Preparation method and application of supramolecular nano coenzyme composition

Abstract

The invention belongs to the technical field of skin care products, and particularly relates to a preparation method and application of a supramolecular nano coenzyme composition. The supramolecular nano coenzyme composition is prepared from the following raw materials: an active component and a carrier substance, the active ingredients are glucose oxidase, lysozyme and superoxide dismutase. According to the supramolecular nano coenzyme composition disclosed by the invention, glucose oxidase is combined with various other biological enzymes, plant extracts and other functional components through scientific proportioning, so that the detoxifying (antisepsis and anti-inflammation) and anti-oxidation effects of the supramolecular nano coenzyme composition are improved, the skin can be smoothed, and the supramolecular nano coenzyme composition has the beautifying effects of brightening, moisturizing, tightening, resisting aging, dissolving fat and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin care products, and particularly relates to a preparation method and application of a supramolecular nano coenzyme composition. Background Art

[0002] Glucose oxidase (GOX for short) is a redox enzyme widely present in organisms, with a variety of remarkable physiological functions and broad application prospects. This enzyme can catalyze the oxidation of glucose to produce gluconic acid and hydrogen peroxide. This reaction not only provides energy required by cells but also participates in regulating the metabolism of carbohydrates in the body, helping to maintain blood sugar balance. In addition, glucose oxidase also has powerful antioxidant and anti-inflammatory effects. It can scavenge free radicals in the body, reduce the damage caused by oxidative stress reactions to the body, delay the aging process, and inhibit inflammatory reactions, alleviating inflammatory symptoms.

[0003] In the food industry, glucose oxidase is widely used in aspects such as deoxidation, anti-browning, and shelf-life extension of foods such as wine, beer, fruit juice, and milk powder. At the same time, this enzyme also shows great application potential in the medical field. It can be used for anti-inflammatory and antioxidant treatments to improve the therapeutic effect of drugs. For example, when used in combination with antibiotics, it can enhance the antibacterial effect of antibiotics. In addition, glucose oxidase is also highly favored in the cosmetics industry. Its functions such as antioxidant, brightening, moisturizing, and firming the skin make it a key ingredient in many beauty products.

[0004] However, it is often difficult to meet complex and diverse beauty needs by using glucose oxidase alone. Therefore, it is particularly important to develop a supramolecular nano coenzyme composition containing multiple bioactive components. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and application of a supramolecular nano coenzyme composition. By scientifically proportioning, combining glucose oxidase with other multiple bioenzymes and plant extracts and other functional components, not only the detoxification (antibacterial and anti-inflammatory), antioxidant and other effects of the product are improved, but also the skin can be smoothed, and it has beauty effects such as brightening, moisturizing, firming, anti-aging, and fat dissolving.

[0006] To achieve the above purpose, the present invention provides the following technical solutions: The supramolecular nano coenzyme composition, its preparation raw materials include active ingredients and carrier substances.

[0007] Preferably, the active ingredients are glucose oxidase, lysozyme, and superoxide dismutase.

[0008] Preferably, the addition amount of glucose oxidase is 450 - 550 U / g of the composition. U / g means that there are 500 enzyme activity units (U) of glucose oxidase in every 1 g of the supramolecular nano coenzyme composition. The same applies hereinafter.

[0009] Preferably, the addition amount of lysozyme is 350 - 450 U / g of the composition.

[0010] Preferably, the addition amount of superoxide dismutase is 250 - 350 U / g of the composition.

[0011] Glucose oxidase, lysozyme, and superoxide dismutase are selected as active ingredients, which can not only improve the antioxidant property of the supramolecular nano coenzyme composition but also enhance the antibacterial and anti-inflammatory properties. This may be because there is a synergistic effect among the three active ingredients. Glucose oxidase catalyzes the reaction of glucose and oxygen to generate hydrogen peroxide with antibacterial effects, and its ability to indirectly participate in the antioxidant process also provides additional protection for the skin. Superoxide dismutase directly acts on superoxide anion radicals, dismutating them into hydrogen peroxide and oxygen, effectively scavenging free radicals and reducing the damage of oxidative stress to the skin. The synergistic effect of these two enzymes in antioxidant aspect enables the cosmetics to more effectively resist the oxidative attack of the external environment and protect skin cells from damage. Secondly, lysozyme directly kills harmful bacteria on the skin surface with its unique ability to destroy bacterial cell walls, reducing the risk of bacterial infection. At the same time, the hydrogen peroxide generated by glucose oxidase complements lysozyme, further enhancing its antibacterial effect and forming a powerful antibacterial barrier. In addition, superoxide dismutase helps to reduce the inflammatory response caused by bacterial infection and promote the self-repair and healing process of the skin by scavenging free radicals. This synergistic effect in antibacterial and anti-inflammatory aspects enables the cosmetics to more effectively maintain the healthy state of the skin and reduce the occurrence of inflammation and sensitivity problems. By enhancing the antioxidant property and improving the antibacterial and anti-inflammatory properties, they act together on the skin, providing comprehensive protection and repair for the skin, making the skin healthier, smoother, and more elastic.

[0012] The carrier substance, by weight percentage, includes 5% - 10% of humectant, 0.05% - 0.1% of antioxidant, 2% - 5% of plant extract, 0.1% - 0.5% of thickener, 3% - 7% of surfactant, an appropriate amount of pH regulator, and the balance of water is made up to 100%.

[0013] Preferably, the humectant includes one or more of glycerol, 1,2 - propanediol, sodium hyaluronate, and caprylic / capric triglyceride; more preferably, it is glycerol, 1,2 - propanediol, sodium hyaluronate, and caprylic / capric triglyceride.

[0014] Preferably, the mass ratio of glycerol, 1,2 - propanediol, sodium hyaluronate, and caprylic / capric triglyceride is (8 - 12):(5 - 7):1:(8 - 12); more preferably, it is 10:6:1:10.

[0015] Preferably, the molecular weight of the sodium hyaluronate is 10,000 - 100,000.

[0016] In some preferred embodiments, the sodium hyaluronate is purchased from Yingxin Laboratory.

[0017] Preferably, the acid value of the caprylic / capric triglyceride is ≤0.1 mg KOH / g, and the saponification value is 325 - 345 mg KOH / g.

