Ascorbic acid peroxidase mimic enzyme and application thereof in anti-aging cosmetics
Technical Field
The invention belongs to the technical field of cosmetics, and particularly relates to an ascorbic acid peroxidase mimic enzyme and application thereof in anti-aging cosmetics.
Background
Aging is one of the most basic natural laws in the biological world, and skin aging refers to the aging damage of skin function, which reduces the protection and regulation abilities of skin to the body. Skin aging mainly includes natural aging, which is an endogenous programmed process, and photoaging, which is the accumulation of damage caused by exposure to other environmental factors or by lifestyle factors, and aging caused by the ultraviolet radiation of the sun is also called photoaging. Ultraviolet rays are the main factors causing skin photoaging, and the skin generates free radicals under the irradiation of ultraviolet rays, so that collagen protein having a skin protecting effect is lost and the content of antioxidant enzymes is reduced, resulting in skin exposed to ultraviolet rays becoming hyperpigmented, wrinkles are increased, roughness and sagging, and telangiectasia, etc., to become aged.
Aging of the skin not only affects the beauty, but also may cause psychological problems such as depression and inferior quality, and even be related to many diseases. Therefore, the search for an ideal anti-skin aging way and the adoption of effective anti-aging measures are one of the hot spots of the current anti-aging cosmetic research.
Peroxidase (PX) is widely present in animals, plants and aerobic microorganisms, and it mainly catalyzes the following types of reactions: h2O2+2H→2H2O, therefore, plays an important role in the metabolism of active oxygen in the body. Peroxidase can be divided into two types according to the difference of catalytic groups, one type uses selenocysteine as a catalytic group, such as glutathione peroxidase; the other uses iron porphyrin as a catalytic group, such as ascorbic acid peroxidase. Although natural peroxidase is an effective antioxidant, it has disadvantages of limited source, difficulty in purification, large molecular weight, and the like.
Disclosure of Invention
In view of the above, the present invention aims to provide an ascorbate peroxidase mimic enzyme and an application thereof in anti-aging cosmetics, wherein the mimic enzyme has high activity of scavenging free radicals and playing an anti-oxidation role, so as to replace ascorbate peroxidase.
The invention provides an ascorbic acid peroxidase mimic enzyme, which comprises heme and milk protein, wherein the mass ratio of the heme to the milk protein is (1-10): (10-100).
Preferably, the mass ratio of the heme to the milk protein is 1: 5 to 50.
Preferably, the mass ratio of the heme to the milk protein is 1: 10.
preferably, the heme comprises hemin or hemin.
Preferably, sodium carbonate solution is also included; the concentration of the sodium carbonate solution is 0.4-0.6 mg/mL;
the concentration of the heme is 1-10 mg/mL; the concentration of the milk protein is 10-100 mg/mL.
Preferably, the mimic enzyme has an ascorbate peroxidase activity of not less than 3.5 × 103U/mL;
The DPPH free radical clearance rate of the mimic enzyme is not less than 65%.
The invention provides application of the ascorbate peroxidase mimic enzyme in anti-aging cosmetics.
Preferably, the volume of the added ascorbate peroxidase mimic enzyme accounts for 1-5% of the volume of the cosmetic.
Preferably, the cosmetic comprises a mask, a cream, a facial cleanser, a serum or an emulsion.
The invention provides a cosmetic containing the ascorbic acid peroxidase mimic enzyme.
The invention provides an ascorbic acid peroxidase mimic enzyme, which comprises heme and milk protein, wherein the mass ratio of the heme to the milk protein is (1-10) - (10-100), the heme (heme) is a complex formed by Protoporphyrin (Protoporphyrin) and iron ions, heme molecules are used as prosthetic groups of the protein, axial ligands (axialigid) generally exist besides the coordination of the central Fe and N atoms on 4 pyrrole rings, common His, Cys, Lys, Met and the like, enzymes containing the heme mostly catalyze redox reactions, such as peroxidase (peroxosidase), are widely researched due to the fact that the peroxidase (peroxosidase) participates in the elimination of free radicals in vivo and eliminates the toxicity of phenols and amines3U/mL, the activity of the commercially available ascorbic acid oxidase is 10000U; the DPPH free radical clearance rate of the mimic enzyme is not lower than 65%, and the DPPH free radical clearance rate of the commercially available ascorbic acid oxidase is 90%, which indicates that the mimic enzyme has higher free radical clearance activity.
