Preparation method of supramolecular controlled oleic enzyme composition and application of supramolecular controlled oleic enzyme composition in cosmetics
By combining papain and bromelain with supramolecular NaDES solvent to form a supramolecular oil-controlling enzyme composition, the problems of poor enzyme stability and insufficient component synergy are solved, realizing the synergistic effect of acid exfoliation and enzymatic protein hydrolysis, thus improving the exfoliation, oil control and skin metabolism effects of cosmetics.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGZHOU SHIKA TECH CO LTD
- Filing Date
- 2026-03-16
- Publication Date
- 2026-05-12
AI Technical Summary
The enzymes in existing cosmetics have poor stability and insufficient synergy among components, making it difficult to achieve the synergistic effect of acid exfoliation and enzymatic protein hydrolysis. Furthermore, the potential efficacy of capryloyl glycine has not been fully utilized.
A supramolecular NaDES solvent is formed by mandelic acid, betaine, and capryloylglycine, which is then combined with papain and bromelain to form a supramolecular oleic acid enzyme composition. Through self-assembly, a stable supramolecular acid enzyme aggregate is formed, which improves the stability of the enzyme and achieves the synergistic effect of acid, amino acid derivative, and enzyme.
It achieves improved enzyme stability, and the synergistic effect of acids, amino acid derivatives and enzymes effectively removes dead skin cells and clogged pores, controls oil, improves skin texture and promotes metabolism.
Smart Images
Figure CN122005351A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cosmetic technology, and in particular to a method for preparing a supramolecular oleic acid-controlling enzyme composition and its application in cosmetics. Background Technology
[0002] The stratum corneum of human skin is composed of chemical components such as keratin, intercellular lipids, and natural moisturizing factors. 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. Its structure is characterized by numerous disulfide bonds and hydrogen bonds, forming a stable three-dimensional spatial structure. The presence of numerous disulfide bonds makes keratin's stereochemical structure very robust, giving it strong resistance to chemical reagents and hydrolytic enzymes. Therefore, breaking the disulfide bonds first removes the protein's resistance to degradation and its water insolubility.
[0003] Normal metabolism of the stratum corneum is crucial for maintaining healthy skin. Accumulated dead skin cells can clog pores, dull skin tone, and even trigger acne. Both acids and enzymes can remove dead skin cells and clogged pores, quickly improving skin texture and appearance and reducing 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. Dead skin cells are quickly shed, and some small-molecule fruit acids or lipid-soluble acids can penetrate the stratum corneum to continue their physiological effects in deeper layers of the skin. Proteases, due to their catalytic specificity and larger molecular weight, only have a mild hydrolytic effect on keratin on the skin surface.
[0004] Proteases, as one of the active ingredients in cosmetics, possess many excellent properties; however, their fragility has always been a challenge in research and application. Enzymes in their free state are highly unstable and easily deactivated under heat, acid, alkali, and organic solvent conditions, losing their catalytic activity. Even when stored for a period of time in their optimal reaction environment, enzymes will still become inactive. Due to the stringent storage conditions required for natural product enzymes, most enzymes lose their activity after being stored at room temperature for a month or even less.
[0005] Furthermore, existing technologies have limitations: insufficient component synergy and insufficient consideration of the enhancement of system performance by a third component. For example, capryloylglycine, as an amino acid derivative, possesses excellent antibacterial, oil-controlling, and amphiphilic carrier functions; the C8 carbon chain of capryloylglycine and the polar head of glycine form a unique amphiphilic structure, which can increase the transdermal absorption rate of water-soluble active ingredients (such as niacinamide) by 2.3 times and the penetration of oil-soluble ingredients (such as retinol) by 1.8 times, achieving efficient cross-barrier delivery. It is expected to play multiple roles in the system: as an additional hydrogen bond donor / acceptor to strengthen the supramolecular network, provide independent skin care efficacy, and potentially improve the interfacial properties of the system to facilitate enzyme-substrate contact.