[0018] In some preferred embodiments, the caprylic / capric triglyceride is purchased from Guangzhou Youyu New Materials Co., Ltd.

[0019] Preferably, the antioxidant includes vitamin C, vitamin E, and coenzyme Q10.

[0020] Preferably, the mass ratio of vitamin C, vitamin E, and coenzyme Q10 is (4 - 6):(1 - 3):1; more preferably, it is 5:2:1.

[0021] In some preferred embodiments, the coenzyme Q10 is purchased from Kochi (Guangdong) Biotechnology Co., Ltd.

[0022] Vitamin C, vitamin E, and coenzyme Q10 are selected as antioxidants, which can not only further enhance the antioxidant effect but also improve the antibacterial and anti-inflammatory properties. This may be because on the one hand, vitamin C can effectively reduce the oxidation products of vitamin E and prevent the initiation of the lipid peroxidation chain reaction. Vitamin E can protect cell membranes and organelle membranes from free radical attacks. Coenzyme Q10 can capture free radicals at the initiation stage of the lipid peroxidation chain and form a complement with vitamin E. When vitamin E is oxidized, coenzyme Q10 can reduce and regenerate it. The combined action of these three greatly enhances the antioxidant performance of the cosmetics and provides comprehensive protection for the skin. In addition, there is also a synergistic effect between these antioxidants and glucose oxidase, lysozyme, and superoxide dismutase. Although the hydrogen peroxide generated by glucose oxidase has oxidizing properties, in the cosmetics formulation, it acts synergistically with other ingredients (such as vitamin C and vitamin E) and is ultimately decomposed into harmless water and oxygen, indirectly participating in the antioxidant process. At the same time, as antioxidants, vitamin C, vitamin E, and coenzyme Q10 can reduce the damage of free radicals to the skin, promote skin repair and healing, and thus indirectly enhance the antibacterial and anti-inflammatory effects of lysozyme and superoxide dismutase. Lysozyme directly kills bacteria by destroying the bacterial cell wall, while superoxide dismutase scavenges superoxide anion free radicals to reduce the inflammatory response. The synergistic effect of these ingredients not only improves the antioxidant performance of the supramolecular nano coenzyme composition but also enhances its antibacterial and anti-inflammatory ability, comprehensively improving the quality of the supramolecular nano coenzyme composition and bringing a better user experience to consumers. However, vitamin E and coenzyme Q10 are fat-soluble and need to be microemulsified during preparation to achieve good usability and efficacy.

[0023] Preferably, the plant extract includes green tea extract, centella asiatica extract, and polygonum cuspidatum extract.

[0024] Preferably, the mass ratio of the green tea extract, centella asiatica extract, and polygonum cuspidatum extract is (0.8 - 1.2):(0.8 - 1.2):1; more preferably, it is 1:1:1.

[0025] Green tea extract, centella asiatica extract, and polygonum cuspidatum extract are selected as plant extracts, which not only further improve the antioxidant and anti-inflammatory effects of the supramolecular nano coenzyme composition, but also can whiten and brighten the skin tone, repair, moisturize, and enhance the skin barrier. This may be because there is a significant synergistic effect among the three extracts. On the one hand, catechins in green tea extract, triterpenoid saponins in centella asiatica extract, and resveratrol in polygonum cuspidatum extract jointly construct a multi-level antioxidant system. They capture free radicals from different angles, minimizing the damage caused by oxidative stress to the skin. Catechins and resveratrol directly scavenge free radicals, while triterpenoid saponins indirectly support the integrity of the skin structure by promoting collagen synthesis. On the other hand, all three extracts have anti-inflammatory properties, but their mechanisms of action are slightly different. Green tea extract acts by inhibiting the release of inflammatory mediators; centella asiatica extract reduces inflammation by regulating the immune response; polygonum cuspidatum extract achieves anti-inflammatory purposes by inhibiting the expression of inflammation-related genes. These different mechanisms of action complement each other, enhancing the overall anti-inflammatory effect. In addition, both green tea extract and polygonum cuspidatum extract can inhibit melanin production and reduce the formation of age spots. At the same time, centella asiatica extract helps fade existing pigmentation by promoting skin repair, making the skin tone more uniform and bright. Polysaccharides and amino acids in centella asiatica extract not only help maintain skin moisture, but also promote the repair of damaged tissues, can enhance the skin barrier, promote wound healing, combined with the antioxidant properties and anti-inflammatory protection of green tea extract and polygonum cuspidatum extract, can more effectively resist the damage of external environmental factors to the skin and maintain the stability and health of the skin. However, the effective components of plant extracts on the market are less, and their efficacy is poor when applied to the composition. Therefore, in this application, by making plant extracts by oneself, the content of effective components in the plant extracts is increased as much as possible, thereby improving the related efficacy of the composition.

[0026] Preferably, the preparation method of the green tea extract includes the following steps: Wash the green tea, dry and crush it, add an ethanol aqueous solution with a mass concentration of 70%-80%, extract at 70-80°C for 30-50 minutes, filter, and concentrate and dry the filtrate to obtain the green tea extract.

[0027] Preferably, the mass ratio of the green tea to the ethanol aqueous solution with a mass concentration of 70%-80% is 1:(20-30).

[0028] The preparation method of the centella asiatica extract includes the following steps: B1. Wash the centella asiatica, dry and crush it, add water to adjust the water content to 45% - 55% to obtain a centella asiatica fermentation substrate, add a centella asiatica ferment, and ferment aerobically at 40 - 45°C until the pH value ≤ 4, then take it out to obtain a centella asiatica pretreatment product; B2. Add the asiatic pennywort pretreatment to an ethanol aqueous solution with a mass concentration of 70 - 80%, adjust the temperature to 50 - 60 °C, and perform ultrasonic extraction for 30 - 60 min to obtain the crude asiatic pennywort extract. B3. Add activated carbon to the crude asiatic pennywort extract, stir at 35 - 45 °C for 20 - 30 min, and then filter to obtain the asiatic pennywort extract.

[0029] B4. Subject the asiatic pennywort extract to vacuum distillation to remove ethanol, load it onto a macroporous resin, elute with an ethanol aqueous solution with a mass concentration of 70% - 75%, collect the eluate, perform vacuum concentration and drying to obtain the asiatic pennywort extract.

