Preparation method of supramolecular acid enzyme composition and application of supramolecular acid enzyme composition in cosmetics

Abstract

The invention relates to the technical field of cosmetics, in particular to a preparation method of a supramolecular acid enzyme composition and application of the supramolecular acid enzyme composition in cosmetics. The preparation method comprises the following steps: preparing a deep eutectic solvent which is in a liquid state at normal temperature, and reacting protease with the deep eutectic solvent to prepare the liquid supramolecular acid enzyme composition. The supramolecular acid enzyme composition is used for preparing cosmetics for removing waste cutin, blocking pores and promoting skin metabolism. According to the stable and reliable enzyme immobilization method, the supramolecular deep eutectic solvent is prepared from the mandelic acid and the betaine, the papain and the bromelain are dissolved and immobilized in the supramolecular deep eutectic solvent, and the method has the advantages of being easy to operate, good in solubility, good in stability and the like; in addition, the finally obtained supramolecular acid enzyme composition also can realize the combination of acid and enzyme, so that the effects of synergistically removing waste cutin and blocked pores of the skin, quickly improving the texture and appearance of the skin and promoting the metabolism of the skin are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a preparation method of a supramolecular acid enzyme composition and its application in cosmetics. Background Art

[0002] The stratum corneum of human skin is composed of chemical components such as keratin, intercellular lipids, and natural moisturizing factors. Among them, keratin accounts for more than 60% of the total weight of the stratum corneum. Keratin is a water-insoluble protein, mainly divided into α-keratin and β-keratin. There are a large number of α-helices and β-sheets in the molecule, and there are a large number of disulfide bonds, hydrogen bonds, etc. between the structures, forming a stable three-dimensional spatial structure. The presence of a large number of disulfide bonds makes the stereochemical structure of keratin very firm, with strong resistance to chemical reagents and hydrolases. Therefore, once the disulfide bonds are broken, the protein no longer has the properties of anti-decomposition and water-insolubility.

[0003] Both acids and enzymes can remove the old cutin and clogged pores of the skin, quickly improve the texture and appearance of the skin, and reduce the occurrence of acne, but their mechanisms of action are quite different. Acid exfoliation works at low pH (pH < 4.5), causing keratin, intercellular lipids, and natural moisturizing factors to rapidly denature and hydrolyze under acidic conditions, and dead skin cells quickly fall off. Some small molecule fruit acids or lipophilic acids can also penetrate the stratum corneum and continue to play a physiological role in the deep skin. Proteases, due to their catalytic specificity and large molecular weight, only play a mild hydrolytic role on the keratin on the skin surface. Currently, the keratinases applied in the daily chemical industry are mainly papain and bromelain.

[0004] Bromelain is a sulfhydryl protease extracted from pineapple juice, skin, etc., which can act on the aging stratum corneum on the human skin, promote its degradation, decomposition, and removal, and promote skin metabolism, making the skin maintenance present a good fair and tender state. Papain is a sulfhydryl protease extracted from papaya milk. Because of its strong protein hydrolysis ability and good hydrolysis effect on various proteins, it has the effects of tenderizing and whitening the skin, promoting blood circulation, and improving skin quality.

[0005] Papain and bromelain, as one of the active cosmetic ingredients, have many excellent properties. However, the fragility of enzymes has always been a difficult problem in research and application. Free enzymes are very unstable and are easily inactivated under conditions of heat, acid, alkali, and organic solvents, losing their catalytic function. Moreover, even when stored in the optimal reaction environment of the enzyme for a period of time, the enzyme will also be inactivated. Due to the harsh storage conditions of natural product enzymes, most enzymes will lose their activity within one month or even a shorter time at room temperature. This has limited the development of enzymes in the daily chemical industry to a certain extent. Among the currently marketed products, there are few products that combine acid and enzymes. Therefore, it is of great significance to develop a reliable method for immobilizing enzymes to increase the stability of enzymes in cosmetics to achieve the synergistic effect of acid and enzyme. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, one of the purposes of the present invention is to provide a preparation method of a supramolecular acid-enzyme composition, and the second purpose of the present invention is to provide an application of the supramolecular acid-enzyme composition in cosmetics.

