A composition for enhancing the antioxidant activity of vitamin c and its derivatives

Abstract

The application discloses a composition for improving the antioxidant activity of vitamin C and derivatives thereof. By testing vitamin C solutions and vitamin C derivative solutions added with different concentrations of mussel myoglobin without a dopa group, it is found that when the mass ratio of vitamin C or vitamin C derivatives and mussel myoglobin without a dopa group is 1:0.001-1, the stability of vitamin C or vitamin C derivatives can be improved, and a new approach for effectively enhancing the antioxidant activity of vitamin C and derivatives thereof in cosmetics is provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of mussel extract, in particular to the use of non-modified recombinant mussel mucin to improve the antioxidant activity of vitamin C and its derivatives. BACKGROUND

[0002] Vitamin C, also known as ascorbic acid, has the chemical formula C6H8O6. In its molecular structure, the two adjacent enolic hydroxyl structures at positions 2 and 3 are unstable and easily dissociate to release H + Therefore, it has strong reducing power and is easily oxidized to dehydroascorbic acid. Vitamin C is widely present in fruits and vegetables and is one of the essential vitamins for the human body. According to epidemiological data analysis, food rich in vitamin C can inhibit the progression of cancer such as laryngeal cancer, esophageal cancer, and gastric cancer, as well as chronic diseases such as coronary heart disease and cataract. Vitamin C can provide reducing power for a series of biochemical reactions in the human body, such as hydroxylating hydroxyproline and lysine by providing electrons to enzymes, and is an essential substance for the synthesis of collagen and connective tissue. In biological fluids, vitamin C is an antioxidant with protective effects, rapidly combining with O 2- , HOO - , OH - , and other oxygen free radicals to form semi-dehydroascorbic acid, protecting cells from active oxygen damage.

[0003] Because vitamin C has strong reducing property, it is easily destroyed by oxidizing agents and heat, especially in neutral or alkaline solution, which limits its application in cosmetics and other fields. Therefore, researchers have developed various ways to reduce the influence of external factors on the overall molecular structure of vitamin C while preserving its active structure, maintain or extend its antioxidant properties: (1) Using liposomes such as lecithin to encapsulate vitamin C, which can block the contact between vitamin C and air, water, etc., thereby achieving the purpose of stabilizing the structure of vitamin C and maintaining its activity. However, the stability of liposomes, their application in cosmetic formulations, and the effect of vitamin C encapsulated by liposomes on the skin are not clear. (2) Introducing other groups to form derivatives while retaining the active dihydroxy structure of vitamin C. However, some derivatives may produce complex or large amounts of by-products during synthesis, such as vitamin C pyrophosphate and double vitamin C phosphate in addition to vitamin C phosphate in the product of phosphonylation. The presence of these by-products, even in small amounts, can affect the viscosity and stability of the cosmetic system. Moreover, the traditional derivatives have limited stability improvement, and may still have stability problems such as discoloration and degradation when added in high amounts or stored for a long time. (3) Forming a composition by compounding other ingredients with vitamin C, such as Chinese patent CN104606392A, which introduces vitamin C, vitamin E, and high-activity oxidizing substances to form a composition. Vitamin C provides hydrogen, allowing the chain-breaking antioxidant effect of vitamin E to continuously protect high-activity oxidizing substances. However, this does not actually improve the antioxidant activity of vitamin C. Chinese patent CN109044871A compiles xanthan gum, ferulic acid, tannic acid, and ginkgo extract with vitamin C, which effectively stabilizes the antioxidant activity of vitamin C for six weeks. However, the experimental groups and results provided do not adequately demonstrate the synergistic effect of antioxidant activity between components.

[0004] Natural mussel adhesive proteins are extracted from the byssus glands of marine mollusks Mytilus edulis. At least 13 mussel adhesive proteins have been studied and identified, including 8 adhesive proteins: Mfp-1, Mfp-2, Mfp-3F, Mfp-3S, Mfp-4, Mfp-5, Mfp-6, Mfp-7, and 5 byssal fiber backbone proteins: preCOL-D, preCOL-P, preCOL-NG, PTMP-1, TMP-1. Natural mussel adhesive proteins can quickly adsorb to the surface of negatively charged cells by using the high positive charge carried by the large amount of lysine (Lys) contained therein at physiological pH, and promote cell adhesion and migration, thereby accelerating wound healing. The inherent DOPA structure in natural mussel adhesive proteins, such as oxidized DOPA and unoxidized DOPA, can cross-link to form a water-resistant adhesive effect with cells and tissues, and form a micro-nano biological scaffold. The anti-wrinkle and anti-spot liquid prepared by combining natural mussel adhesive proteins with vitamin C at a ratio of 0.5:1 in Chinese Patent CN104323927A does not exhibit a protective and synergistic effect on the antioxidant activity of vitamin C.

[0005] According to the "Cosmetic Ingredient Directory", the so-called mussel extract in the industry includes modified recombinant mussel adhesive proteins. Such recombinant mussel adhesive proteins are obtained by using genetic engineering technology to construct genetically engineered bacteria expressing mussel adhesive proteins, and using the fermentation of the genetically engineered bacteria (to obtain mussel adhesive proteins without DOPA groups) and downstream processing (including processing to modify DOPA groups). Since it is originally derived from mussel adhesive proteins without DOPA groups extracted from the fermentation system, it is also collectively referred to as mussel extract. There is no report on using mussel adhesive proteins without DOPA groups to enhance the antioxidant activity of vitamin C and its derivatives. SUMMARY

[0006] The purpose of the present application is to provide a composition for enhancing the antioxidant activity of vitamin C and its derivatives.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] A method for enhancing the antioxidant activity of vitamin C, comprising the following steps:

[0009] Adding mussel adhesive proteins without DOPA groups to a dispersion system containing vitamin C, or adding mussel adhesive proteins without DOPA groups to a dispersion system containing vitamin C derivatives.

[0010] Preferably, the mussel adhesive proteins without DOPA groups are one or more types of mussel adhesive proteins formed by fermentation of genetically engineered bacteria (such as constructed by Pichia pastoris, etc.).