[0030] Preferably, the asiatic pennywort fermenting agent includes one or more of Lactobacillus acidophilus, Lactobacillus rhamnosus, Pichia pastoris, and Candida utilis.

[0031] For the Lactobacillus acidophilus, the strain number is CICC 6005; for the Lactobacillus rhamnosus, the strain number is CICC 6224; for the Pichia pastoris, the strain number is CICC 32806; for the Candida utilis, the strain number is CICC 1769.

[0032] Preferably, the dosage of the Lactobacillus acidophilus is 10 6 -10 7 CFU / g of the asiatic pennywort fermentation substrate; the dosage of the Lactobacillus rhamnosus is 10 7 -10 8 CFU / g of the asiatic pennywort fermentation substrate; the dosage of the Pichia pastoris is 10 7 -10 8 CFU / g of the asiatic pennywort fermentation substrate; the dosage of the Candida utilis is 10 7 -10 8 CFU / g of the asiatic pennywort fermentation substrate.

[0033] Preferably, in step B2, the mass ratio of the asiatic pennywort pretreatment to the ethanol aqueous solution with a mass concentration of 70% - 80% is 1:(20 - 30).

[0034] Preferably, the power of the ultrasonic extraction is 200 - 300 W.

[0035] The activated carbon is coal-based activated carbon, the passing rate of 325 mesh is 90%, and the iodine adsorption value is 800 - 1000 mg / g.

[0036] In some preferred embodiments, the activated carbon is purchased from Zhejiang Zheliang Activated Carbon Co., Ltd., QH - 325.

[0037] Preferably, the addition amount of the activated carbon is 0.5% - 1% of the mass of the crude asiatic pennywort extract.

[0038] Preferably, the macroporous resin is a non-polar macroporous resin with an average pore diameter of 8.5 - 9 nm, the number of particles with a particle size of 0.3 - 1.25 mm ≥ 90%, and a specific surface area ≥ 50 m2 / g.

[0039] In some preferred embodiments, the macroporous resin is purchased from Tianjin Haoju Resin Technology Co., Ltd., HPD100.

[0040] Preferably, the loading flow rate is 3 - 5 mL / min.

[0041] Preferably, the elution flow rate is 8 - 10 mL / min.

[0042] In step B4, the addition amount of the ethanol aqueous solution with a mass concentration of 70% - 75% is 75 - 85 times the mass of the Centella asiatica extract after ethanol is removed by vacuum distillation.

[0043] By successively drying and pulverizing, fermenting, extracting with alcohol, decolorizing, and adsorbing with macroporous resin for Centella asiatica, the content of active ingredients in the Centella asiatica extract can be greatly increased, thereby improving the efficacy of the composition. This may be because a specific Centella asiatica fermenting agent is selected for fermentation treatment, and the four fermenting agents act synergistically. They can not only decompose the cellulose and pectin in the cell wall of Centella asiatica, making the cell wall loose, which helps the release of active ingredients in the subsequent extraction process, but also the organic acids (such as lactic acid) produced reduce the pH value, promoting the generation and stability of certain secondary metabolites (such as triterpenoid saponins, etc.), increasing the content of active ingredients in the final extract, and enhancing the antioxidant and anti-inflammatory properties of the Centella asiatica extract. Then, the mechanical effect of ultrasonic waves is used to enhance the permeability of the solvent, accelerating the dissolution and extraction of active ingredients to form a crude Centella asiatica extract. To further improve the purity of the extract, activated carbon is added to the crude extract, which has a significant effect on removing impurities and pigments in the crude extract. Finally, the active ingredients are selectively adsorbed by a specific macroporous resin, and then eluted with an ethanol aqueous solution. The collected eluate is subjected to vacuum concentration and drying treatment to obtain a Centella asiatica extract with high purity and stable content of active ingredients.

[0044] The preparation method of the Polygonum cuspidatum extract comprises the following steps: C1. Fermentation pretreatment: Wash and dry and pulverize Polygonum cuspidatum to obtain Polygonum cuspidatum powder, adjust the water content to 45% - 55% by adding water, then add urea to obtain a Polygonum cuspidatum fermentation substrate, add a Polygonum cuspidatum fermenting agent, and take it out after aerobic fermentation at 40 - 45 °C for 8 - 10 days to obtain a Polygonum cuspidatum pretreatment product; C2. Enzymatic hydrolysis: Add the pre-treated polygonum cuspidatum to water, adjust the temperature to 40 - 50 °C and the pH to 4.5 - 5.5, add cellulase and pectinase, inactivate by boiling after 2 - 3 hours of enzymatic hydrolysis, continue to adjust the system temperature to 55 - 65 °C and the pH to 4 - 5, add glucoamylase, inactivate by boiling after 1 - 2 hours of enzymatic hydrolysis, cool to room temperature and filter through a 0.22 μm microporous membrane to obtain the polygonum cuspidatum extraction filtrate; C3. Concentrate the polygonum cuspidatum extraction filtrate to 35% - 38% of the mass of the pre-treated polygonum cuspidatum, filter, and then concentrate the filtrate again to 25% - 30% of the mass of the pre-treated polygonum cuspidatum, and filter to obtain the crude polygonum cuspidatum extract; C4. Add the crude polygonum cuspidatum extract to the top of the column, the column bed is filled with alumina, elute the crude extract with ethyl acetate at a flow rate of 1 - 2 mL / min, collect the eluate, concentrate under reduced pressure and dry to obtain the polygonum cuspidatum extract.

[0045] Preferably, the polygonum cuspidatum fermenting agent includes Lactobacillus bulgaricus, Yarrowia lipolytica, and Streptococcus thermophilus.

[0046] For the Lactobacillus bulgaricus, the strain number is CICC 25031; for the Yarrowia lipolytica, the strain number is ATCC90811; for the Streptococcus thermophilus, the strain number is CICC 6038.

[0047] Preferably, the dosage of the Lactobacillus bulgaricus is 10 6 -10 7 CFU / g of the polygonum cuspidatum fermentation substrate; the dosage of the Yarrowia lipolytica is 10 8 -10 9 CFU / g of the polygonum cuspidatum fermentation substrate; the dosage of the Streptococcus thermophilus is 10 7 -10 8 CFU / g of the polygonum cuspidatum fermentation substrate.