The invention provides application of the ascorbate peroxidase mimic enzyme in anti-aging cosmetics. After the mimic enzyme is prepared into the cosmetics, the mimic enzyme has obvious advantages in the aspect of the activity of the ascorbic acid peroxidase, the activity reaches 2310-3100U/mL, and the mimic enzyme is remarkably improved compared with the cosmetics prepared by commercially available ascorbic acid oxidase; in terms of DPPH free radical clearance, the clearance of cosmetics prepared by mimic enzyme is 56% -72%, while the clearance of cosmetics prepared by commercial ascorbic acid oxidase is only 51%. The cosmetic containing the heme-milk protein composition is applied to the faces of women of different ages, and the result shows that the facial wrinkles have obvious improvement effect which is better than that of an ascorbate peroxidase control group.
Detailed Description
The invention provides an ascorbic acid peroxidase mimic enzyme, which comprises heme and milk protein, wherein the mass ratio of the heme to the milk protein is (1-10): (10 to 100), preferably 1:2 to 1:90, more preferably 1: 5-50, most preferably 1: 10. in the present invention, the heme preferably comprises hemin or hemin. The sources of the heme and the milk protein are not particularly limited in the present invention, and heme and milk protein which are well known in the art can be used. In the examples of the present invention, the heme was obtained from Aladdin reagent, and the milk protein was obtained from Aladdin reagent.
In the present invention, the mimetic enzyme preferably further comprises a solvent; the solvent is preferably a sodium carbonate solution; the concentration of the sodium carbonate solution is 0.5 mg/mL. In the mimic enzyme, the concentration of the heme is 1-10 mg/mL, preferably 2-8 mg/mL, more preferably 3-7 mg/mL, and most preferably 5 mg/mL; the concentration of the milk protein is 10-100 mg/mL, preferably 20-90 mg/mL, more preferably 30-70 mg/mL, further preferably 40-60 mg/mL, and most preferably 50 mg/mL.
In the present invention, the ascorbic acid peroxidase activity of the mimetic enzyme is measured by the ascorbic acid-hydrogen peroxide method, and the ascorbic acid peroxidase activity of the mimetic enzyme is preferably not less than 3.9 × 103U/mL. The DPPH radical clearance of the mimic enzyme is measured by a conventional method, and is preferably not less than65%。
The invention provides application of the ascorbate peroxidase mimic enzyme in anti-aging cosmetics.
In the present invention, the volume of the ascorbate peroxidase mimic enzyme is preferably 1% to 5%, more preferably 2% to 4%, and most preferably 3% of the volume of the cosmetic. The kind of the cosmetic is not particularly limited in the present invention, and may be any known cosmetic, such as a mask, a cream, a face lotion, an essence, an emulsion, etc. The method for preparing the cosmetic is not particularly limited, and a method for preparing the cosmetic known in the art may be used. The cosmetic has antiaging effect, and can be used for improving facial wrinkle.
The invention provides a cosmetic containing the ascorbic acid peroxidase mimic enzyme. The addition amount, the type and the preparation method of the mimic enzyme in the cosmetic are the same as those of the cosmetic prepared by the application, and are not repeated herein. Experiments show that the cosmetic has advantages in the aspects of the activity of the ascorbic acid peroxidase and the DPPH clearance compared with the cosmetic added with the commercially available ascorbic acid peroxidase.
The following examples are provided to illustrate the ascorbate peroxidase mimic enzyme and its use in anti-aging cosmetics, but should not be construed as limiting the scope of the invention.
Example 1
Preparation method of heme-milk protein mimic enzyme
Dissolving 5mg heme in 5mL of 0.5mg/mL sodium carbonate solution, adding 50mg milk protein powder, and stirring at 40 deg.C to obtain heme-milk protein solution.