[0011] Preferably, the kind of the mussel adhesive protein expressed by the genetically engineered bacteria comprises at least any one of Mfp-5 and Mfp-1.

[0012] Preferably, the vitamin C derivative is selected from one or more of vitamin C phosphate, vitamin C glucoside, and vitamin C palmitate.

[0013] Preferably, in the dispersion system containing vitamin C, the mass ratio of vitamin C to mussel adhesive protein without dopa group is 1:0.001-1; and in the dispersion system containing vitamin C derivative, the mass ratio of vitamin C derivative to mussel adhesive protein without dopa group is 1:0.001-1.

[0014] Preferably, in the dispersion system containing vitamin C, the mass ratio of vitamin C to mussel adhesive protein without dopa group is 1:0.01-1; and in the dispersion system containing vitamin C derivative, the mass ratio of vitamin C derivative to mussel adhesive protein without dopa group is 1:0.01-1.

[0015] Preferably, in the dispersion system containing vitamin C, the mass ratio of vitamin C to mussel adhesive protein without dopa group is 1:0.02-0.4; and in the dispersion system containing vitamin C derivative, the mass ratio of vitamin C derivative to mussel adhesive protein without dopa group is 1:0.02-0.4.

[0016] Preferably, in the dispersion system containing vitamin C, the mass ratio of vitamin C to mussel adhesive protein without dopa group is 1:0.02-0.2; and in the dispersion system containing vitamin C derivative, the mass ratio of vitamin C derivative to mussel adhesive protein without dopa group is 1:0.02-0.2.

[0017] Preferably, in the dispersion system containing vitamin C, the mass ratio of vitamin C to mussel adhesive protein without dopa group is 1:0.02-0.1; and in the dispersion system containing vitamin C derivative, the mass ratio of vitamin C derivative to mussel adhesive protein without dopa group is 1:0.02-0.1.

[0018] Preferably, the dispersion system containing vitamin C comprises a vitamin C solution, and the dispersion system containing vitamin C derivative comprises a vitamin C derivative solution.

[0019] Preferably, the solvent used in the vitamin C solution or the vitamin C derivative solution is selected from one or more of water (e.g. purified water), polyhydric alcohol, oil, and the like.

[0020] Preferably, the concentration of vitamin C in the dispersion system containing vitamin C is 0.1% to 5.0%, and the concentration of vitamin C derivative in the dispersion system containing vitamin C derivative is 0.1% to 5.0%.

[0021] A composition for improving antioxidant activity, the composition comprising vitamin C and the mussel myoglobin without dopa group, wherein the mass ratio of vitamin C: mussel myoglobin without dopa group is 1:0.001 to 1; or the composition comprising vitamin C derivative and the mussel myoglobin without dopa group, wherein the mass ratio of vitamin C derivative: mussel myoglobin without dopa group is 1:0.001 to 1.

[0022] Preferably, the mass ratio of vitamin C or vitamin C derivative: mussel myoglobin without dopa group is 1:0.01 to 1.

[0023] Preferably, the mass ratio of vitamin C or vitamin C derivative: mussel myoglobin without dopa group is 1:0.02 to 0.4.

[0024] Preferably, the mass ratio of vitamin C or vitamin C derivative: mussel myoglobin without dopa group is 1:0.02 to 0.2.

[0025] Preferably, the mass ratio of vitamin C or vitamin C derivative: mussel myoglobin without dopa group is 1:0.02 to 0.1.

[0026] The present application has the following advantages:

[0027] The present application combines vitamin C or vitamin C derivative (such as vitamin C phosphate, vitamin C glucoside, vitamin C palmitate) with mussel extract (mussel myoglobin without dopa group), so that the stability of vitamin C or vitamin C derivative can be significantly improved when the content of vitamin C or vitamin C derivative in the system (such as water phase system, oil phase system, emulsion system) is 0.001 to 1 times the unit of the added mussel extract (mussel myoglobin without dopa group). The present application does not introduce complex compound components and does not change the molecular structure of vitamin C and its corresponding derivatives, and protects the activity of such antioxidant components by adding mussel extract (mussel myoglobin without dopa group), so that the antioxidant performance of vitamin C and its derivatives in the system is improved, and the application prospect in the development of cosmetics and other products is broad. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1Residual content of VC after addition of different concentrations of mussel extract (mussel mucus without dopa groups) to a 0.1% VC solution at 15°C in Example 1.

[0029] Figure 2 pH of the solution after addition of different concentrations of mussel extract (mussel mucus without dopa groups) to a 0.1% VC solution at 15°C in Example 1.

[0030] Figure 3 Residual content of VC after addition of different concentrations of mussel extract (mussel mucus without dopa groups) to a 5.0% VC solution at 15°C in Example 1.

[0031] Figure 4 pH of the solution after addition of different concentrations of mussel extract (mussel mucus without dopa groups) to a 5.0% VC solution at 15°C in Example 1.

[0032] Figure 5 Residual content of VC after addition of different concentrations of mussel extract (mussel mucus without dopa groups) to a 0.5% VC solution at 40°C in Example 1.

[0033] Figure 6 pH of the solution after addition of different concentrations of mussel extract (mussel mucus without dopa groups) to a 0.5% VC solution at 40°C in Example 1.

[0034] Figure 7 Residual content of VC after addition of different concentrations of mussel extract (mussel mucus without dopa groups) to a 1.0% VC solution at 40°C in Example 1.

[0035] Figure 8 pH of the solution after addition of different concentrations of mussel extract (mussel mucus without dopa groups) to a 1.0% VC solution at 40°C in Example 1.

[0036] Figure 9 , Figure 10 OH radical scavenging rate (15°C) of 0.1%, 5.0% VC at a gradient concentration of mussel extract (mussel mucus without dopa groups) in Example 1, respectively.

[0037] Figure 11 , Figure 12 OH radical scavenging rate (40°C) of 0.5%, 1.0% VC at a gradient concentration of mussel extract (mussel mucus without dopa groups) in Example 1, respectively.

[0038] Figure 13 , Figure 14OH radical scavenging rate (15°C) of 0.1%, 5.0% AA2G in gradient Mytilus extract (mussel mucin without dopa group) concentration respectively in Example 1.