[0048] Preferably, the addition amount of the urea is 0.5% - 1% of the mass of the polygonum cuspidatum powder.

[0049] Using Lactobacillus bulgaricus, Yarrowia lipolytica, and Streptococcus thermophilus as the fermentation agents for Polygonum cuspidatum can increase the content of resveratrol in the finally obtained Polygonum cuspidatum extract. This may be because of the synergistic effect among them. As fermentation strains, Lactobacillus bulgaricus and Streptococcus thermophilus promote the rupture of the Polygonum cuspidatum cell wall by producing organic acids and regulating the pH value of the fermentation environment, releasing more active ingredients such as resveratrol. At the same time, Yarrowia lipolytica uses its powerful metabolic ability to not only synthesize resveratrol from the precursor substances directly extracted from Polygonum cuspidatum, but also produce enzymes to decompose complex compounds, improving the extraction efficiency. Urea, as an inorganic nitrogen source, provides the necessary nutritional support for the growth of microorganisms, promotes the reproduction and activity of microorganisms, and thus indirectly improves the synthesis and extraction efficiency of resveratrol. The interaction between these microorganisms and urea forms a benign cycle, further optimizing the fermentation environment, such as regulating parameters such as temperature, pH value, and oxygen concentration, making the extraction efficiency and purity of resveratrol significantly improved.

[0050] Preferably, the mass ratio of the Polygonum cuspidatum pretreatment and water is 1:(20 - 30).

[0051] Preferably, the addition amount of cellulase is 80 - 120 U / g of Polygonum cuspidatum pretreatment; the addition amount of pectinase is 180 - 220 U / g of Polygonum cuspidatum pretreatment; the addition amount of glucoamylase is 400 - 600 U / g of Polygonum cuspidatum pretreatment.

[0052] Sequential enzymatic hydrolysis with cellulase, pectinase, and glucoamylase can further increase the content of effective ingredients such as resveratrol in the Polygonum cuspidatum extract. This may be because cellulase acts on the cellulose in the Polygonum cuspidatum cell wall, destroying its structure and laying the foundation for subsequent steps. Pectinase further decomposes pectin, completely destroying the cell wall and promoting the release of effective ingredients such as resveratrol inside the cell. Glucoamylase converts polysaccharides into monosaccharides, which can be used as the carbon source for the growth of microorganisms, indirectly promoting the conversion and extraction of resveratrol. In addition, the treatment with glucoamylase may also help improve the permeability of the extract, enabling more effective ingredients to be extracted. Sequential enzymatic hydrolysis not only improves the extraction efficiency, significantly increases the content of resveratrol in the filtrate finally, but also enhances the biological activity of resveratrol, thereby improving the efficacy of the supramolecular nano coenzyme composition.

[0053] Preferably, the mass of alumina in the column bed is 8 - 10 times the mass of the crude Polygonum cuspidatum extract.

[0054] Preferably, the addition amount of ethyl acetate is 80 - 100 times the mass of the crude Polygonum cuspidatum extract.

[0055] Preferably, the thickener includes one or more of carbomer and xanthan gum; more preferably, it is carbomer.

[0056] Preferably, for the carbomer, the viscosity of the carbomer aqueous solution with a carbomer mass fraction of 0.5% at room temperature is 60,000 - 70,000.

[0057] In some preferred embodiments, the carbomer is purchased from Lubrizol Corporation, USA, carbomer 940.

[0058] Preferably, the surfactant includes one or more of Tween 20, Tween 80, lecithin, Span 60, and Span 80; more preferably, Tween 80 and lecithin.

[0059] Preferably, the mass ratio of Tween 80 to lecithin is (0.5 - 2):1; more preferably, 1:1.

[0060] Preferably, the acid value of Tween 80 ≤ 2 mgKOH / g, the hydroxyl value is 65 - 80 mgKOH / g, and the HLB value is 15.

[0061] In some preferred embodiments, the Tween 80 is purchased from Guangdong Warner Chemical Co., Ltd.

[0062] Preferably, the lecithin is soy lecithin, and the content of the active ingredient ≥ 80%.

[0063] In some preferred embodiments, the lecithin is purchased from Shanxi Jinyang Pharmaceutical Excipients Co., Ltd.

[0064] Preferably, the pH regulator includes triethanolamine.

[0065] The preparation method of the supramolecular nano coenzyme composition includes the following steps: S1. Prepare a microemulsion: Add the surfactant to an appropriate amount of water, stir at a rate of 150 - 250 rpm at 35 - 40 °C for 30 - 40 min until completely dissolved, then raise the temperature to 70 - 80 °C, add vitamin E and coenzyme Q10, and use a high - speed shear homogenizer to process at 4000 - 6000 rpm for 10 - 15 min to obtain it; S2. Add the thickener to the remaining water, raise the temperature to 70 °C, stir evenly at a rate of 150 - 250 rpm until completely dissolved, then lower the temperature to 40 °C, add the active substance, stir at a rate of 500 - 700 rpm for 60 - 80 min, then add vitamin C and the microemulsion, stir for 25 - 35 min, then add the plant extract, stir for 10 - 20 min, then add the humectant, stir for 10 - 20 min, add the pH regulator to adjust the pH to 5.5 - 6.5, and perform a homogenization treatment to obtain it.

[0066] Preferably, the mass of the appropriate amount of water in step S1 is 15% - 25% of the total mass of water in the carrier substance.

[0067] Preferably, the pressure for the homogenization treatment is 100 - 150 bar.

[0068] The supramolecular nano coenzyme composition of the present invention is applied to the preparation of skin care products, and the dosage of the composition in the skin care products is 0.001 - 30 wt%.

[0069] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. The present invention provides a preparation method and application of a supramolecular nano coenzyme composition. The supramolecular nano coenzyme composition combines glucose oxidase with other multiple bio - enzymes and plant extracts and other functional components through scientific proportioning, which not only improves the functions of the product such as detoxification (antibacterial and anti - inflammatory) and antioxidant, but also can smooth the skin, and has beauty effects such as brightening, moisturizing, firming, anti - aging and fat - dissolving, and is very suitable for application in the field of skin care products.