Example 2
Preparation method of heme-milk protein mimic enzyme
Dissolving 5mg heme in 5mL of 0.5mg/mL sodium carbonate solution, adding 5mg milk protein powder, and stirring at 60 deg.C to obtain heme-milk protein solution.
Example 3
Preparation method of heme-milk protein mimic enzyme
Dissolving 5mg heme in 5mL of 0.5mg/mL sodium carbonate solution, adding 500mg milk protein powder, and stirring at 50 deg.C to obtain heme-milk protein solution.
Example 4
Preparation method of heme-milk protein mimic enzyme
Dissolving 5mg heme in 5mL of 0.5mg/mL sodium carbonate solution, adding 450mg of milk protein powder, and stirring at 45 ℃ to dissolve to obtain the heme-milk protein solution.
Example 5
Preparation method of heme-milk protein mimic enzyme
Dissolving 5mg heme in 5mL sodium carbonate solution with concentration of 0.5mg/mL, adding 250mg milk protein powder, and stirring at 55 deg.C to obtain heme-milk protein solution.
Example 6
Preparation method of heme-milk protein mimic enzyme
Dissolving 25mg heme in 5mL of 0.5mg/mL sodium carbonate solution, adding 250mg milk protein powder, and stirring at 55 deg.C to obtain heme-milk protein solution.
Example 7
Preparation method of heme-milk protein mimic enzyme
Dissolving 10mg heme in 5mL of 0.5mg/mL sodium carbonate solution, adding 100mg milk protein powder, and stirring at 55 deg.C to obtain heme-milk protein solution.
Comparative example 1
Preparation method of heme-milk protein mimic enzyme
Dissolving 25mg heme in 5mL of sodium chloride solution with concentration of 0.5mg/mL, adding 250mg of milk protein powder, and stirring at 55 ℃ to dissolve to obtain heme-milk protein solution.
Example 8
Determination of the Activity of ascorbic acid peroxidase Using the classical ascorbic acid-Hydrogen peroxide method
3ml the reaction mixture contained 50mmol/LK2HPO4-KH2PO4Buffer (pH 7.0), 0.1mmol/LEDTA-Na20.3mmol/L ascorbic acid, 0.06mmol/LH2O2And 0.1ml of the heme-milk protein solution prepared in the above example, with commercially available ascorbate oxidase as a positive control. Addition of H2O2Immediately measuring the change of light absorption at 290nm with UV2501 UV-visible spectrophotometry system of Shimadzu corporation at 25 deg.C within 10-30 s at 20 deg.C to obtain A290The value is obtained. The amount of enzyme required to oxidize 1 μ M ascorbic acid per minute is defined as one activity unit.
The results of the ascorbate peroxidase activity of the heme-milk protein solution are shown in Table 1.
TABLE 1 examples 1-7 and positive controls for ascorbate peroxidase activity
Example 9
Antioxidant activity assay of heme-milk protein composition solutions
The method for measuring the antioxidant activity of the substance by adopting a DPPH free radical scavenging experiment is a simple, effective, economic and reliable method, and has good result repeatability and small influence by environmental factors.
95% ethanol is used as a solvent to prepare a DPPH free radical solution with the concentration of 0.1mmol/L, and the solution needs to be prepared in situ. 2.0mL of the heme-milk protein solution prepared in examples 1 to 7 was mixed with 2.0mL of the LDPPH solution, incubated at 25 ℃ in the dark for 30min, the absorbance of the reaction solution was measured at 517nm, 95% ethanol was used as a blank control, and commercially available ascorbic acid oxidase was used as a positive control. The DPPH radical clearance was calculated as follows: DPPH free radical clearance (%) (1-absorbance blank) x 100% formula I was determined and the DPPH free radical clearance results for the heme-milk protein solution are shown in table 2.