[0007] One of the purposes of the present invention is achieved through the following technical solutions:

[0008] A preparation method of a supramolecular acid-enzyme composition, comprising the following steps:

[0009] Step 1, prepare a deep eutectic solvent that is liquid at room temperature;

[0010] Step 2, react the protease with the deep eutectic solvent to prepare a liquid supramolecular acid-enzyme composition.

[0011] Further, the preparation method of Step 1 is: put mandelic acid and betaine into a reactor in a molar ratio of (1-2):(1-2), heat at 30-50 °C for 4 hours to obtain a mandelic acid-betaine supramolecular NaDES solvent (light yellow); the mandelic acid-betaine supramolecular NaDES solvent is a natural deep eutectic solvent formed with mandelic acid as the hydrogen bond donor and betaine as the hydrogen bond acceptor, and is liquid at room temperature.

[0012] Further, the protease in Step 2 is a papain and bromelain complex (obtained by stirring papain and bromelain evenly), and the mass ratio of papain to bromelain is (1-2):(1-2). Papain is a sulfhydryl protease extracted from papaya latex. Because of its strong protein hydrolysis ability and good hydrolysis effect on a variety of proteins, it has the effects of tenderizing and whitening the skin, promoting blood circulation, and improving skin texture. Bromelain is a sulfhydryl protease extracted from pineapple juice, peel, etc. It can act on the aged cutin layer on the human skin, promote its degradation, decomposition, and removal, and promote skin metabolism, making the skin maintenance present a good fair and tender state. And under the above ratio, a better synergistic effect can be produced between the two enzymes.

[0013] Further, the preparation method of step two is as follows: Put the papain and bromelain complex and the mandelic acid betaine supramolecular NaDES solvent into the reactor at a mass ratio of (0.1-1):(99.0-99.9), heat at 30-50 °C for 2 hours to obtain a light yellow transparent homogeneous liquid, which is the supramolecular acid-enzyme composition.

[0014] The second object of the present invention is achieved through the following technical solutions:

[0015] A supramolecular acid-enzyme composition for preparing cosmetics for removing waste cutin, clogging pores and promoting skin metabolism.

[0016] The beneficial effects of the present invention are as follows: The present invention provides a stable and reliable enzyme immobilization method. By using mandelic acid and betaine to prepare a supramolecular deep eutectic solvent, and dissolving and immobilizing papain and bromelain in the supramolecular deep eutectic solvent, it has the characteristics of simple operation, good solubility and good stability; in addition, the finally obtained supramolecular acid-enzyme composition can also achieve the combination of acid and enzyme, achieve the synergistic effect of removing waste cutin and clogged pores on the skin, quickly improve the texture and appearance of the skin, and promote skin metabolism. Description of the Drawings

[0017] Figure 1 1H NMR spectra of mandelic acid monomer, betaine monomer and mandelic acid betaine supramolecular NaDES solvent;

[0018] Figure 2 1 is the enzyme activity decay rate diagram;

[0019] Figure 3 2 is the detection result diagram of BCA of each group of keratinocytes; *P<0.05; **P<0.01; ***P<0.001 in the figure; Note: * indicates that there is a significant difference between the tested sample and the blank group under the action of this concentration. Detailed Embodiments

[0020] The following is a further description in conjunction with the specific embodiments:

[0021] Example 1

[0022] A preparation method of a supramolecular acid-enzyme composition, comprising the following steps:

[0023] Step A: Preparation of mandelic acid betaine supramolecular NaDES solvent: Put mandelic acid and betaine into the reactor at a molar ratio of 2:1, heat at 40 °C for 4 hours to obtain a light yellow mandelic acid betaine supramolecular NaDES solvent.

[0024] The described mandelic acid betaine supramolecular NaDES solvent is a natural deep eutectic solvent formed with mandelic acid as the hydrogen bond donor and betaine as the hydrogen bond acceptor. It is liquid at room temperature. The 1H NMR spectrum is shown in the appendix of the specification Figure 1 .

[0025] Step B: Preparation of the supramolecular acid-enzyme composition: Put the papain and bromelain complex (the mass ratio of papain to bromelain is 1:1) and the mandelic acid betaine supramolecular NaDES solvent obtained in Step A into the reactor at a mass ratio of 0.5:99.5, and heat at 40 °C for 2 hours to obtain a light yellow, transparent and homogeneous liquid, which is the supramolecular acid-enzyme composition.