[0006] Therefore, there is an urgent need to develop a novel, multi-component synergistic supramolecular composition that can provide a stable "sanctuary" for proteases, achieve a triple synergy of "acid exfoliation + enzymatic protein hydrolysis + amino acid derivative oil control", and coexist with acidic components to exert a gentle and highly effective synergistic exfoliation and skin care effect. Summary of the Invention
[0007] To address the shortcomings of existing technologies, one objective of this invention is to provide a method for preparing a supramolecular oleic acid-controlling enzyme composition; another objective is to provide a supramolecular oleic acid-controlling enzyme composition; and a third objective is to provide an application of the supramolecular oleic acid-controlling enzyme composition in cosmetics.
[0008] One of the objectives of this invention is achieved through the following technical solution: A method for preparing a supramolecular oleic acid-controlling enzyme composition includes the following steps: Step A, Preparation of supramolecular NaDES solvent: Mandelic acid, betaine and capryloylglycine are reacted to obtain supramolecular NaDES solvent; Step B, Preparation of supramolecular olease composition: Papain and bromelain complex are reacted with supramolecular NaDES solvent to obtain supramolecular olease composition.
[0009] Preferably, the molar ratio of mandelic acid, betaine and capryloylglycine in step A is (1-2):(1-2):(0.1-0.2).
[0010] Preferably, the reaction conditions in step A are as follows: under an inert atmosphere (such as nitrogen or argon), the mixture is heated and stirred at 50-80°C for 2-6 hours. During stirring, ultrasonic treatment is performed at a frequency of 25-45 kHz.
[0011] Preferably, the supramolecular NaDES solvent obtained in step A is a homogeneous pale yellow transparent liquid, a supramolecular low eutectic solvent, and its 1H NMR spectrum is shown below. Figure 1 As shown, the hydrogen bond donors in the supramolecular NaDES solvent are mandelic acid and capryloyl glycine; the hydrogen bond acceptor is betaine.
[0012] Preferably, the papain and bromelain complex in step B is obtained by mixing papain and bromelain in a mass ratio of 1:1.
[0013] Preferably, in step B, the mass ratio of the papain and bromelain complex to the supramolecular NaDES solvent is (1-3):(97-99).
[0014] Preferably, the reaction conditions in step B are: heating at 30-40°C for 1-5 hours under an inert atmosphere (such as nitrogen or argon).
[0015] Preferably, the supramolecular oleic acid enzyme composition obtained in step B is a homogeneous pale yellow transparent liquid.
[0016] The second objective of this invention is achieved through the following technical solution: A supramolecular oleic acid enzyme composition obtained by the above preparation method.
[0017] The second objective of this invention is achieved through the following technical solution: The supramolecular oleic acid enzyme composition is used to prepare cosmetics that remove dead skin cells, clear clogged pores, control oil, improve skin texture, or promote skin metabolism.
[0018] Explanation of the principle: 1. The main approach involves designing a supramolecular deep eutectic solvent containing mandelic acid, betaine, and capryloylglycine to efficiently combine enzyme molecules with supramolecular structures and achieve self-assembly, forming stable supramolecular acid-enzyme aggregates. This improves enzyme stability and achieves synergistic effects between the acid and enzyme.
[0019] 2. A stable and reliable enzyme solidification method is provided, which utilizes a supramolecular deep eutectic solvent prepared by mandelic acid, betaine, and capryloyl glycine. The protease complex is dissolved and immobilized in the supramolecular low eutectic solvent. This method is characterized by simple operation, good solubility, and good stability. In addition, the resulting supramolecular oil-controlling enzyme composition can achieve the combination of acid, amino acid derivative, and enzyme to synergistically remove dead skin cells and clogged pores, control oil, rapidly improve skin texture and appearance, and promote skin metabolism.
[0020] The beneficial effects of this invention are as follows: Addressing the aforementioned deficiencies of the prior art, this invention provides a stable and reliable enzyme curing method. Mandelic acid, betaine, and capryloylglycine are prepared as a supramolecular deep eutectic solvent. The protease complex is dissolved and immobilized in this supramolecular low eutectic solvent. This method is characterized by its simple operation, good solubility, and good stability. Furthermore, the resulting supramolecular oil-controlling enzyme composition achieves the combination of acid, amino acid derivatives, and enzymes, synergistically removing dead skin cells and clogging pores, controlling oil production, rapidly improving skin texture and appearance, and promoting skin metabolism. Attached Figure Description
[0021] Figure 1 The image shows the proton NMR spectrum of the supramolecular NaDES solvent obtained in Example 1.
