to prepare anti-wrinkle skin care products, the total concentration of chrysanthemum notoginseng extract and VC ethyl ether in the composition is ≥ 20 mg / mL, the inhibition rate on elastase activity is higher, and the anti-wrinkle effect of the skin care products is also better. Combined with the data of Example 11, it can be seen that the total concentration of the anti-wrinkle composition in the skin care product is increased to 190 mg / mL, and the anti-wrinkle performance of the composition is still excellent, indicating that the composition provided by the present invention can be used in a larger concentration range. At the same time, by comparing the data of Examples 1 to 11 with Comparative Examples 5 to 12, it can be seen that too much or too little mass fraction of each component in the composition provided by the present invention (Comparative Examples 5 to 12) will lead to a decrease in synergistic synergy, CI>1.

[0073] Figure 1 The activity curve of inhibiting elastase by Panax notoginseng extract, VC ethyl ether, hydrolyzed collagen and ginseng essential oil is shown in Figure 2. Figure 1 As shown, the IC50 of the inhibition rate of Panax notoginseng extract, VC ethyl ether, hydrolyzed collagen and ginseng essential oil on elastase activity are 102 mg / mL, 94.4 mg / mL, 200 mg / mL and 78.87 mg / mL, respectively. It can be seen that each component has a certain activity in inhibiting elastase. At the same time, the IC50 values ​​of Rhodiola rosea extract, vitamin A liposome, recombinant collagen, and Dendrobium officinale in Comparative Examples 13 to 16 were measured to be 114.26 mg / mL, 97.82 mg / mL, 218.49 mg / mL, and 74.65 mg / mL, respectively, indicating that the components in Comparative Examples 13 to 16 also have a certain effect of inhibiting the activity of elastase, and the elastase activity inhibition rate of a single component is similar to the inhibition rate corresponding to the similar efficacy component of Example 1 when used alone. However, it is difficult for the components in Comparative Examples 13 to 16 to exhibit a synergistic effect. Therefore, after the composition is formed, the activity inhibition rate of the composition as a whole on elastase is not as good as that of the present invention, indicating that the selection of components has a crucial influence on the synergistic anti-wrinkle composition of the present invention.

[0074] 3. Free Radical Scavenging Rate Test

[0075] The sample solution in the elastase activity inhibition test was diluted 10 times with deionized water to obtain a diluted sample solution, 100 μL of which was added to a 96-well plate, and then 100 μL of 0.2 mmol / L DPPH anhydrous ethanol solution was added. After shaking evenly, it was placed in a dark place at 25°C to avoid light for 30 minutes, and then the absorbance A1 of the mixed solution was measured at a wavelength of 517 nm; 100 μL of anhydrous ethanol was used to replace the DPPH anhydrous ethanol solution and the same operation was performed; 100 μL of anhydrous ethanol was used to replace the sample and the same operation was performed; vitamin C solution was used as a positive control, and 3 parallel experiments were set up in each group to calculate the average value, and the free radical scavenging ability of different compositions was calculated according to the DPPH scavenging rate formula. DPPH scavenging rate calculation formula:

[0076] DPPH clearance rate = [1-(A1-A2) / A3] × 100%

[0077] Where:

[0078] A1 is the absorbance value of 100 μL DPPH working solution + 100 μL sample;

[0079] A2 is the absorbance value of 100 μL anhydrous ethanol + 100 μL sample;

[0080] A3 is the absorbance value of 100 μL DPPH working solution + 100 μL anhydrous ethanol.

[0081] The specific data of the above determination are shown in Table 3 below:

[0082] Table 3. DPPH removal rates of the examples and comparative examples

[0083] Group DPPH clearance Example 1 95.45％ Example 5 93.57％ Example 6 92.32％ Comparative Example 7 78.39％ Comparative Example 12 86.88％ Comparative Example 15 81.34％

[0084] As can be seen from Table 3, the compositions obtained in Example 1, Example 5, and Example 6 all have strong DPPH free radical scavenging ability, and the free radical scavenging rate of Example 1 even reaches 95.45%. The DPPH scavenging rate is decreased in both Comparative Examples 7 and 12 in which the component ratio is not appropriate and Comparative Example 15 in which the components in the present invention are replaced by substances with similar efficacy.

[0085] 4. Zebrafish anti-wrinkle efficacy test

[0086] The structure of zebrafish skin is similar to that of human skin, so it can be used as a model to evaluate the anti-wrinkle effect of samples. UV irradiation will cause changes in collagen and elastin in the skin structure, causing the fish fins to shrink and deform. The anti-wrinkle effect of the test substance is evaluated by evaluating the degree to which the test substance slows down the shrinkage of the zebrafish tail fin.