[0026] Example 2

[0027] A preparation method of a supramolecular acid-enzyme composition includes the following steps:

[0028] Step A: Preparation of the mandelic acid betaine supramolecular NaDES solvent: Put mandelic acid and betaine into the reactor at a molar ratio of 1:2, and heat at 30 °C for 4 hours to obtain a light yellow mandelic acid betaine supramolecular NaDES solvent.

[0029] The described mandelic acid betaine supramolecular NaDES solvent is a natural deep eutectic solvent formed with mandelic acid as the hydrogen bond donor and betaine as the hydrogen bond acceptor. It is liquid at room temperature.

[0030] Step B: Preparation of the supramolecular acid-enzyme composition: Put the papain and bromelain complex and the mandelic acid betaine supramolecular NaDES solvent obtained in Step A into the reactor at a mass ratio of 0.1:99.9, and heat at 30 °C for 2 hours to obtain a light yellow, transparent and homogeneous liquid, which is the supramolecular acid-enzyme composition.

[0031] Example 3

[0032] A preparation method of a supramolecular acid-enzyme composition includes the following steps:

[0033] Step A: Preparation of the mandelic acid betaine supramolecular NaDES solvent: Put mandelic acid and betaine into the reactor at a molar ratio of 1:1, and heat at 50 °C for 4 hours to obtain a light yellow mandelic acid betaine supramolecular NaDES solvent.

[0034] The described mandelic acid betaine supramolecular NaDES solvent is a natural deep eutectic solvent formed with mandelic acid as the hydrogen bond donor and betaine as the hydrogen bond acceptor. It is liquid at room temperature.

[0035] Step B: Preparation of supramolecular acid-enzyme composition: Papain and bromelain complex and the mandelic acid betaine supramolecular NaDES solvent obtained in Step A were put into a reactor at a mass ratio of 1:99.0, heated at 50 °C for 2 hours to obtain a light yellow transparent and homogeneous liquid, which is the supramolecular acid-enzyme composition.

[0036] Physicochemical property experiments

[0037] 1. Solubility test:

[0038] The supramolecular acid-enzyme composition obtained in Example 1 and the untreated complex enzyme (i.e., papain and bromelain complex) were dissolved in water at the concentrations (mass concentrations) recorded in Table 1, and their states after dissolution were observed and compared.

[0039] Table 1 Solubility record table of supramolecular acid-enzyme composition or untreated complex enzyme at each concentration

[0040] Concentration (in terms of enzyme) Supramolecular acid enzyme composition Untreated complex enzyme 0.2％ Transparent and clear liquid Turbid and milky liquid 0.1％ Transparent and clear liquid Turbid and milky liquid 0.05％ Transparent and clear liquid Slightly milky liquid 0.025％ Transparent and clear liquid Very slightly milky liquid 0.0125％ Transparent and clear liquid Transparent and clear liquid

[0041] As can be seen from Table 1, when the concentration of the untreated complex enzyme in water was 0.0125%, the solution was clear and transparent; when the concentration was higher than 0.0125%, the untreated complex enzyme showed a slight whitening phenomenon, and when the concentration was 0.1%, the untreated complex enzyme showed a turbid whitening phenomenon, while the supramolecular acid-enzyme composition treated by the supramolecular technology still showed a clear and transparent state, proving that the solubility of the enzyme can be increased after being treated by the supramolecular technology.

[0042] 2. Protective enzyme activity test:

[0043] Test principle: Under certain temperature and pH, hydrolyzing the substrate casein can produce amino acids containing phenolic groups (tyrosine, tryptophan, etc.). Therefore, under alkaline conditions, the Folin-Ciocalteu reagent is reduced to molybdenum blue and tungsten blue, and the absorbance of the solution is measured with a spectrophotometer at a wavelength of 680 nm. The enzyme activity is proportional to the absorbance, so the enzyme activity of the product can be calculated.

[0044] Drawing of the standard curve: Weigh the L-tyrosine standard product and prepare solutions with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL.

[0045] Specific operation process: Add 1.00 mL of enzyme solution to the sample and the blank tube, place it at 40 ± 0.2 °C, incubate for 2 min. Add 1.00 mL of casein solution to the sample tube (shake well), add 2.00 mL of trichloroacetic acid solution to the blank tube (shake well), place it at 40 ± 0.2 °C, after incubating for 10 min, add 2.00 mL of trichloroacetic acid to the sample tube, add casein solution to the blank tube, let it stand for 10 min, filter, take 1.00 mL of the filtrate, add 5.0 mL of sodium carbonate solution, 1.0 mL of Folin reagent solution, at 40 ± 0.2 °C, after color development for 20 min, measure the absorbance value with a UV spectrophotometer at a wavelength of 680 nm and calculate the protease activity. Three parallel tubes need to be set up for each tube.