TABLE 2 DPPH radical clearance in examples 1-7 and positive controls
Group of
|
DPPH radical scavenging ratio (%)
|
Example 1
|
80
|
Example 2
|
78
|
Example 3
|
75
|
Example 4
|
68
|
Example 5
|
79
|
Example 6
|
82
|
Example 7
|
83
|
Blank control
|
0
|
Positive control
|
90
|
Comparative example 1
|
15 |
As can be seen from Table 2, the DPPH free radical clearance of the heme-milk protein solution can reach 68% -83%, and the DPPH free radical clearance of the purchased ascorbic acid oxidase is 90%, which indicates that the mimic enzyme provided by the invention has higher activity of clearing free radicals.
Example 10
Preparation method of mask containing heme-milk protein mimic enzyme and ascorbic acid peroxidase activity
The composition of the mask liquid is as follows: 15g of polyvinyl alcohol, 5g of propylene glycol, 0.1g of platinum methyl ester, 0.05g of essence, 5.0g of the heme-milk protein solution prepared in the embodiments 1 to 7, adding deionized water to 100g, and fully mixing to obtain the facial mask liquid. Two control mask solutions were prepared simultaneously, replacing the heme-milk protein solution with commercially available ascorbate peroxidase or comparative example 1.
The above-prepared facial mask solutions were subjected to the measurement of ascorbate peroxidase activity according to the method of example 8, and the results are shown in Table 3. The antioxidant activity of the mask solution was measured according to the method of example 9, and the results are shown in Table 4.
TABLE 3 examples 1-7, Positive control and comparative example 1 Activity of ascorbic acid peroxidase
Group of
|
Ascorbic acid peroxidase Activity (U/mL)
|
Example 1
|
2870
|
Example 2
|
2650
|
Example 3
|
2310
|
Example 4
|
2580
|
Example 5
|
2960
|
Example 6
|
3100
|
Example 7
|
2880
|
Positive control
|
1220
|
Comparative example 1
|
-160 |
TABLE 4 DPPH radical scavenging rates in examples 1-7, positive control and comparative example 1
Example 11
Wrinkle-improving effect test
An experimental group (the mask solution prepared in example 10, to which the mimetic enzyme solution prepared in example 1 was added) and a blank control group (the above-mentioned mask solution to which the heme-milk protein solution was not added) were selected as test samples, and the effect of improving wrinkles by each test sample was examined.
100 women of 30-50 years old are selected and randomly divided into 1 group and 2 groups, 50 people in each group are used, each person in the 1 group respectively uses the facial masks of the experimental group and the control group on the left and right faces every day, each person in the 2 groups respectively uses the facial masks of the experimental group and the ascorbic acid peroxidase control group on the left and right faces every day for 8 weeks continuously, and the variation of each index of the skin before and after the marking point of a subject uses the sample is measured by a Visinirane VL650 wrinkle analysis system, so that the influence of the sample on each index of the skin is evaluated.
The principle of the Visinilone VL650 wrinkle analysis system is that shadows are formed at positions with wrinkles on a silica gel copy model of the skin by virtue of oblique illumination, a high-resolution camera is arranged above a host platform, and the change of the area, the length and the wrinkle depth value of the shadow parts is analyzed by special software to reflect the wrinkle change. Results are expressed as total wrinkle area (mm)2) The average wrinkle length (mm) and the average wrinkle depth (μm) are shown, and the photograph taken at the first time is used as a positioning reference in the subsequent analysis.
When the wrinkle model is copied, the subject lies down, the left side of the head faces upwards, the eyes are closed, and the positioning glue is stuck to the mark points. Pouring into a certain amount of silica gel and a small plastic box, adding a proper amount of catalyst coagulant, quickly and uniformly stirring, uniformly coating the silica gel on the region defined by the positioning gel, and removing the silica gel after 10min and pasting on a positioning paper board special for a Visioline VL650 wrinkle analysis system. The change in wrinkles of the eye corner fine-line silica gel replication model was analyzed by visioline vl650 wrinkle analysis system before and after 2 weeks, 4 weeks, 6 weeks, and 8 weeks of continuous use. Placing the prepared silica gel replica on a Visiline VL650 platform, adjusting the focus and light rays which are vertical to the wrinkle lines, and analyzing the total wrinkle area (mm) after photographing2) As a result, when the wrinkle area before and after use is compared, the reduction in area is regarded as significant, and the invariance in area is regarded as ineffective.