[0039] Figure 15 , Figure 16 OH radical scavenging rate (40°C) of 0.5%, 1.0% AA2G in gradient Mytilus extract (mussel mucin without dopa group) concentration respectively in Example 1.

[0040] Figure 17 , Figure 18 OH radical scavenging rate (15°C) of 0.1%, 5.0% MAP in gradient Mytilus extract (mussel mucin without dopa group) concentration respectively in Example 1.

[0041] Figure 19 , Figure 20 OH radical scavenging rate (40°C) of 0.5%, 1.0% MAP in gradient Mytilus extract (mussel mucin without dopa group) concentration respectively in Example 1. DETAILED DESCRIPTION

[0042] The application will be further described in conjunction with the accompanying drawings and examples. The examples are only used to explain the application, and are not intended to limit the scope of protection of the application.

[0043] Example 1

[0044] (I) Composition for increasing antioxidant activity of antioxidant substances

[0045] 1. Source of composition ingredients

[0046] (1) Vitamin C (VC) is a commercially available product, i.e. L-ascorbic acid (L-Ascorbic acid).

[0047] (2) Vitamin C glucoside (AA2G) is a commercially available product, i.e. ascorbyl glucoside (Ascorbyl Glucoside).

[0048] (3) Vitamin C phosphate magnesium (MAP) is a commercially available product, i.e. magnesium ascorbyl phosphate (Magnesium Ascorbyl Phosphate).

[0049] (4) Mytilus extract is a fusion protein of mussel mucin Mfp-5 and Mfp-1 provided by Xi'an Deno Haisi Medical Technology Co., Ltd. Since it is directly extracted after being expressed by Pichia pastoris, it belongs to mussel mucin without dopa group.

[0050] 2. First type of verification group formula and experimental test conditions

[0051] (1) According to the composition of each group, the solution for verifying the effect of mussel extract (mussel mucus without dopa group) was prepared; the composition and ratio of each group are shown in Table 1, wherein 1-0, 2-0 are blank control groups of mussel extract (mussel mucus without dopa group), 1-1, 2-1 are blank control groups of vitamin C, and the rest are experimental groups:

[0052] Table 1. Formula of the first type of verification combination (wt%)

[0053]

[0054] Solution preparation: taking group 1-2 and group 2-2 as examples, 0.002g of the above mussel extract (mussel mucus without dopa group) was added to 100g of 0.1% and 5.0% VC aqueous solution respectively, and mixed uniformly.

[0055] (2) Test conditions

[0056] After the preparation of each group of solutions, they were stored at room temperature (15°C) and protected from light.

[0057] 3. Formula of the second type of verification combination and experimental test conditions

[0058] (1) According to the composition of each group, the solution for verifying the effect of mussel extract (mussel mucus without dopa group) was prepared; the composition and ratio of each group are shown in Table 2, wherein 3-0, 4-0 are blank control groups of mussel extract (mussel mucus without dopa group), 3-1, 4-1 are blank control groups of vitamin C, and the rest are experimental groups:

[0059] Table 2. Formula of the second type of verification combination (wt%)

[0060]

[0061] Solution preparation: taking group 3-2 and group 4-2 as examples, 0.002g of the above mussel extract (mussel mucus without dopa group) was added to 100g of 0.5% and 1.0% VC aqueous solution respectively, and mixed uniformly.

[0062] (2) Test conditions

[0063] After the preparation of each group of solutions, they were stored in a constant temperature box (40°C±1°C) and protected from light.

[0064] 4. Formula of the third type of verification combination and experimental test conditions

[0065] (1) Preparation of solutions for verifying the effect of mussel extract (mussel mucin without dopa group) according to the formula of each composition; the ingredients and proportions of each composition are shown in Table 3, wherein 5-1 and 6-1 are ascorbic acid glucoside blank control groups, and the rest are experimental groups:

[0066] Table 3. Formula of the third type of verification combination (wt%)

[0067]

[0068] Preparation of solutions: taking the 5-2 group and the 6-2 group as examples, 0.002 g of the above-mentioned mussel extract (mussel mucin without dopa group) was added to 100 g of 0.1% and 5.0% ascorbic acid glucoside aqueous solution respectively, and mixed well.

[0069] (2) Test conditions

[0070] After the preparation of each group of solutions, they were stored at room temperature (15°C) and protected from light.

[0071] 5. Formula of the fourth type of verification combination and experimental test conditions

[0072] (1) Preparation of solutions for verifying the effect of mussel extract (mussel mucin without dopa group) according to the formula of each composition; the ingredients and proportions of each composition are shown in Table 4, wherein 7-1 and 8-1 are ascorbic acid glucoside blank control groups, and the rest are experimental groups:

[0073] Table 4. Formula of the fourth type of verification combination (wt%)

[0074]

[0075] Preparation of solutions: taking the 7-2 group and the 8-2 group as examples, 0.002 g of the above-mentioned mussel extract (mussel mucin without dopa group) was added to 100 g of 0.5% and 1.0% ascorbic acid glucoside aqueous solution respectively, and mixed well.

[0076] (2) Test conditions

[0077] After the preparation of each group of solutions, they were stored in a constant temperature box (40°C ± 1°C) and protected from light.

[0078] 6. Formula of the fifth type of verification combination and experimental test conditions

[0079] (1) Preparation of solutions for verifying the effect of mussel extract (mussel mucin without dopa group) according to the formula of each composition; the ingredients and proportions of each composition are shown in Table 5, wherein 9-1 and 10-1 are ascorbic acid phosphate magnesium blank control groups, and the rest are experimental groups:

[0080] Table 5. The fifth type of verification combination formula (wt%)

[0081]

[0082] Solution preparation: for example, in the 9-2 group and the 10-2 group, 0.002 g of the mussel extract (mussel mucin without a dopa group) was added to 100 g of a 0.1% and 5.0% ascorbic acid magnesium phosphate aqueous solution, respectively, and mixed well.

[0083] (2) Test conditions

[0084] After the preparation of each group of solutions, they were stored at room temperature (15°C) and in the dark.