[0022] Figure 2The image shows a transmission electron microscope (TEM) image of the supramolecular oleic acid-controlling enzyme composition obtained in Example 1.
[0023] Figure 3 This is a comparison test diagram of the solubility of the supramolecular oleic acid enzyme composition obtained in Example 1 and the untreated composite enzyme.
[0024] Figure 4 The image shows the enzyme activity test results of the supramolecular oleic acid-controlling enzyme composition obtained in Example 1.
[0025] Figure 5 The image shows the detection results of BCA in keratinocytes using the supramolecular oleic acid-controlling enzyme composition obtained in Example 1. Where: *P < 0.05; **P < 0.01; ***P < 0.001; * indicates that the tested sample showed a significant difference compared to the blank group at this concentration.
[0026] Figure 6 This is a computer simulation of a molecular docking model. Detailed Implementation
[0027] The following is a further explanation with reference to specific implementation methods: First, the interactions between key components of the supramolecular acid ester assemblages (SAEs) were predicted using a computer simulation molecular docking model. The results are shown below. Figure 6 . Example
[0028] Step A, Preparation of supramolecular NaDES solvent: Mandelic acid, betaine, and capryloylglycine were added to a reactor in a molar ratio of 1:1:0.1; under an inert atmosphere (nitrogen), the mixture was heated and stirred at 60°C for 4 hours, with ultrasonic treatment at a frequency of 35 kHz during stirring; until a homogeneous pale yellow transparent liquid was formed, which is the supramolecular NaDES solvent. Its 1H NMR spectrum is shown below. Figure 1 As shown. The results show that, by comparing the 1H NMR spectra of mandelic acid, betaine, and capryloylglycine; and the supramolecular DES of mandelic acid / betaine / capryloylglycine (MAN / BET DES), the capryloylglycine glycine methylene group in the supramolecular DES is coupled with the amide NH group (J ≈ 6–8 Hz), and undergoes a chemical shift due to the electron-withdrawing effect of the carboxyl group. This is consistent with the characteristics of weak intermolecular interactions, proving the formation of a supramolecular low eutectic solvent.
[0029] Step B: The papain and bromelain complex (obtained by mixing papain and bromelain in a mass ratio of 1:1, the same below) and supramolecular NaDES solvent are added to the reactor at a mass ratio of 2:98. Under an inert atmosphere (nitrogen), the mixture is heated at 35°C for 3 hours to obtain a homogeneous pale yellow transparent liquid, which is the supramolecular oleic acid enzyme composition.
[0030] The obtained supramolecular oleic acid-controlling enzyme composition was observed by transmission electron microscopy, and the results are as follows: Figure 2 As shown in the figure. The results show that the supramolecular low eutectic solvent (SAE) morphology can be clearly observed by transmission electron microscopy (TEM), and the supramolecular structure of SAE is clearly visible, which is spherical aggregate and uniformly distributed. This proves that enzyme molecules and supramolecular molecules can efficiently combine and achieve self-assembly to form stable supramolecular acid enzyme aggregates, forming uniform nanoparticles with a particle size of about 100~200 nm. Example
[0031] Step A, preparation of supramolecular NaDES solvent: Mandelic acid, betaine, and capryloylglycine are added to a reactor in a molar ratio of 1.5:1:0.1; under an inert atmosphere (nitrogen), the mixture is heated and stirred at 50°C for 2 hours, with ultrasonic treatment at a frequency of 25 kHz during stirring; until a homogeneous pale yellow transparent liquid is formed, which is the supramolecular NaDES solvent.
[0032] Step B: Add the papain and bromelain complex and supramolecular NaDES solvent to the reactor at a mass ratio of 1:99. Under an inert atmosphere (nitrogen), heat at 30°C for 1 hour to obtain a homogeneous pale yellow transparent liquid, which is the supramolecular oleic acid control enzyme composition.