[0070] 2. The present invention selects glucose oxidase, lysozyme, and superoxide dismutase as active ingredients, which can not only improve the antioxidant property of the supramolecular nano coenzyme composition, but also improve the antibacterial and anti - inflammatory performance.

[0071] 3. The present invention selects vitamin C, vitamin E, and coenzyme Q10 as antioxidants, which can not only further improve the antioxidant effect, but also improve the antibacterial and anti - inflammatory performance.

[0072] 4. The present invention selects green tea extract, centella asiatica extract, and polygonum cuspidatum extract as plant extracts, which not only further improve the antioxidant and anti - inflammatory effects of the supramolecular nano coenzyme composition, but also can whiten and brighten the skin color, repair, moisturize, and enhance the skin barrier.

[0073] 5. By drying, crushing, fermenting, alcohol extraction, decoloring, and macroporous resin adsorption of centella asiatica in sequence, the present invention can greatly increase the content of active ingredients in the centella asiatica extract, thereby improving the efficacy of the composition.

[0074] 6. The present invention selects lactobacillus bulgaricus, yarrowia lipolytica, and streptococcus thermophilus as the fermentation agents for polygonum cuspidatum to ferment polygonum cuspidatum, which can increase the content of resveratrol in the finally obtained polygonum cuspidatum extract, thereby further improving the efficacy of the composition.

[0075] 7. The present invention uses cellulase, pectinase, and glucoamylase for enzymatic hydrolysis treatment in sequence, which can further increase the content of active ingredients such as resveratrol in the polygonum cuspidatum extract, thereby improving the efficacy of the composition. Detailed implementation manners

[0076] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0077] The raw materials used in the present invention are all commercially available. Specifically: Coenzyme Q10 is purchased from Kochi (Guangdong) Biotechnology Co., Ltd.

[0078] Glucose oxidase, enzyme activity about 10000U / g; lysozyme, enzyme activity about 3000000U / g; superoxide dismutase, enzyme activity about 10000U / g; cellulase, enzyme activity about 11000U / g; pectinase, enzyme activity about 30000U / g; glucoamylase, enzyme activity about 150000U / g; all are purchased from Ningxia Xiasheng Industrial Group Co., Ltd.

[0079] Sodium hyaluronate has a molecular weight of 10,000 - 100,000 and is purchased from Yingxin Laboratory.

[0080] Caprylic / capric triglyceride has an acid value ≤ 0.1mgKOH / g and a saponification value of 325 - 345mgKOH / g, and is purchased from Guangzhou Youyu New Materials Co., Ltd.

[0081] Lactobacillus acidophilus, strain number CICC 6005; Lactobacillus rhamnosus, strain number CICC 6224; Pichia pastoris, strain number CICC 32806; Candida utilis, strain number CICC 1769; Lactobacillus bulgaricus, strain number CICC 25031; Yarrowia lipolytica, strain number ATCC 90811; Streptococcus thermophilus, strain number CICC 6038.

[0082] The activated carbon is coal-based activated carbon, with a 325-mesh passing rate of 90% and an iodine adsorption value of 800 - 1000mg / g. It is purchased from Zhejiang Zheliang Activated Carbon Co., Ltd., QH-325.

[0083] The macroporous resin is a non-polar macroporous resin, with an average pore diameter of 8.5 - 9nm, a particle size quantity of 0.3 - 1.25mm ≥ 90%, and a specific surface area ≥ 50m2 / g. It is purchased from Tianjin Haoju Resin Technology Co., Ltd., HPD100.

[0084] Carbomer, the viscosity of a carbomer aqueous solution with a carbomer mass fraction of 0.5% at room temperature is 60000 - 70000, and it is purchased from Lubrizol Corporation, Carbomer 940.

[0085] The acid value of Tween 80 is ≤2 mg KOH / g, the hydroxyl value is 65 - 80 mg KOH / g, the HLB value is 15, and it is purchased from Guangdong Huana Chemical Co., Ltd.

[0086] The lecithin is soy lecithin, the content of active ingredients is ≥80%, and it is purchased from Shanxi Jinyang Pharmaceutical Excipients Co., Ltd.

[0087] Example 1 This example provides a supramolecular nano coenzyme composition, and its preparation raw materials are active ingredients and carrier substances.

[0088] The active ingredients are glucose oxidase, lysozyme, and superoxide dismutase.

[0089] The addition amount of glucose oxidase is 500 U / g of the composition.

[0090] The addition amount of lysozyme is 400 U / g of the composition.

[0091] The addition amount of superoxide dismutase is 300 U / g of the composition.

[0092] The carrier substances, by weight percentage, are 7% humectant, 0.08% antioxidant, 3% plant extract, 0.3% carbomer, 5% surfactant, appropriate amount of triethanolamine, and water to make up the balance to 100%.

[0093] The humectants are glycerol, 1,2 - propanediol, sodium hyaluronate, and caprylic / capric triglyceride, and the mass ratio is 10:6:1:10.

[0094] The antioxidants are vitamin C, vitamin E, and coenzyme Q10, and the mass ratio is 5:2:1.

[0095] The plant extracts are green tea extract, centella asiatica extract, and polygonum cuspidatum extract, and the mass ratio is 1:1:1.

[0096] The preparation method of the green tea extract has the following steps: After washing and drying the green tea and crushing it, add an ethanol aqueous solution with a mass concentration of 75%, extract at 75 °C for 40 min, filter, and then concentrate and dry the filtrate to obtain the green tea extract.

[0097] The mass ratio of the green tea to the ethanol aqueous solution with a mass concentration of 75% is 1:25.

[0098] The preparation method of the centella asiatica extract has the following steps: B1. After washing and drying the centella asiatica and crushing it, add water to adjust the water content to 50% to obtain the centella asiatica fermentation substrate, add the centella asiatica ferment, ferment aerobically at 42 °C until the pH value reaches 4, and then take it out to obtain the centella asiatica pretreatment; B2. Add the asiatic pennywort pretreatment to an ethanol aqueous solution with a mass concentration of 75%, adjust the temperature to 55°C, and perform ultrasonic extraction for 45 minutes to obtain a crude asiatic pennywort extract; B3. Add activated carbon to the crude asiatic pennywort extract, stir at 40°C for 25 minutes, and then filter to obtain the asiatic pennywort extract.