[0085] 7. The sixth type of verification combination formula and experimental test conditions

[0086] (1) According to the composition of each group, the solution for verifying the effect of mussel extract (mussel mucin without a dopa group) was prepared; the composition and ratio of each group are shown in Table 6, 11-1 and 12-1 are blank control groups of ascorbic acid magnesium phosphate, and the rest are experimental groups:

[0087] Table 6. The sixth type of verification combination formula (wt%)

[0088]

[0089] Solution preparation: for example, in the 11-2 group and the 12-2 group, 0.002 g of the mussel extract (mussel mucin without a dopa group) was added to 100 g of a 0.5% and 1.0% ascorbic acid magnesium phosphate aqueous solution, respectively, and mixed well.

[0090] (2) Test conditions

[0091] After the preparation of each group of solutions, they were stored in an incubator (40°C ± 1°C) and in the dark.

[0092] (II) Specific methods and experimental data of the experimental test

[0093] 1. Determination of the remaining content of vitamin C in the solution and the pH of the solution

[0094] 1.1 Sample handling method

[0095] Each group of solution samples was stored in a light-proof sample bottle and stored in the dark at room temperature (15°C) and under high-temperature accelerated conditions (40°C).

[0096] The content of vitamin C was detected every month under room temperature conditions (15°C), and the content of vitamin C was detected every week under high-temperature accelerated conditions (40°C).

[0097] 1.2 Experimental instruments and materials for determination of vitamin C content

[0098] 1.2.1 Preparation of standard solution

[0099] Accurately weigh 10 mg of L-ascorbic acid standard, place it in a 10 mL volumetric flask, dissolve with water and dilute to the mark to prepare a 1000 ppm L-ascorbic acid standard solution, and dilute to 1 ppm, 5 ppm, 10 ppm, 50 ppm, 100 ppm.

[0100] 1.2.2 Chromatographic conditions

[0101] Chromatographic column: C18 column (250 mm x 4.6 mm, 5 μm)

[0102] Mobile phase: methanol-0.1% phosphoric acid solution (5:95, V / V)

[0103] Column temperature: 25.0 ± 0.5°C

[0104] Flow rate: 0.8 mL / min

[0105] Detection wavelength: 242 nm

[0106] Injection volume: 10 μL

[0107] Analysis of standard and sample: prepare the standard curve of L-ascorbic acid, obtain the linear equation and correlation coefficient, and calculate the concentration of L-ascorbic acid in each sample by the linear equation combined with the peak area of L-ascorbic acid in each sample.

[0108] Reference: Chen, P. J., et al. Determination of vitamin C and its derivatives in cosmetics by high performance liquid chromatography. Chromatography, 2015, 33(07): 771-776 (The absorption wavelengths of vitamin C and vitamin C derivatives are different, and the peak state and position under high performance liquid chromatography are different).

[0109] 1.3 pH detection

[0110] Each group of solution samples was taken out and placed at room temperature, and a Reichert PHS-3E pH meter was used for testing. During testing, the detection probe was cleaned with purified water and excess water was removed, then it was immersed in the solution sample. After the detection value was stable, the value was read and recorded as the detection value at that time.

[0111] 1.4 Vitamin C content and solution pH detection results (15°C)

[0112] The results are shown in Tables 7, 8, and Figures 1 to 4

[0113] Table 7. pH and residual content of 0.1% VC at gradient mussel extract concentration at 15°C​

[0114]

[0115] Note: Compared with the vitamin C blank control group, "*" means p <0.05, "**" means p <0.01, "***" means p <0.001

[0116] Table 8. pH and residual content of 5.0% VC at gradient Mytilus extract concentration at 15°C

[0117]

[0118]

[0119] Note: Compared with the vitamin C blank control group, "*" means p <0.05, "**" means p <0.01, "***" means p <0.001

[0120] The results show that after 4 months of 15°C normal temperature test, some experimental groups can achieve different degrees of increase in the residual content of vitamin C compared with the vitamin C blank control group. For example, in the experimental group of 0.1% vitamin C, when the Mytilus extract (mussel mucin without dopa group) is added to a concentration of about 0.002%-0.1%, i.e. the mass ratio of vitamin C to Mytilus extract (mussel mucin without dopa group) is 1:0.02-1, the residual content of vitamin C is significantly improved; and in the experimental group of 5.0% vitamin C, when the Mytilus extract (mussel mucin without dopa group) is added to a concentration of about 0.005%-2.0%, i.e. the mass ratio of vitamin C to Mytilus extract (mussel mucin without dopa group) is 1:0.001-0.4, the residual content of vitamin C is significantly improved. The above results confirm that the Mytilus extract (mussel mucin without dopa group) can improve the stability of vitamin C and reduce its digestion rate at normal temperature (15°C).

[0121] 1.5 Vitamin C content in solution, solution pH test results (40°C)

[0122] The results are shown in Tables 9, 10, and Figures 5 to 8

[0123] Table 9. pH and residual content of 0.5% VC at gradient Mytilus extract concentration at 40°C

[0124]

[0125] Note: Compared with the vitamin C blank control group, "*" means p <0.05, "**" means p <0.01, "***" means p <0.001 ​

[0126] Table 10. pH and residual content of 1.0% VC at gradient concentrations of mussel extract at 40°C

[0127]

[0128]

[0129] Note: Compared with the vitamin C blank control group, "*" indicates p < 0.05, "**" indicates p < 0.01, and "***" indicates p < 0.001

[0130] The results show that after 4 weeks of high-temperature acceleration test at 40°C, the experimental groups compared with the vitamin C blank control group can still maintain the residual content of vitamin C to a certain extent or even increase the residual content of vitamin C within the mass ratio range of vitamin C to mussel extract (mussel mucin without dopa group) obtained in the above normal temperature experiment results, for example, 1:0.1-0.2. In particular, in the experimental group of 0.5% vitamin C, when the concentration of mussel extract (mussel mucin without dopa group) is about 0.1%, i.e., the mass ratio of vitamin C to mussel extract is 1:0.2, the residual content of vitamin C in the 4th week can be increased by 10.30%; and in the experimental group of 1.0% vitamin C, when the concentration of mussel extract (mussel mucin without dopa group) is about 0.1%, i.e., the mass ratio of vitamin C to mussel extract (mussel mucin without dopa group) is 1:0.1, the residual content of vitamin C in the 4th week can be increased by 22.70%. The above results confirm that mussel extract (mussel mucin without dopa group) can improve the stability of vitamin C and reduce its digestion rate under high temperature (40°C).