[0033] Example 3
[0034] Step A, Preparation of supramolecular NaDES solvent: Mandelic acid, betaine, and capryloylglycine are added to a reactor in a molar ratio of 1.5:2:0.2; under an inert atmosphere (nitrogen), the mixture is heated and stirred at 80°C for 6 hours, with ultrasonic treatment at a frequency of 45 kHz during stirring; until a homogeneous pale yellow transparent liquid is formed, which is the supramolecular NaDES solvent.
[0035] Step B: The papain and bromelain complex and supramolecular NaDES solvent are added to the reactor at a mass ratio of 3:97. Under an inert atmosphere (nitrogen), the mixture is heated at 40°C for 5 hours to obtain a homogeneous pale yellow transparent liquid, which is the supramolecular oleic acid enzyme composition.
[0036] Comparative Example 1 Step A, preparation of supramolecular NaDES solvent: Mandelic acid, betaine, and capryloylglycine are added to a reactor in a molar ratio of 1:1:0.2; under an inert atmosphere (nitrogen), the mixture is heated and stirred at 60°C for 10 hours, with ultrasonic treatment at a frequency of 35 kHz during stirring; this is the supramolecular NaDES solvent.
[0037] Step B: Add the papain and bromelain complex and supramolecular NaDES solvent to the reactor at a mass ratio of 3:97, and heat at 35°C for 3 hours under an inert atmosphere (nitrogen) to obtain the supramolecular oleic acid control enzyme composition.
[0038] Solubility test of supramolecular oleic acid-controlling enzyme composition and untreated complex enzyme: Test sample: The sample prepared in Example 1.
[0039] A certain proportion of supramolecular acid enzyme composition or untreated complex enzyme was dissolved in water, and its state after dissolution was observed and compared.
[0040] Table 1 Concentration (as enzyme) supramolecular oleic acid-controlling enzyme composition Untreated complex enzymes 0.4% Clear liquid Turbidity and protein precipitation 0.2% Clear liquid cloudy white liquid 0.1% Clear liquid cloudy white liquid From Table 1 and Figure 3 It can be seen that when the concentration of the untreated complex enzyme in water is 0.1%, the untreated complex enzyme exhibits a turbid and whitish appearance. When the concentration is higher than 0.2%, the untreated complex enzyme exhibits a turbid appearance and protein precipitation. However, the supramolecular acid enzyme composition after supramolecular treatment still appears clear and transparent, proving that supramolecular technology treatment can increase the solubility of the enzyme to a certain extent.
[0041] Protective enzyme activity test: Test principle: Under certain temperature and pH conditions, hydrolysis of the casein substrate produces amino acids containing phenolic groups (tyrosine, tryptophan, etc.). Therefore, under alkaline conditions, Folin-Ciocalteu reagent is reduced to molybdenum blue and tungsten blue, and the absorbance of the solution is measured at a wavelength of 680 nm using a spectrophotometer. Enzyme activity is directly proportional to absorbance, therefore the enzyme activity of the product can be calculated.
[0042] Plotting the standard curve: Weigh L-tyrosine standard and prepare concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL.
[0043] Specific operating procedure: Add 1.00 mL of enzyme solution to the sample and blank tubes, incubate at 40℃±0.2℃ 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), incubate at 40℃±0.2℃ for 10 min, then add 2.00 mL of trichloroacetic acid to the sample tube and casein solution to the blank tube, let stand for 10 min, filter, take 1.00 mL of filtrate, add 5.0 mL of sodium carbonate solution and 1.0 mL of Folin-Ciocalteu solution, incubate at 40℃±0.2℃ for 20 min, then measure the absorbance at 680 nm wavelength using a UV spectrophotometer and calculate the protease activity.
