A traditional Chinese medicine protein peptide composition with whitening, anti-aging and moisturizing effects, a preparation method thereof and an application thereof in cosmetics

Abstract

The present invention discloses a traditional Chinese medicine protein peptide composition with whitening, anti-aging and moisturizing effects, a preparation method thereof and its application in cosmetics. The traditional Chinese medicine protein peptide composition is composed of polygonatum protein peptide and odoratum protein peptide with a molecular weight below 10 KDa obtained by an enzymatic hydrolysis method, and the two protein peptides have a synergistic effect on whitening, anti-aging and moisturizing effects. The present invention also provides a preparation method for the above-mentioned polygonatum protein peptide and odoratum protein peptide. Through multiple enzymatic hydrolysis techniques and optimization of the preparation process, a mild and environmentally friendly production process is realized, the biological activity of the traditional Chinese medicine protein peptide is maintained to the greatest extent, and at the same time the purity of the protein peptide is improved to ensure no irritation. The traditional Chinese medicine protein peptide composition is applied to cosmetics of different dosage forms such as aqueous solutions, lotions, creams, essences, etc., with wide applications, providing a multifunctional, non-irritating and natural mild raw material choice for the cosmetics industry, and meeting the urgent needs of consumers for safe, efficient and environmentally friendly cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics. Specifically, it relates to a traditional Chinese medicine protein peptide composition with whitening, anti-aging and moisturizing effects, a preparation method thereof, and its application in cosmetics. Background Art

[0002] In the rapidly changing beauty and health market, consumers' demands for cosmetics have far exceeded basic cleansing and moisturizing, and instead they pursue safer, more efficient, multi-functional and environmentally friendly products. This trend has prompted the cosmetics industry to continuously explore new raw material sources and preparation technologies to meet the growing personalized needs of consumers. Traditional Chinese medicine, a natural resource rich in bioactive ingredients and with a long application history, is gradually becoming a new hot spot in cosmetics research and development.

[0003] According to the "National Food Safety Standard Plant Protein Peptides for Food Processing" (GB 31611-2023), traditional Chinese medicine protein peptides are products mainly composed of peptides with a relative molecular mass ranging from 189 to 10,000, which are produced from traditional Chinese medicine materials rich in protein through processes such as extraction, enzymatic hydrolysis and / or microbial fermentation, filtration, sterilization, and drying. These peptides are usually composed of 2 to 50 amino acid residues, with small molecular weights, rich varieties, and characteristics such as easy cell transport, high absorption and utilization rate, short half-life, low toxicity and immunogenicity, and diverse biological activities. Therefore, they show excellent application potential in the fields of food and cosmetics. For example, protein peptides extracted from traditional Chinese medicines such as ginseng, astragalus, and Chinese yam can be used to develop products that enhance the body's resistance due to their function of regulating the immune system. Protein peptides extracted from medicinal materials such as wolfberry and hawthorn can not only provide additional nutritional supplements but also improve the taste and flavor of food; protein peptide substances extracted from medicinal materials such as ganoderma lucidum, ginseng, and schisandra chinensis have antioxidant activity and can effectively resist the damage of free radicals to the skin and delay the skin aging process. In short, traditional Chinese medicine protein peptides, extracted and enzymatically hydrolyzed from traditional Chinese medicine materials through modern biotechnology, show great application potential in the fields of food and cosmetics due to their small molecular weight, easy absorption, and biological activities such as immune regulation, antioxidant, and free radical elimination.

[0004] Polygonatum sibiricum Red., Polygonatum kingianum Coll. et Hemsl., or Polygonatum cyrtonema Hua of the Liliaceae family, the dry rhizomes of which are used as Huangjing (Polygonatum). Polygonatum odoratum (Mill.) Druce of the Liliaceae family, the dry rhizome of which is used as Yuzhu (Polygonatum odoratum). As traditional Chinese medicines, Huangjing and Yuzhu have the effects of nourishing yin and moistening the lungs, beautifying the face and other effects in traditional Chinese medicine theory. Modern research shows that both Huangjing and Yuzhu contain rich active ingredients such as polysaccharides, proteins, and amino acids, and these ingredients play an important role in improving skin condition and enhancing skin immunity. In particular, Huangjing protein peptide and Yuzhu protein peptide obtained by modern biotechnology extraction and enzymatic hydrolysis have reduced molecular weight through enzymatic hydrolysis technology, improving skin permeability and bioavailability.