[0131] 2. Composition scavenging OH effect determination

[0132] 2.1 Experimental materials and instruments

[0133] 2.1.1 Experimental solution

[0134] Liquid preparation solution: 2.0 mmol / L FeSO4 solution, 1.0 mmol / L H2O2 solution, 6.0 mmol / L salicylic acid solution

[0135] Background sample: 10% of the solution to be tested (sample) is added to 90% distilled water

[0136] 2.1.2 Experimental instruments

[0137] 722 grating spectrophotometer (Shanghai Third Analytical Instrument Factory)

[0138] 2.2 Sample treatment method and detection of OH scavenging rate

[0139] 15℃ normal temperature condition, 0 month after preparation and every 1 month, 40℃ high temperature acceleration condition, 0 week after preparation and every 1 week, the detection process is as follows:

[0140] Take 2.0mmol / L FeSO4 solution 3.0mL, add 1.0mmol / L H2O2 solution 3.0mL, then add 6.0mmol / L salicylic acid solution 3.0mL, shake immediately after 37℃ water bath heating for 15min, then add 1.0mL test liquid, continue water bath heating for 15min, measure its absorbance A at wavelength 510nm X ;

[0141] Take 1.0mL distilled water instead of test liquid, according to the above steps, measure the absorbance A0.

[0142] The test liquid is diluted to 1:10 concentration as the background sample, water bath heating for 15min, measure the absorbance A X0 .

[0143] According to formula (1), calculate the ·OH clearance rate:

[0144] ·OH clearance rate % = (1-(A X -A X0 ) / A0) x 100% (1)

[0145] 2.3 detection results of vitamin C on ·OH clearance rate (15℃)

[0146] The results are shown in Table 11, Table 12, Table 13, and Figure 9 、 Figure 10

[0147] Table 11. ·OH clearance rate of mussel extract blank control group (15℃)

[0148]

[0149] Table 12. ·OH clearance rate of 0.1% VC at gradient mussel extract concentration (15℃)

[0150]

[0151] Note: Compared with the vitamin C blank control group, "*" indicates p<0.05, "**" indicates p<0.01, "***" indicates p<0.001

[0152] Table 13. ·OH clearance rate of 5.0% VC at gradient mussel extract concentration (15℃)

[0153] ​

[0154]

[0155] Note: Compared with the Vitamin C blank control group, "*" means p<0.05, "**" means p<0.01, and "***" means p<0.001

[0156] The results show that, after 4 months of normal temperature test at 15°C, the corresponding experimental groups have higher OH scavenging rate compared with the Vitamin C blank control group. For example, in the experimental group of 0.1% Vitamin C, when the mussel extract (mussel mucin without dopa group) is added to a concentration of about 0.002%-0.1%, i.e. the mass ratio of Vitamin C to mussel extract (mussel mucin without dopa group) is 1:0.02-1, the OH scavenging rate can be significantly improved; and in the experimental group of 5.0% Vitamin C, when the mussel extract (mussel mucin without dopa group) is added to a concentration of about 0.005%-2.0%, i.e. the mass ratio of Vitamin C to mussel extract (mussel mucin without dopa group) is 1:0.001-0.4, the OH scavenging rate can be significantly improved. The OH scavenging rate results of the control groups 1-0 and 2-0 show that the aqueous solution containing only mussel extract (mussel mucin without dopa group) does not show OH scavenging effect, and this result is related to the fact that the mussel extract added in the experimental groups is mussel mucin without dopa group. The results of this control group are representative under the gradient concentration of mussel extract used in each experimental group. The above results confirm that Vitamin C and mussel extract (mussel mucin without dopa group) synergistically enhance the antioxidant effect at normal temperature (15°C).

[0157] 2.4 Detection results of Vitamin C OH scavenging rate (40°C)

[0158] The results are shown in Tables 14, 15, 16, and Figure 11 , Figure 12

[0159] Table 14. OH scavenging rate of mussel extract blank control group (40°C)

[0160] Verification group number 0 weeks 1 week 2 weeks 3 weeks 4 weeks 3-0 0.00％ 0.00％ 0.00％ 0.00％ 0.00％ 4-0 0.02％ 0.02％ 0.02％ 0.02％ 0.02％

[0161] Table 15. OH scavenging rate of 0.5% VC at gradient concentration of mussel extract (40°C)

[0162] Verification group number 0 weeks 1 week 2 weeks 3 weeks 4 weeks 3-1 80.64％ 72.57％ 48.47％ 47.10％ 40.24％ 3-2 80.64％ 69.44％ 45.42％ 44.02％ 36.41％ 3-3 80.61％ 79.32% ＊＊＊ ]]> 59.21% ＊＊＊ ]] 57.97% ＊＊＊ ]] 44.26% ＊＊＊ ]] 3-4 80.64％ 67.44％ 31.37％ 29.26％ 23.23％ 3-5 80.66％ 52.30％ 26.07％ 22.32％ 15.66％

[0163] Note: Compared with the Vitamin C blank control group, "*" means p<0.05, "**" means p<0.01, and "***" means p<0.001

[0164] ​Table 16. ·OH scavenging rate of 1.0% VC at gradient concentrations of mussel extract (40°C)

[0165] Verification group number 0 weeks 1 week 2 weeks 3 weeks 4 weeks 4-1 89.90％ 85.54％ 65.24％ 62.70％ 52.10％ 4-2 89.90％ 84.98％ 64.92％ 61.21％ 39.55％ 4-3 89.93％ 87.77% ＊＊＊ ]] 74.32% ＊＊＊ ]]> 71.46% ＊＊＊ ]]> 61.98% ＊＊＊ ]] 4-4 89.89％ 83.10％ 60.74％ 55.01％ 48.86％ 4-5 89.92％ 73.30％ 51.38％ 46.24％ 9.78％

[0166] Note: Compared with the vitamin C blank control group, "*" means p<0.05, "**" means p<0.01, and "***" means p<0.001