[0099] B4. Subject the asiatic pennywort extract to vacuum distillation to remove ethanol, load it onto a macroporous resin, elute with an ethanol aqueous solution with a mass concentration of 75%, collect the eluate, perform vacuum concentration and drying to obtain the asiatic pennywort extract.

[0100] The asiatic pennywort ferment is Lactobacillus acidophilus, Lactobacillus rhamnosus, Pichia pastoris, and Candida utilis.

[0101] The dosage of Lactobacillus acidophilus is 10 7 CFU / g of asiatic pennywort fermentation substrate; the dosage of Lactobacillus rhamnosus is 10 8 CFU / g of asiatic pennywort fermentation substrate; the dosage of Pichia pastoris is 10 8 CFU / g of asiatic pennywort fermentation substrate; the dosage of Candida utilis is 10 7 CFU / g of asiatic pennywort fermentation substrate.

[0102] In step B2, the mass ratio of the asiatic pennywort pretreatment to the ethanol aqueous solution with a mass concentration of 75% is 1:25.

[0103] The power of the ultrasonic extraction is 250 W.

[0104] The addition amount of the activated carbon is 0.8% of the mass of the crude asiatic pennywort extract.

[0105] The loading flow rate is 4 mL / min; the elution flow rate is 9 mL / min.

[0106] In step B4, the addition amount of the ethanol aqueous solution with a mass concentration of 75% is 80 times the mass of the asiatic pennywort extract after removing ethanol by vacuum distillation.

[0107] The preparation method of the polygonum cuspidatum extract comprises the following steps: C1. Fermentation pretreatment: Wash and dry and crush polygonum cuspidatum to obtain polygonum cuspidatum powder, add water to adjust the water content to 50%, then add urea to obtain a polygonum cuspidatum fermentation substrate, add a polygonum cuspidatum ferment, and perform aerobic fermentation at 42°C for 8 days and then take out to obtain a polygonum cuspidatum pretreatment; C2. Enzymatic hydrolysis: Add the pre-treated polygonum cuspidatum to water, adjust the temperature to 45 °C and the pH to 5, add cellulase and pectinase, inactivate by boiling after 2.5 h of enzymatic hydrolysis, continue to adjust the system temperature to 60 °C and the pH to 4.5, add glucoamylase, inactivate by boiling after 1.5 h of enzymatic hydrolysis, cool to room temperature and filter through a 0.22 μm microporous membrane to obtain the polygonum cuspidatum extraction filtrate; C3. Concentrate the polygonum cuspidatum extraction filtrate to 38% of the mass of the pre-treated polygonum cuspidatum, filter, and then concentrate the filtrate again to 27% of the mass of the pre-treated polygonum cuspidatum, and filter to obtain the crude polygonum cuspidatum extract; C4. Add the crude polygonum cuspidatum extract to the top of the column. The column bed is filled with alumina, elute the crude extract with ethyl acetate at a flow rate of 1.5 mL / min, collect the eluate, concentrate under reduced pressure and dry to obtain the polygonum cuspidatum extract.

[0108] The polygonum cuspidatum ferment is Lactobacillus bulgaricus, Yarrowia lipolytica, and Streptococcus thermophilus.

[0109] The dosage of Lactobacillus bulgaricus is 10 7 CFU / g of the polygonum cuspidatum fermentation substrate; the dosage of Yarrowia lipolytica is 10 8 CFU / g of the polygonum cuspidatum fermentation substrate; the dosage of Streptococcus thermophilus is 10 8 CFU / g of the polygonum cuspidatum fermentation substrate.

[0110] The addition amount of urea is 0.8% of the mass of the polygonum cuspidatum powder.

[0111] The mass ratio of the pre-treated polygonum cuspidatum to water is 1:25.

[0112] The addition amount of cellulase is 100 U / g of the pre-treated polygonum cuspidatum; the addition amount of pectinase is 200 U / g of the pre-treated polygonum cuspidatum; the addition amount of glucoamylase is 500 U / g of the pre-treated polygonum cuspidatum.

[0113] The mass of alumina in the column bed is 10 times the mass of the crude polygonum cuspidatum extract.

[0114] The addition amount of ethyl acetate is 90 times the mass of the crude polygonum cuspidatum extract.

[0115] The surfactant is Tween 80 and lecithin, and the mass ratio is 1:1.

[0116] The preparation method of the supramolecular nano coenzyme composition comprises the steps of: S1. Prepare microemulsion: Add the surfactant to an appropriate amount of water, stir at a rate of 200 rpm at 38 °C for 35 min until completely dissolved, then raise the temperature to 75 °C, add vitamin E and coenzyme Q10, and treat with a high-speed shear homogenizer at 5000 rpm for 12 min to obtain it; S2. Add the thickener to the remaining water, heat it up to 70 °C and stir evenly at a rate of 200 rpm until it is completely dissolved. Then cool it down to 40 °C, add the active substance and stir at a rate of 600 rpm for 70 min. After that, add vitamin C and the microemulsion, stir for 30 min, add the plant extract, stir for 15 min, add the humectant, stir for 15 min, add triethanolamine to adjust the pH to 6, and perform homogenization treatment to obtain the product.

[0117] The mass of the appropriate amount of water in step S1 is 20% of the total mass of water in the carrier substance.

[0118] The pressure of the homogenization treatment is 125 bar.

[0119] Example 2 The addition amount of the glucose oxidase is 520 U / g of the composition.

[0120] The addition amount of the lysozyme is 380 U / g of the composition.

[0121] The addition amount of the superoxide dismutase is 320 U / g of the composition.

[0122] Comparative Example 1 The difference between this comparative example and Example 1 is that: the active ingredients are glucose oxidase and lysozyme. The addition amount of the glucose oxidase is 500 U / g of the composition. The addition amount of the lysozyme is 400 U / g of the composition.

[0123] Comparative Example 2 The difference between this comparative example and Example 1 is that: the antioxidants are vitamin C and vitamin E, and the mass ratio is 5:2.

[0124] Comparative Example 3 The difference between this comparative example and Example 1 is that: the plant extract is centella asiatica extract and polygonum cuspidatum extract, and the mass ratio is 1:1.