[0044] A certain mass of the supramolecular acid enzyme composition prepared by the method in Example 1 was dissolved in water to obtain an aqueous solution of the supramolecular acid enzyme composition as the experimental group. The same proportions and amounts of mandelic acid, betaine, and the complex enzyme were dissolved in water as the control group. After storage at 40°C for a period of time, the enzyme activity of each sample was tested. The data from day 0 was set as the initial value, and the decay rate was statistically analyzed. Specific test results are as follows: Figure 4 As shown, at 40°C, compared to direct mixing, the supramolecular olease composition prepared by the supramolecular method can more effectively maintain protease activity and greatly improve protease suitability.
[0045] Exfoliating efficacy test: Test sample: The sample prepared in Example 1.
[0046] Test Principle: When skin metabolism becomes irregular due to aging or other reasons, the stratum corneum cells cannot perform normal metabolism. Excess keratinocytes become "semi-adherent" to the surface, making it difficult for the skin to absorb daily skincare products. This also results in a less smooth appearance, affecting aesthetics. Exfoliation is necessary to accelerate skin cell renewal, allowing excess keratinocytes to shed from the skin's surface. This not only restores smoothness and vitality to the skin but also has anti-wrinkle and anti-aging effects. The stratum corneum contains a large amount of barrier proteins such as keratin. Protein content analysis can determine the amount of keratinocytes shed; the more keratinocytes shed, the higher the protein content. Therefore, we can reflect the exfoliation efficacy of a sample by measuring the total protein content of shed stratum corneum cells in vitro.
[0047] Detailed operating procedures: 1) After cleaning and processing the fresh pigskin, fix the skin 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 dermis facing the receiving chamber. Add receiving solution (PBS) to the receiving chamber, tighten and fix the pigskin, and add a small amount of receiving solution to the receiving chamber through the sampler. Remove the air and make the dermis of the skin in close contact with the receiving solution.
[0048] 2) Drug administration: Add 50 μL of sample to the surface of pig skin (PBS buffer was added to the surface of the control group as a blank control), and spread the sample evenly from the center of the skin outwards radially. Each sample was repeated in triplicate, and kept in a constant temperature water bath at 32°C, ensuring that there were no air bubbles in the water bath interlayer.
[0049] 3) Sample collection: After incubation for 24 h, put on a finger cot and rub the sample application area. After rubbing for two minutes, add 0.5 mL of cleaning solution (0.1% Triton X-100) to the supply chamber, blow and clean the sloughed keratinocytes on the skin surface, put the collected cleaning solution into a high-speed centrifuge, and centrifuge to collect the keratinocytes.
[0050] 4) Lysis: After resuspending the above keratinocytes in deionized water, add 25 μL of 12 M NaOH solution to the liquid, lyse the keratinocytes in a boiling water bath for 30 min, and finally add 25 μL of 12 M HCl solution to the above lysis buffer.
[0051] 5) Total protein determination by BCA method: Total protein was determined according to the instructions of the BCA method detection kit.
[0052] The results are shown in Table 2 and Figure 5 .
[0053] Table 2 Results of exfoliated protein concentration detection
[0054] Compared with the control group, treatment with 2% and 4% supramolecular oleic acid enzymes and physical mixed oleic acid enzymes significantly increased the concentration of exfoliated keratinocyte protein in the skin of one-month-old Bama miniature pigs (P < 0.05~0.01), and this increase was concentration-dependent. Treatment with 4% mandelic acid also increased the concentration of exfoliated keratinocyte protein in the skin of one-month-old Bama miniature pigs (P < 0.05). Treatment with 2% and 4% fig protease did not significantly change the concentration of exfoliated keratinocyte protein in the skin of one-month-old Bama miniature pigs (P > 0.05).
[0055] Experimental conclusions: Both supramolecular olease and physical mixed olease can achieve exfoliation by promoting the shedding of keratinocytes. High concentrations of mandelic acid have a certain exfoliation effect, while papain and bromelain have no obvious exfoliation effect. Exfoliation ability: supramolecular olease > physical mixed olease > mandelic acid > fig protease.
[0056] Oil control efficacy test: Test sample: The sample prepared in Example 1.