[0167] The results show that after 4 weeks of high-temperature acceleration test at 40°C, the experimental group compared with the vitamin C blank control group, when the mass ratio of vitamin C to mussel extract (mussel mucin without dopa group) is 1:0.1-0.2, can play a role in improving the ·OH scavenging rate of vitamin C. Especially in the experimental group of 0.5% vitamin C, when the mussel extract (mussel mucin without dopa group) is added to a concentration of about 0.1%, that is, the mass ratio of vitamin C to mussel extract (mussel mucin without dopa group) is 1:0.2, the ·OH scavenging rate of vitamin C in the 4th week is improved by 9.99%; while in the experimental group of 1.0% vitamin C, when the mussel extract (mussel mucin without dopa group) is added to a concentration of about 0.1%, that is, the mass ratio of vitamin C to mussel extract (mussel mucin without dopa group) is 1:0.1, the ·OH scavenging rate of vitamin C in the 4th week can be improved by 18.96%. The results of the ·OH scavenging rate of the control groups 3-0 and 4-0 show that the aqueous solution containing only mussel extract (mussel mucin without dopa group) does not show ·OH scavenging effect, and this result is related to the mussel extract added in the experimental group, which is mussel mucin without dopa group. The results of this control group are representative at the gradient concentrations of mussel extract used in each experimental group. The above results confirm that vitamin C and mussel extract (mussel mucin without dopa group) can synergistically enhance the antioxidant effect at high temperature (40°C).

[0168] 2.5 Detection results of ·OH scavenging rate of ascorbic acid glucoside (15°C)

[0169] The results are shown in Tables 17, 18, and Figure 13 , Figure 14

[0170] Table 17. ·OH scavenging rate of 0.1% AA2G at gradient concentrations of mussel extract (15°C)

[0171]

[0172]

[0173] ​Note: "*" means p<0.05, "**" means p<0.01, "***" means p<0.001 compared with ascorbic acid glucoside blank control group

[0174] Table 18. OH radical scavenging rate of 5.0% AA2G at gradient Mytilus extract concentration (15°C)

[0175] Verification group number 0 months 1 month 2 months 3 months 4 months 6-1 89.34％ 82.03％ 76.18％ 64.65％ 51.57％ 6-2 89.68％ 79.43％ 71.91％ 63.40％ 50.63％ 6-3 90.01％ 77.57％ 69.65％ 65.24％ 52.63% ＊＊＊ ]]> 6-4 89.79％ 79.11％ 73.04％ 69.59% ＊＊＊ ]]> 56.14% ＊＊＊ ]]> 6-5 89.67％ 84.83% ＊＊＊ ]]> 78.47% ＊＊＊ ]]> 65.76% ＊＊＊ ]]> 48.36％ 6-6 89.71％ 84.08% ＊＊＊ ]]> 77.68% ＊＊＊ ]] 65.71％ 49.57％ 6-7 89.64％ 80.21％ 63.74％ 44.02％ 37.88％ 6-8 88.99％ 75.55％ 57.77％ 43.87％ 26.99％ 6-9 89.57％ 72.21％ 53.15％ 36.77％ 20.30％ 6-10 89.15％ 68.47％ 47.32％ 32.13％ 18.67％

[0176] Note: "*" means p<0.05, "**" means p<0.01, "***" means p<0.001 compared with ascorbic acid glucoside blank control group

[0177] The results show that after 4 months of 15°C normal temperature test, some experimental groups have higher OH radical scavenging rate compared with ascorbic acid glucoside blank control group. For example, in the experimental group of 0.1% ascorbic acid glucoside, when Mytilus extract (Mytilus mucus without dopa group) is added to a concentration of about 0.002% to 0.1%, i.e. the mass ratio of ascorbic acid glucoside to Mytilus extract (Mytilus mucus without dopa group) is 1:0.02 to 1, the OH radical scavenging rate can be significantly improved; and in the experimental group of 5.0% ascorbic acid glucoside, when Mytilus extract (Mytilus mucus without dopa group) is added to a concentration of about 0.005% to 0.01% (4th month), i.e. the mass ratio of ascorbic acid glucoside to Mytilus extract (Mytilus mucus without dopa group) is 1:0.001 to 0.02, the OH radical scavenging rate can be significantly improved. The above results confirm that ascorbic acid glucoside and Mytilus extract (Mytilus mucus without dopa group) synergistically enhance the antioxidant effect at normal temperature (15°C).

[0178] 2.7 Detection results of ascorbic acid glucoside OH radical scavenging rate (40°C)

[0179] The results are shown in Tables 19, 20, and Figure 15 , Figure 16

[0180] Table 19. OH radical scavenging rate of 0.5% AA2G at gradient Mytilus extract concentration (40°C)

[0181] Verification group number 0 months 1 month 2 months 3 months 4 months 7-1 79.25％ 67.75％ 53.33％ 42.53％ 35.33％ 7-2 80.25％ 64.40％ 46.13％ 35.69％ 23.26％ 7-3 80.17％ 76.72% ＊＊＊ ]]> 58.07% ＊＊ ]] 52.45% ＊＊＊ ]]> 46.54% ＊＊＊ ]]> 7-4 80.09％ 62.85％ 36.93％ 26.79％ 19.42％ 7-5 80.19％ 56.56％ 27.86％ 21.37％ 12.17％

[0182] Note: "*" means p<0.05, "**" means p<0.01, "***" means p<0.001 compared with ascorbic acid glucoside blank control group

[0183] Table 20. OH radical scavenging rate of 1.0% AA2G at gradient Mytilus extract concentration (40°C)

[0184] ​ Verification group number 0 months 1 month 2 months 3 months 4 months 8-1 89.25％ 72.51％ 59.44％ 49.72％ 36.34％ 8-2 89.87％ 70.25％ 58.65％ 40.51％ 31.80％ 8-3 91.05％ 81.25% ＊＊＊ ]]> 70.31% ＊＊＊ ]]> 59.66% ＊＊＊ ]]> 47.26% ＊＊＊ ]]> 8-4 90.27％ 72.25％ 54.70％ 42.07％ 29.37％ 8-5 89.88％ 64.18％ 51.39％ 36.59％ 27.47％