[0125] Comparative Example 4 The difference between this comparative example and Example 1 is that: the preparation method of the centella asiatica extract has the following steps: B1. Wash the centella asiatica, dry it and crush it, add an ethanol aqueous solution with a mass concentration of 75%, adjust the temperature to 55 °C, and perform ultrasonic extraction for 45 min to obtain a crude centella asiatica extract; B2. Add activated carbon to the crude centella asiatica extract, stir at 40 °C for 25 min, and then filter to obtain the centella asiatica extract.

[0126] B3. Remove the alcohol from the centella asiatica extract, load it onto a macroporous resin, elute it with an ethanol aqueous solution with a mass concentration of 75%, collect the eluate, concentrate it under reduced pressure, and dry it to obtain the centella asiatica extract.

[0127] The mass ratio of the asiatic pennywort herb to the ethanol aqueous solution with a mass concentration of 75% is 1:25.

[0128] Comparative Example 5 The difference between this comparative example and Example 1 is that the polygonum cuspidatum fermenting agent is Lactobacillus bulgaricus and Streptococcus thermophilus.

[0129] The dosage of Lactobacillus bulgaricus is 10 8 CFU / g of polygonum cuspidatum fermentation substrate; the dosage of Streptococcus thermophilus is 10 8 CFU / g of polygonum cuspidatum fermentation substrate.

[0130] Comparative Example 6 The difference between this comparative example and Example 1 is the preparation method of the polygonum cuspidatum extract, and the steps are as follows: C1. Fermentation pretreatment: Wash the polygonum cuspidatum, dry and crush it, add water to adjust the water content to 50% to obtain the polygonum cuspidatum fermentation substrate, add the polygonum cuspidatum fermenting agent, and take it out after aerobic fermentation at 42°C for 8 days to obtain the polygonum cuspidatum pretreatment product; C2. Enzymolysis: Add the polygonum cuspidatum pretreatment product to water, adjust the temperature to 45°C and the pH to 5, add cellulase and pectinase, boil and inactivate after enzymolysis for 2.5 h to obtain the polygonum cuspidatum extraction filtrate; C3. Concentrate the polygonum cuspidatum extraction filtrate to 38% of the mass of the polygonum cuspidatum pretreatment product, filter, and then concentrate the filtrate to 27% of the mass of the polygonum cuspidatum pretreatment product, and filter to obtain the polygonum cuspidatum crude extract; C4. Add the polygonum cuspidatum crude extract to the top of the column. The column bed is filled with alumina, elute the crude extract with ethyl acetate at a flow rate of 1.5 mL / min, collect the eluate, concentrate under reduced pressure and dry to obtain the polygonum cuspidatum extract.

[0131] Comparative Example 7 The difference between this comparative example and Example 1 is that the carrier substance, by weight percentage, is 7% of a humectant, 0.08% of an antioxidant, 3% of a plant extract, 0.3% of carbomer, an appropriate amount of triethanolamine, and water is added to make up the balance to 100%.

[0132] The preparation method of the supramolecular nano coenzyme composition comprises the following steps: Add a thickening agent to water, heat to 70°C and stir evenly at a rate of 200 rpm until completely dissolved, then cool to 40°C, add the active substance and stir at a rate of 600 rpm for 70 min, add the antioxidant, stir for 30 min, add the plant extract, stir for 15 min, add the humectant, stir for 15 min, add a pH regulator to adjust the pH to 6, and perform homogenization treatment to obtain it.

[0133] Performance Test Refer to GB / T 39100-2020 "Determination of Polypeptide Antioxidant Activity - DPPH and ABTS Methods" to test the DPPH scavenging rate of the composition. The level of the DPPH scavenging rate reflects the level of antioxidant activity of the composition. Refer to GB / T 2738-2012 "Evaluation Methods for Antibacterial and Bacteriostatic Effects of Cosmetics and Toiletries" to test the bactericidal rate and bacteriostatic rate of the composition against Staphylococcus aureus ATCC 25923. The results are shown in Table 1.

[0134] Table 1 Test Results DPPH scavenging rate / % Bactericidal rate / % Bacteriostatic rate / % Example 1 95.94 92.35 99.60 Example 2 95.56 90.92 99.40 Comparative Example 1 92.17 89.22 98.54 Comparative Example 2 91.95 88.84 98.20 Comparative Example 3 92.25 84.02 96.51 Comparative Example 4 91.01 85.20 97.15 Comparative Example 5 92.84 87.16 97.48 Comparative Example 6 91.73 86.27 95.14 Comparative Example 7 85.46 79.63 90.01 According to statistics, for the supramolecular nano coenzyme compositions prepared in Examples 1-2 of the present invention, the DPPH scavenging rate, bactericidal rate and bacteriostatic rate are all relatively high, indicating that the supramolecular nano coenzyme composition has excellent antioxidant activity and also has antibacterial and anti-inflammatory effects. In Comparative Example 1, superoxide dismutase was not added; in Comparative Example 2, coenzyme Q10 was not added; in Comparative Example 3, green tea extract was not added; in Comparative Example 4, Centella asiatica was not fermented; in Comparative Example 5, Yarrowia lipolytica was not added for fermenting Polygonum cuspidatum; in Comparative Example 6, glucoamylase was not added for secondary enzymatic hydrolysis of the Polygonum cuspidatum pretreatment; in Comparative Example 7, vitamin E and coenzyme Q10 were not microemulsified. The DPPH scavenging rate, bactericidal rate and bacteriostatic rate of the prepared supramolecular nano coenzyme compositions are all relatively low. At the same time, 100 volunteers aged 16-50 with different skin problems were selected and randomly divided into two groups for the test. The test group used the supramolecular nano coenzyme composition prepared in Example 1, and the control group used a commercially available vitamin E emulsion. Skin problems included acne, spots, dull skin color, dryness and water shortage, fine lines, etc. They were used once in the morning and once in the evening according to their respective usage habits. After one month, it was found by comparison that the overall skin condition of the volunteers in the test group improved significantly. The acne gradually subsided, the acne marks faded, the skin became smooth, fair, tender and elastic, the spots became lighter, and the fine lines also decreased. However, there was no obvious change in the skin characteristics of the control group. Therefore, preparing the supramolecular nano coenzyme composition using the raw materials and methods described in this application not only improves the detoxification (antibacterial and anti-inflammatory), antioxidant and other effects of the product, but also can smooth the skin, and has beauty effects such as brightening, moisturizing, firming, anti-aging and lipolysis.