[0046] Take a certain mass of the supramolecular acid enzyme composition obtained by the preparation method of Example 1 and dissolve it in water to obtain an aqueous solution of the supramolecular acid enzyme composition (0.5 wt% in this example) as the experimental group, dissolve mandelic acid, betaine, and the complex enzyme with the same ratio and content in water as the control group. Adjust the pH value of the two groups of solutions to 4.5 with a buffer solution, store them at 40 °C for a period of time, then conduct enzyme activity tests on each sample, set the detection data on the 0th day as the initial value, and calculate the decay rate. The specific test results are as Figure 2 shown.

[0047] It can be Figure 2 seen that under the conditions of pH value 4.5 and 40 °C, compared with the direct mixing method, the supramolecular acid enzyme composition prepared by the supramolecular method can more effectively maintain the protease activity and greatly improve the applicability of the protease.

[0048] 3. Exfoliating efficacy test:

[0049] Test principle: When the skin metabolism becomes irregular due to aging or other reasons, the stratum corneum cells cannot carry out normal metabolism. Excessive stratum corneum cells "semi-adhere" to the surface layer, which makes it difficult for the skin to absorb daily skin care products. Secondly, it will also make the skin look less smooth and affect the beauty. At this time, exfoliation is needed to accelerate the speed of skin cell renewal and make the excess stratum corneum on the skin surface fall off. This not only restores the smooth and energetic state of the skin but also has the effects of removing wrinkles and anti-aging. The stratum corneum contains a large amount of barrier proteins such as keratin. The number of exfoliated stratum corneum can be obtained through protein content analysis. The more the stratum corneum exfoliates, the higher the protein content. Therefore, we can reflect the exfoliating efficacy of the sample by measuring the total protein content of the exfoliated stratum corneum cells in vitro.

[0050] Specific operation process: 1) After thawing the pigskin, measure the trans-epidermal water loss rate (TEWL) value to verify the integrity of the skin barrier (the TEWL value of the skin with an intact stratum corneum < 15 g / m 2·h), and then fixed between the supply chamber and the receiving chamber of the Franz cell diffusion cell, with the stratum corneum of the skin facing the supply chamber and the dermal layer side facing the receiving chamber.

[0051] 2) Drug administration: Add 50 μL of the sample to the surface of the porcine skin, and spread the sample evenly from the central part of the skin radially towards the edge. Use PBS buffer as the blank control, set 3 parallel replicates for each group, maintain a constant water bath at 32 °C, and ensure that there are no bubbles in the water bath sandwich.

[0052] 3) Sample collection: After incubation for 24 h, wear finger cots and rub the area where the sample was used. After rubbing for two minutes, add 0.5 mL of cleaning solution (0.1% Triton X-100) to the supply chamber, pipette to wash the keratinocytes shed from the skin surface, and put the collected cleaning solution into a high-speed centrifuge to centrifuge and collect the keratinocytes.

[0053] 4) Lysis: After resuspending the above-mentioned keratinocytes with deionized water, add 25 μL of 12 M NaOH solution to the liquid, and perform a boiling water bath for 30 min to lyse the keratinocytes. Finally, add 25 μL of 12 M HCl solution to neutralize the above-mentioned lysate.

[0054] 5) Determination of total protein by BCA method: Perform the determination of total protein according to the operation manual of the BCA method detection kit.

[0055] Test results:

[0056] Table 2 Detection results of the concentration of exfoliated proteins

[0057]

[0058]

[0059] Group and sample description: Group 1 is PBS buffer, Group 2 is aqueous salicylic acid solution, Group 3 is the aqueous solution of the supramolecular acid enzyme composition obtained in Example 1, Group 4 is the aqueous solution of a mixture of mandelic acid, betaine, papain and bromelain with the same proportion and content as the supramolecular acid enzyme composition obtained in Example 1, Group 5 is aqueous mandelic acid solution, Group 6 is the aqueous solution of the papain and bromelain complex obtained in Example 1, and Group 7 is aqueous lactic acid solution.