[0185] Note: Compared with ascorbic acid glucoside blank control group, "*" means p<0.05, "**" means p<0.01, "***" means p<0.001

[0186] The results show that after 4 weeks of high temperature acceleration test at 40°C, the experimental group compared with the ascorbic acid glucoside blank control group, when the mass ratio of ascorbic acid glucoside to mussel extract (mussel mucin without dopa group) is 1:0.1-0.2, can play a role in improving the clearance rate of ascorbic acid glucoside to ·OH. Especially in the 0.5% ascorbic acid glucoside experimental group, the mussel extract (mussel mucin without dopa group) is added to a concentration of about 0.1%, that is, the mass ratio of ascorbic acid glucoside to mussel extract (mussel mucin without dopa group) is 1:0.2, the clearance rate of ascorbic acid glucoside to ·OH in the 4th week is improved by 31.72%; while in the 1.0% ascorbic acid glucoside experimental group, the mussel extract (mussel mucin without dopa group) is added to a concentration of about 0.1%, that is, the mass ratio of ascorbic acid glucoside to mussel extract (mussel mucin without dopa group) is 1:0.1, the clearance rate of ascorbic acid glucoside to ·OH in the 4th week can be improved by 30.05%. The above results confirm that ascorbic acid glucoside and mussel extract (mussel mucin without dopa group) can synergistically enhance the antioxidant effect under high temperature (40°C).

[0187] 2.8 Detection results of the clearance rate of MAP to ·OH (15°C)

[0188] The results are shown in Tables 21, 22, and Figure 17 , Figure 18

[0189] Table 21. ·OH clearance rate of 0.1% MAP under gradient mussel extract concentration (15°C)

[0190]

[0191] Note: Compared with ascorbic acid glucoside blank control group, "*" means p<0.05, "**" means p<0.01, "***" means p<0.001

[0192] Table 22. ·OH clearance rate of 5.0% MAP under gradient mussel extract concentration (15°C)

[0193] Verification group number 0 months 1 month 2 months 3 months 4 months 10-1 85.75％ 72.89％ 67.15％ 51.82％ 39.58％ 10-2 85.46％ 74.25% ＊ ]]> 67.18％ 52.86% ＊＊ ]]> 41.57% ＊＊＊ ]]> 10-3 85.41％ 76.85% ＊＊ ]] 68.17% ＊＊ ]]> 54.82% ＊＊＊ ]] 44.47% ＊＊＊ ]]> 10-4 85.45％ 75.89% ＊＊ ]]> 64.84％ 58.28% ＊＊＊ ]]> 44.12% ＊＊＊ ]]> 10-5 85.77％ 80.28% ＊＊＊ ]]> 71.66% ＊＊＊ ]] 62.58% ＊＊＊ ]]> 41.58% ＊＊＊ ]] 10-6 85.26％ 80.84% ＊＊＊ ]]> 72.80% ＊＊＊ ]] 61.82% ＊＊＊ ]] 37.85％ 10-7 85.68％ 76.11% ＊＊ ]]> 59.44％ 50.67％ 32.68％ 10-8 85.57％ 74.69% ＊＊ ]] 53.75％ 40.25％ 21.07％ 10-9 85.67％ 70.42％ 48.47％ 32.74％ 16.85％ 10-10 85.67％ 64.68％ 41.66％ 30.25％ 15.35％

[0194] ​Note: Compared with the ascorbic acid magnesium phosphate blank control group, "*" means p < 0.05, "**" means p < 0.01, and "***" means p < 0.001

[0195] The results show that after 4 months of 15°C normal temperature test, some experimental groups have higher ·OH removal rate compared with the ascorbic acid magnesium phosphate blank control group. For example, in the experimental group of 0.1% ascorbic acid magnesium phosphate, when the mussel extract (mussel mucin without dopa group) is added to a concentration of about 0.002% to 0.1%, i.e. the mass ratio of ascorbic acid magnesium phosphate to mussel extract (mussel mucin without dopa group) is 1:0.02 to 1, the ·OH removal rate can be significantly improved. In the experimental group of 5.0% ascorbic acid magnesium phosphate, when the mussel extract (mussel mucin without dopa group) is added to a concentration of about 0.002% to 0.05% (4th month), i.e. the mass ratio of ascorbic acid magnesium phosphate to mussel extract (mussel mucin without dopa group) is 1:0.0004 to 0.01, the ·OH removal rate can be significantly improved. The above results confirm that ascorbic acid magnesium phosphate and mussel extract (mussel mucin without dopa group) synergistically enhance the antioxidant effect at normal temperature (15°C).

[0196] 2.9 Detection results of ascorbic acid magnesium phosphate ·OH removal rate (40°C)

[0197] The results are shown in Tables 23, 24, and Figure 19 , Figure 20

[0198] Table 23. 0.5% MAP ·OH removal rate at gradient mussel extract concentration (40°C)

[0199] Verification group number 0 months 1 month 2 months 3 months 4 months 11-1 76.58％ 61.25％ 49.38％ 35.82％ 30.10％ 11-2 76.98％ 60.28％ 49.40％ 32.58％ 23.81％ 11-3 76.91％ 69.58% ＊＊＊ ]] 56.58% ＊＊＊ ]] 49.28% ＊＊＊ ]] 39.54% ＊＊＊ ]] 11-4 76.04％ 59.85％ 37.19％ 23.41％ 16.38％ 11-5 75.98％ 54.71％ 28.74％ 19.28％ 11.28％

[0200] Note: Compared with the ascorbic acid magnesium phosphate blank control group, "*" means p < 0.05, "**" means p < 0.01, and "***" means p < 0.001

[0201] Table 24. 1.0% MAP ·OH removal rate at gradient mussel extract concentration (40°C)

[0202] Verification group number 0 months 1 month 2 months 3 months 4 months 12-1 85.35％ 65.83％ 59.44％ 48.25％ 29.58％ 12-2 85.27％ 65.98％ 55.69％ 40.51％ 28.98％ 12-3 84.95％ 75.82% ＊＊＊ ]] 65.86% ＊＊＊ ]] 53.28% ＊＊＊ ]]> 35.82% ＊＊＊ ]] 12-4 85.59％ 73.83％ 54.25％ 41.26％ 26.84％ 12-5 85.46％ 61.25％ 50.25％ 32.58％ 21.47％