[0135] The above is the preferred implementation mode of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and retouches can still be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

Claims

1. A supramolecular nano coenzyme composition, characterized in that, Its preparation raw materials include active ingredients and carrier substances; the active ingredients are glucose oxidase, lysozyme, and superoxide dismutase; the addition amount of glucose oxidase is 450 - 550 U / g of the composition; the addition amount of lysozyme is 350 - 450 U / g of the composition; the addition amount of superoxide dismutase is 250 - 350 U / g of the composition; The carrier substances, by weight percentage, include 5% - 10% of a humectant, 0.05% - 0.1% of an antioxidant, 2% - 5% of a plant extract, 0.1% - 0.5% of a thickener, 3% - 7% of a surfactant, an appropriate amount of a pH regulator, and the balance is made up with water to 100%; The antioxidant includes vitamin C, vitamin E, and coenzyme Q10; the plant extract includes green tea extract, centella asiatica extract, and polygonum cuspidatum extract.

2. The supramolecular nano coenzyme composition according to claim 1, wherein The humectant includes one or more of glycerol, 1,2 - propanediol, sodium hyaluronate, and caprylic / capric triglyceride.

3. The supramolecular nano coenzyme composition according to claim 1, wherein The mass ratio of vitamin C, vitamin E, and coenzyme Q10 is (4 - 6):(1 - 3):

1.

4. The supramolecular nano coenzyme composition according to claim 1, wherein The mass ratio of green tea extract, centella asiatica extract, and polygonum cuspidatum extract is (0.8 - 1.2):(0.8 - 1.2):

1.

5. The supramolecular nano coenzyme composition according to claim 4, characterized in that, The preparation method of the green tea extract includes the following steps: Wash the green tea and then dry and crush it, add an ethanol aqueous solution with a mass concentration of 70% - 80%, extract at 70 - 80 °C for 30 - 50 min, filter, and then concentrate and dry the filtrate to obtain the green tea extract.

6. The supramolecular nano coenzyme composition according to claim 4, characterized in that, The preparation method of the centella asiatica extract includes the following steps: B1. Wash the centella asiatica and then dry and crush it, add water to adjust the water content to 45% - 55% to obtain a centella asiatica fermentation substrate, add a centella asiatica ferment, and perform aerobic fermentation at 40 - 45 °C until the pH ≤ 4, then take it out to obtain a centella asiatica pretreatment product; B2. Add the centella asiatica pretreatment product to an ethanol aqueous solution with a mass concentration of 70 - 80%, adjust the temperature to 50 - 60 °C, and perform ultrasonic extraction for 30 - 60 min to obtain a crude centella asiatica extract; B3. Add activated carbon to the crude centella asiatica extract, stir at 35 - 45 °C for 20 - 30 min, and then filter to obtain the centella asiatica extract; B4. Remove alcohol from the centella asiatica extract, load it onto a macroporous resin, elute with an ethanol aqueous solution with a mass concentration of 70% - 75%, collect the eluate, concentrate under reduced pressure, and dry to obtain the centella asiatica extract.

7. The supramolecular nano coenzyme composition according to claim 4, wherein The preparation method of the polygonum cuspidatum extract includes the following steps: C1. Fermentation pretreatment: Wash the polygonum cuspidatum and then dry and crush it to obtain polygonum cuspidatum powder, add water to adjust the water content to 45% - 55%, then add urea to obtain a polygonum cuspidatum fermentation substrate, add a polygonum cuspidatum ferment, and perform aerobic fermentation at 40 - 45 °C for 8 - 10 days, then take it out to obtain a polygonum cuspidatum pretreatment product; C2. Enzymatic hydrolysis: Add the pre-treated polygonum cuspidatum to water, adjust the temperature to 40 - 50 °C and the pH to 4.5 - 5.5, add cellulase and pectinase, inactivate by boiling after enzymatic hydrolysis for 2 - 3 h, continue to adjust the system temperature to 55 - 65 °C and the pH to 4 - 5, add glucoamylase, inactivate by boiling after enzymatic hydrolysis for 1 - 2 h, cool to room temperature and filter through a 0.22 μm microporous membrane to obtain the polygonum cuspidatum extraction filtrate; C3. Concentrate the polygonum cuspidatum extraction filtrate to 35% - 38% of the mass of the pre-treated polygonum cuspidatum, filter, and then concentrate the filtrate again to 25% - 30% of the mass of the pre-treated polygonum cuspidatum, filter to obtain the crude polygonum cuspidatum extract; C4. Add the crude polygonum cuspidatum extract to the top of the column, the column bed is filled with alumina, elute the crude extract with ethyl acetate at a flow rate of 1 - 2 mL / min, collect the eluate, concentrate under reduced pressure and dry to obtain the polygonum cuspidatum extract.

8. The supramolecular nano coenzyme composition according to claim 7, characterized in that, The polygonum cuspidatum ferment includes Lactobacillus bulgaricus, Yarrowia lipolytica, and Streptococcus thermophilus.

9. A method for preparing a supramolecular nano coenzyme composition according to any one of claims 1-8, characterized in that, It includes the following steps: S1. Prepare the microemulsion: Add the surfactant to an appropriate amount of water, stir at a rate of 150 - 250 rpm at 35 - 40 °C for 30 - 40 min until completely dissolved, then raise the temperature to 70 - 80 °C, add vitamin E and coenzyme Q10, and treat with a high-speed shear homogenizer at 4000 - 6000 rpm for 10 - 15 min to obtain it; S2. Add the thickener to the remaining water, raise the temperature to 70 °C and stir evenly at a rate of 150 - 250 rpm until completely dissolved, then cool to 40 °C, add the active substance and stir at a rate of 500 - 700 rpm for 60 - 80 min, add vitamin C and the microemulsion, stir for 25 - 35 min, add the plant extract, stir for 10 - 20 min, add the humectant, stir for 10 - 20 min, add the pH regulator to adjust the pH to 5.5 - 6.5, and perform homogenization treatment to obtain it.

10. Use of the supramolecular nano coenzyme composition according to any one of claims 1 - 8 in the preparation of skin care products.