[0060] From Table 2, Figure 3It can be seen that, compared with the blank group, after treating the ex vivo skin of one-month-old Bama mini-pigs with 2% and 4% salicylic acid, supramolecular acid enzyme composition, physical mixed acid enzyme, and lactic acid, the protein concentration of exfoliated cells in the stratum corneum was significantly increased (P < 0.05), and it showed a concentration-dependent relationship; after treating the ex vivo skin of one-month-old Bama mini-pigs with 4% mandelic acid, the protein concentration of exfoliated cells in the stratum corneum increased to some extent (P < 0.05); after treating the ex vivo skin of one-month-old Bama mini-pigs with 2% and 4% papain & bromelain, the protein concentration of exfoliated cells in the stratum corneum did not change significantly (P > 0.05). Salicylic acid, supramolecular acid enzyme combination, physical mixed acid enzyme combination, and lactic acid can all achieve the exfoliating effect by promoting the exfoliation of keratinocytes. High-concentration mandelic acid has a certain exfoliating effect, while papain & bromelain does not have an obvious exfoliating effect. The exfoliating ability: supramolecular acid enzyme combination > salicylic acid ≈ physical mixed acid enzyme > lactic acid > mandelic acid > papain & bromelain. It should be noted that: the average value of the protein concentration exfoliated by papain & bromelain in Table 2 is lower than that of the blank control group. The main reason is that the sample collection process is manual rubbing, so there is a small amount of error. However, this does not affect the evaluation of the exfoliating effect of papain & bromelain (the complex of papain and bromelain).

[0061] Experimental conclusion:

[0062] It is common knowledge that papain or bromelain alone has the function of exfoliating, but the present invention proves that the combined use of the two does not have an obvious exfoliating effect. When mandelic acid, betaine, papain, and bromelain are simply mixed and used, they have a significant exfoliating effect, and their exfoliating ability is stronger than that of lactic acid and mandelic acid; as is well known, lactic acid and mandelic acid are both commonly used exfoliating substances in this field. Therefore, it can be proved that mandelic acid and betaine have a certain synergistic effect on the complex of papain and bromelain.

[0063] After obtaining the supramolecular acid enzyme composition from mandelic acid, betaine, papain, and bromelain through the preparation method of the present invention, compared with the simple mixing method, its exfoliating effect is further improved, and it is even better than salicylic acid with a relatively strong recognized exfoliating ability. Thus, it can be inferred that the preparation method of the present invention promotes the synergistic effect of mandelic acid, betaine, papain, and bromelain.

[0064] What is described in the above embodiments and the specification only illustrates the principle and the best embodiments of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A method for preparing a supramolecular acid enzyme composition, characterized in that: The following steps are involved: Step 1, preparing a deep eutectic solvent that is liquid at room temperature; Step 2: reacting the protease with a deep eutectic solvent to prepare a liquid supramolecular acid enzyme composition.

2. The method for preparing the supramolecular acid enzyme composition according to claim 1, characterized in that: The preparation method of step 1 is as follows: mandelic acid and betaine are added into a reactor at a molar ratio of (1-2): (1-2), and heated at 30-50° C. for 4 hours to obtain a mandelic acid betaine supramolecular NaDES solvent; the mandelic acid betaine supramolecular NaDES solvent is a natural deep eutectic solvent formed with mandelic acid as a hydrogen bond donor and betaine as a hydrogen bond acceptor, and is liquid at room temperature.

3. The method for preparing the supramolecular acid enzyme composition according to claim 1, characterized in that: The protease in step 2 is a papain and bromelain complex, and the mass ratio of papain to bromelain is (1-2): (1-2).

4. The method for preparing the supramolecular acid enzyme composition according to claim 1, characterized in that: The preparation method of step 2 is as follows: putting the papain and bromelain complex and the mandelic acid betaine supramolecular NaDES solvent into a reactor at a mass ratio of (0.1-1): (99.0-99.9), heating at 30-50° C. for 2 hours to obtain a light yellow transparent uniform liquid, which is the supramolecular acid enzyme composition.

5. An application of the supramolecular acid enzyme composition according to any one of claims 1 to 4, characterized in that: Used to prepare cosmetics that remove dead skin cells, clogged pores and promote skin metabolism.

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