The total wrinkle area of the subjects after using the test group mask for 5 weeks was from 82mm2Reduced to 68mm2The average wrinkle length is reduced from 1.5mm to 0.7mm, and the average wrinkle depth is reduced from 69 μm to 57 μm, which shows that the experimental group mask has more obvious anti-wrinkle effect than the commercial enzyme group mask. The mask usage statistics are shown in table 5.
TABLE 5 use of different groups of masks
From the results in table 5, it can be seen that the facial mask containing the heme-milk protein mimic enzyme has a significant improvement effect on female facial wrinkles, the improvement effect is better than that of the ascorbate peroxidase control group, and it is presumed that the components in the facial mask have a greater influence on the activity of commercially available enzymes.
Example 12
1) A method for preparing cosmetic lotion containing heme-milk protein mimic enzyme
The toning lotion comprises the following components: 0.5g of sodium pyrrolidone carboxylate, 0.1g of hyaluronic acid, 5.0g of glycerin, 0.05g of methyl nicotinate, 0.05g of essence, 5.0g of the heme-milk protein solution prepared in example 2, and deionized water to 100 g.
2) Wrinkle-improving effect test
The experimental group (the lotion prepared above) and the control group (the lotion without the heme-milk protein composition solution added) were selected, and the ascorbate peroxidase control group (the lotion prepared by replacing the heme-milk protein solution with commercially available ascorbate peroxidase) was tested for the wrinkle-improving effect of each group of lotions.
100 women of 30-50 years old are selected and randomly divided into 1 group and 2 groups, wherein 50 people in each group are used, the experimental group lotion and the control group lotion are respectively used on the left face and the right face of each person in the 1 group every day, the experimental group lotion and the ascorbic acid peroxidase control group lotion are respectively used on the left face and the right face of each person in the 2 groups every day and are continuously used for 8 weeks, the method of the embodiment 11 is adopted to judge the obvious effect condition of the lotion, and the wrinkle improvement effect of the experimental group and the control group is compared. The results are shown in Table 6.
TABLE 6 use of lotions of different groups
As can be seen from the results in Table 6, the astringent containing the heme-milk protein mimetic enzyme has a remarkable improvement effect on female facial wrinkles, which is better than that of the ascorbate peroxidase control group.
Example 13
1) Preparation method of essence containing heme-milk protein mimic enzyme
The essence comprises the following components: 4g of polyethylene glycol, 5g of propylene glycol, 4.0g of tea oil, 2.0g of hydrogenated castor oil, 0.05g of niplatinumma, 0.05g of essence, 1.0g of sodium pyrrolidone carboxylate, 10g of the heme-milk protein solution prepared in example 3, and deionized water added to 100 g.
2) Wrinkle-improving effect test
An experimental group (the essence prepared above) and a control group (the essence without the heme-milk protein solution) were selected, and an ascorbate peroxidase control group (the essence prepared by replacing the heme-milk protein solution with commercially available ascorbate peroxidase) was used to test the wrinkle improvement effect of each group of the essences.
Selecting 100 women of 30-50 years old, randomly dividing the women into 1 group and 2 groups, wherein each group comprises 50 people, using the essence of the experimental group and the essence of the control group on the left face and the right face of each person of the 1 group respectively every day, using the essence of the experimental group and the essence of the ascorbic acid peroxidase control group on the left face and the right face of each person of the 2 group respectively every day, continuously using for 8 weeks, judging the significant effect condition of the essence by adopting the method of the embodiment 11, and comparing the wrinkle improvement effect of the experimental group and the control group. The results are shown in Table 7.
TABLE 7 use of different groups of essences
As can be seen from the results in table 7, the serum containing the heme-milk protein mimetic enzyme has a significant improvement effect on female facial wrinkles, which is better than that of the ascorbate peroxidase control group.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.