[0203] Note: Compared with the ascorbic acid magnesium phosphate blank control group, "*" means p < 0.05, "**" means p < 0.01, and "***" means p < 0.001

[0204] ​The results show that after 4 weeks of high temperature acceleration test at 40℃, compared with the ascorbic acid magnesium phosphate blank control group, the experimental group can improve the clearance rate of ascorbic acid magnesium phosphate to ·OH when the mass ratio of ascorbic acid magnesium phosphate to mussel extract (mussel mucin without dopa group) is 1:0.1-0.2. Especially in the 0.5% ascorbic acid magnesium phosphate experimental group, when the concentration of mussel extract (mussel mucin without dopa group) is about 0.1%, that is, the mass ratio of ascorbic acid magnesium phosphate to mussel extract (mussel mucin without dopa group) is 1:0.2, the clearance rate of ascorbic acid magnesium phosphate to ·OH in the fourth week is improved by 31.36%; while in the 1.0% ascorbic acid magnesium phosphate experimental group, when the concentration of mussel extract (mussel mucin without dopa group) is about 0.1%, that is, the mass ratio of ascorbic acid magnesium phosphate to mussel extract (mussel mucin without dopa group) is 1:0.1, the clearance rate of ascorbic acid magnesium phosphate to ·OH in the fourth week can be improved by 21.20%. The above results confirm that ascorbic acid magnesium phosphate and mussel extract (mussel mucin without dopa group) can synergistically improve the antioxidant effect under high temperature (40℃).

[0205] Example 2

[0206] The mussel extract used in the experiment is mussel mucin Mfp-5 pure product provided by Xi'an Deno Haisi Medical Technology Co., Ltd. Since it is expressed by Pichia pastoris, it belongs to mussel mucin without dopa group.

[0207] The experimental process refers to Example 1, and the results (similar to Example 1) also show that the mussel extract can improve the stability of vitamin C and its corresponding derivatives and reduce their digestion rate under normal temperature (15℃) and high temperature (40℃); and can synergistically improve the antioxidant effect with vitamin C and its corresponding derivatives under normal temperature (15℃) and high temperature (40℃).

[0208] Example 3

[0209] The mussel extract is mussel mucin Mfp-1 pure product provided by Xi'an Deno Haisi Medical Technology Co., Ltd. Since it is expressed by Pichia pastoris, it belongs to mussel mucin without dopa group.

[0210] The experimental process refers to Example 1, and the results (similar to Example 1) also show that the mussel extract can improve the stability of vitamin C and its corresponding derivatives and reduce their digestion rate under normal temperature (15℃) and high temperature (40℃); and can synergistically improve the antioxidant effect with vitamin C and its corresponding derivatives under normal temperature (15℃) and high temperature (40℃).

[0211] In summary, the present application tests vitamin C solutions containing different concentration gradients of mussel extract (mussel myoglobin without dopa group), and finds that when the mass ratio of vitamin C or vitamin C derivative and mussel extract (mussel myoglobin without dopa group) is 1:0.001-1, the mussel extract (mussel myoglobin without dopa group) can improve the stability of vitamin C or vitamin C derivative in the solution. Thus, the present application proposes a new way to effectively enhance the activity of antioxidants (vitamin C, corresponding vitamin C derivative) in cosmetics in addition to liposome wrapping and derivative modification.

Claims

1. A method of enhancing the antioxidant activity of vitamin C and its derivatives, characterized by: The method comprises the following steps: adding mussel adhesive protein without dopa group into a dispersion system containing vitamin C or vitamin C derivative; the mussel adhesive protein without dopa group is formed by fermentation of genetically engineered bacteria; the mass ratio of vitamin C or vitamin C derivative to mussel adhesive protein without dopa group is 1:0.001-1; the concentration of vitamin C in the dispersion system containing vitamin C is 0.1%-5.0%, or the concentration of vitamin C derivative in the dispersion system containing vitamin C derivative is 0.1%-5.0%; the mussel adhesive protein expressed by the genetically engineered bacteria at least contains any one of Mfp-5 and Mfp-1; the vitamin C derivative is selected from one or more of vitamin C phosphate and vitamin C glucoside.

2. The method of enhancing the antioxidant activity of vitamin C and its derivatives as claimed in claim 1, wherein: the mass ratio of vitamin C or vitamin C derivative to mussel adhesive protein without dopa group is 1:0.01-1.

3. The method of enhancing the antioxidant activity of vitamin C and its derivatives as claimed in claim 1, wherein: the mass ratio of vitamin C or vitamin C derivative to mussel adhesive protein without dopa group is 1:0.02-0.

4.

4. The method of enhancing the antioxidant activity of vitamin C and its derivatives as claimed in claim 1, wherein: the mass ratio of vitamin C or vitamin C derivative to mussel adhesive protein without dopa group is 1:0.02-0.

2.

5. The method of claim 1, wherein the antioxidant activity of vitamin C is enhanced. the mass ratio of vitamin C or vitamin C derivative to mussel adhesive protein without dopa group is 1:0.02-0.

1.

6. A composition for enhancing antioxidant activity, characterized by: the mass ratio of vitamin C or vitamin C derivative to mussel adhesive protein without dopa group is 1:0.001-1; the mussel adhesive protein without dopa group is formed by fermentation of genetically engineered bacteria; the concentration of vitamin C in the dispersion system containing vitamin C is 0.1%-5.0%, or the concentration of vitamin C derivative in the dispersion system containing vitamin C derivative is 0.1%-5.0%; the mussel adhesive protein expressed by the genetically engineered bacteria at least contains any one of Mfp-5 and Mfp-1; the vitamin C derivative is selected from any one of vitamin C phosphate and vitamin C glucoside.

7. A cosmetic product characterized in that: The cosmetic contains the composition with improved antioxidant activity as claimed in claim 6. The cosmetic contains the composition with improved antioxidant activity as claimed in claim 6.

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