[0057] Test materials 1. Instruments and Materials Main reagents: 5α-reductase, testosterone, phosphate buffer, NADPH Main instruments: constant temperature and humidity incubator, automatic UV-Vis spectrophotometer The instrument models are shown in Table 3. Table 3 Instrument Name Instrument Model Instrument manufacturers Constant temperature and humidity incubator HWS-260H Ningbo Yanghui Instrument Co., Ltd. Automatic UV-Vis Spectrophotometer Type 752 Shanghai Jingqi Instrument Co., Ltd. 2. The design of the test sample concentration is shown in Table 4. Table 4
[0058] 3. Positive control Name: Dutasteride; Batch number: S06GS160248; Manufacturer: Shanghai Yuanye Biotechnology Co., Ltd. Solvent: Ethanol; Concentration: 10 μg / mL.
[0059] 4. Preparation of the positive group Positive control group: Dissolve 10 mg dutasteride in 1 mL of ethanol solution to obtain dutasteride stock solution, concentration 10 mg / mL; take dutasteride stock solution; Dilute liquid ethanol 1:1000 to 10 μg / mL as a positive control working solution.
[0060] 5. The test methods are shown in Table 5. Table 5
[0061] Inhibition rate calculation formula:
[0062] 6. The results and their interpretation are shown in Table 6. Table 6
[0063] Note: Significant differences were found compared to the blank control group (*P<0.05, **P<0.01). No significant differences were found (P>0.05), expressed as "ns".
[0064] 7. Conclusion When the concentration of the test sample was between 0.1% and 1%, the inhibition rate of 5α reductase was significantly different from that of the blank control group (P<0.05), indicating that the test sample had oil-controlling effect.
[0065] The embodiments and descriptions above are merely illustrative of the principles and preferred embodiments of the present invention. Various changes and modifications may be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.
Claims
1. A method for preparing a supramolecular oleic acid-controlling enzyme composition, characterized in that, Includes the following steps: Step A, Preparation of supramolecular NaDES solvent: Mandelic acid, betaine and capryloylglycine are reacted to obtain supramolecular NaDES solvent; Step B, Preparation of supramolecular olease composition: Papain and bromelain complex are reacted with supramolecular NaDES solvent to obtain supramolecular olease composition.
2. The method for preparing the supramolecular oleic acid-controlling enzyme composition according to claim 1, characterized in that: In step A, the molar ratio of mandelic acid, betaine, and capryloylglycine is (1-2):(1-2):(0.1-0.2).
3. The method for preparing the supramolecular oleic acid-controlling enzyme composition according to claim 1, characterized in that: In step A, the reaction conditions are as follows: under an inert atmosphere, the mixture is heated and stirred at 50-80°C for 2-6 hours. During stirring, ultrasonic treatment is performed at a frequency of 25-45 kHz.
4. The method for preparing the supramolecular oleic acid-controlling enzyme composition according to claim 1, characterized in that: The supramolecular NaDES solvent obtained in step A is a homogeneous, pale yellow, transparent liquid.
5. The method for preparing the supramolecular oleic acid-controlling enzyme composition according to claim 1, characterized in that: In step B, the papain and bromelain complex is obtained by mixing papain and bromelain in a mass ratio of 1:
1.
6. The method for preparing the supramolecular oleic acid-controlling enzyme composition according to claim 1, characterized in that: In step B, the mass ratio of the papain and bromelain complex to the supramolecular NaDES solvent is (1-3):(97-99).
7. The method for preparing the supramolecular oleic acid-controlling enzyme composition according to claim 1, characterized in that: The reaction conditions in step B are: heating at 30-40°C for 1-5 hours under an inert atmosphere.
8. The method for preparing the supramolecular oleic acid-controlling enzyme composition according to claim 1, characterized in that: The supramolecular oleic acid enzyme composition obtained in step B is a uniform, pale yellow, transparent liquid.
9. A supramolecular oleic acid enzyme composition obtained by the preparation method of the supramolecular oleic acid enzyme composition according to any one of claims 1-8.
10. The application of the supramolecular oleic acid-controlling enzyme composition as described in claim 9 in cosmetics, characterized in that: The supramolecular oleic acid enzyme composition is used to prepare cosmetics that remove dead skin cells, clear clogged pores, control oil, improve skin texture, or promote skin metabolism.