[0087] The test method for zebrafish anti-wrinkle efficacy is as follows:

[0088] Select 2dpf zebrafish fry with normal development and place them in a six-well cell culture plate, with 15 fish in each well, and divide them into normal control group, model group, positive control group and test group (the sample solution in the elastase activity inhibition test was diluted 1600 times with embryo culture medium to obtain the test dilution solution for testing). Remove the standard dilution water in the six-well plate without harming the fry, and quickly add 3mL of the test dilution solution of the corresponding concentration to each well. Wrap the six-well plate with aluminum foil and incubate it in a biochemical incubator (28.5℃±1.0℃) in the dark for 2h, and place it under an irradiance of 2.0~2.5Mw / cm 2 Under the condition of , the ultraviolet light was irradiated three times, each irradiation time was 15 minutes, and the interval was 30 minutes. The cells were incubated in a biochemical incubator (28.5℃±0.5℃) in the dark for 22 hours (3dpf) and then observed. The death of zebrafish and other toxic effects were recorded. Among them, the normal control group contained zebrafish embryos and embryo culture medium, and no model was established; the model group contained zebrafish embryos and embryo culture medium, and the model was established by ultraviolet irradiation; the positive control group contained zebrafish embryos and 200μg / mL dibenzophenone-4 (CAS No.: 4065-45-6, purity 99%) solution prepared with embryo culture medium, and the model was established by ultraviolet irradiation; the test group of the test substance contained the test substance and zebrafish embryos, and the model was established by ultraviolet irradiation. After the modeling incubation is completed, at least 8 zebrafish are randomly selected from zebrafish with normal phenotypes and behaviors, fixed with 3% methylcellulose, observed and photographed under a stereo microscope. When taking pictures, the zebrafish should face left, side down, and keep the body horizontal. All zebrafish photography results must be completed under the same instrument and environmental conditions, and the body position of the zebrafish should be consistent. After the photo is taken, the image analysis software is used to analyze the obtained zebrafish pictures, and the quantitative area is selected as the zebrafish tail fin. Set the analysis parameter to area, analyze the picture to obtain data, and take 8 valid data for each group. The tail fin wrinkle recovery rate calculation formula:

[0089]

[0090] The specific data of the above determination are shown in Table 4 and Figure 2 As shown:

[0091] Table 4. Anti-wrinkle effects of Example 1 and Comparative Examples 1 to 4 on zebrafish

[0092]

[0093]

[0094] Note: Compared with the blank group, “####” indicates P﹤0.0001; compared with the model group, “ns” indicates no significant difference, P>0.05, “**” indicates P﹤0.01, “***” indicates P﹤0.001, and “****” indicates P﹤0.0001.

[0095] Depend on Figure 2 As shown in Table 4, statistical analysis shows that: compared with the blank group, the tail fin area of ​​zebrafish in the model group was significantly reduced (P<0.0001), indicating that the model was successfully established; and compared with the model group, the tail fin area of ​​zebrafish in the positive control group was significantly increased after administration (P<0.0001), indicating that the positive drug (benzophenone) has anti-wrinkle effect. The tail fin area of ​​zebrafish in Example 1 and Comparative Examples 1-4 was increased compared with the model group. Among them, the tail fin wrinkle recovery rate of Example 1 with four components was much higher than that of any three-component Comparative Examples 1-4, up to 59.69%, and had a good anti-wrinkle effect. It can be seen that the combination of the four components of the composition has a good synergistic anti-wrinkle effect.

[0096] 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. A synergistic anti-wrinkle composition, characterized in that: It includes the following components calculated by mass: 5-35 parts of Chrysanthemum procumbentum notoginseng extract, 5-30 parts of VC ethyl ether, 5-30 parts of hydrolyzed collagen, and 5-20 parts of ginseng essential oil.

2. The synergistic anti-wrinkle composition according to claim 1, characterized in that: The notoginseng procumbens extract is prepared by extracting the notoginseng procumbens with water and / or an organic solvent.

3. The synergistic anti-wrinkle composition according to claim 1, characterized in that: The content of ginsenoside in the ginseng essential oil is 0.1-1 mg / g.

4. The synergistic anti-wrinkle composition according to claim 1, characterized in that: It includes the following components calculated by mass: 25-35 parts of Chrysanthemum procumbentum extract, 10-30 parts of VC ethyl ether, 5-30 parts of hydrolyzed collagen and 5-20 parts of ginseng essential oil.

5. The synergistic anti-wrinkle composition according to claim 4, characterized in that: The mass ratio of the notoginseng extract, VC ethyl ether, hydrolyzed collagen and ginseng essential oil is 1: (0.83-1): (0.67-1.2): (0.27-0.6).

6. The synergistic anti-wrinkle composition according to any one of claims 1 to 5, characterized in that: The synergistic anti-wrinkle composition has an elastase activity inhibition rate of ≥15.43%.

7. The synergistic anti-wrinkle composition according to any one of claims 1 to 5, characterized in that: The combination index of the synergistic anti-wrinkle composition is <1.

8. The synergistic anti-wrinkle composition according to any one of claims 1 to 5, characterized in that: The DPPH scavenging rate of the synergistic anti-wrinkle composition is ≥92.32%.

9. Use of the synergistic anti-wrinkle composition according to any one of claims 1 to 5 in the preparation of a product for inhibiting the activity of elastase.

10. An anti-wrinkle skin care product, characterized in that: The synergistic anti-wrinkle composition comprises the synergistic anti-wrinkle composition according to any one of claims 1 to 5.

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