[0005] Both Huangjing and Yuzhu contain a lot of protein and polysaccharide components, and the two are often combined into glycoproteins. Glycoproteins are biological macromolecules formed by the combination of sugars and proteins. To obtain the protein peptides of Huangjing and Yuzhu, the glycoproteins need to be decomposed into sugars and proteins, the sugars removed, and the resulting proteins further enzymatically hydrolyzed. At present, there is little research on the extraction and preparation process of Huangjing protein peptide and Yuzhu protein peptide, and there are certain problems. For example, extreme conditions such as high temperature, high pressure, strong acid and strong base chemical hydrolysis, and electrification are used in the preparation process, which cannot guarantee the activity of the protein peptide; or the crude protein is not extracted in advance, but the traditional Chinese medicine liquid is directly enzymatically hydrolyzed and then filtered, and the final product contains a large number of other components with molecular weights close to those of the protein peptides, resulting in a low purity of the obtained protein peptides.

[0006] CN 110934837A discloses a Polygonatum sibiricum polypeptide composite tablet and its preparation method. The preparation method of the Polygonatum sibiricum polypeptide involves adding a Polygonatum sibiricum extract to an expansion solvent composed of CO2 and ethanol, pressurizing it to 1.5-2.5 MPa to form an expansion system for high-pressure extraction. CN 106083990A discloses a Polygonatum sibiricum peptide extraction method. The preparation method of the Polygonatum sibiricum peptide involves passing an electric current through a Polygonatum sibiricum stock solution to obtain a precipitate, which is then prepared using a specific method to obtain the Polygonatum sibiricum peptide. Whether the current passing method can improve preparation efficiency and ensure the activity of the peptide remains to be discussed. CN 108034682 A discloses a process for extracting a Polygonatum sibiricum solid small molecule peptide, which includes not only dual-frequency composite ultrasound and microwave radiation alternating assisted extraction, but also supercritical CO2 high-pressure extraction. The extraction conditions are relatively extreme. CN 116649571 A discloses a Polygonatum sibiricum peptide oral solution and its preparation method. However, according to the preparation method described in the patent application, the resulting extract should be a mixture of Polygonatum odoratum polysaccharides and Polygonatum odoratum polypeptides, primarily composed of polysaccharides, with relatively low levels of proteins and peptides. Studies have examined the microwave extraction and antioxidant effects of Polygonatum odoratum glycoprotein, yielding an 8.85% yield. Studies have shown that Polygonatum odoratum glycoprotein possesses significant reducing power and the ability to scavenge DPPH free radicals. However, glycoproteins are macromolecular complexes composed of sugars and proteins linked by some mechanism, significantly different from smaller protein peptides.

[0007] In summary, current preparation technologies for TCM protein peptides and their application in cosmetics face several challenges. For one thing, the harsh conditions commonly used in the preparation process, such as high temperature, high pressure, acid-base hydrolysis, and electrical current, often damage the peptide's natural structure and bioactivity, thereby weakening its skincare benefits. Furthermore, most TCM protein peptide products on the market contain numerous impurities and lack purity. Furthermore, the functional limitations of individual TCM protein peptides make it difficult to meet consumers' expectations for multifunctional cosmetics. Furthermore, the safety of cosmetic raw materials has always been a focus of the industry, and ensuring that TCM protein peptides are both safe and non-irritating is a key issue that needs to be addressed.

[0008] In view of the above background, it is of great significance to provide a multifunctional Chinese herbal protein peptide composition. At the same time, it is particularly urgent to develop a Chinese herbal protein peptide composition that can overcome the shortcomings of existing technologies, adopt a mild and environmentally friendly preparation process, and retain the biological activity of Chinese herbal protein peptides to the greatest extent. Summary of the Invention

[0009] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a traditional Chinese medicine protein peptide composition with whitening, anti-aging and moisturizing effects.

[0010] The second object of the present invention is to provide a preparation method of the traditional Chinese medicine protein peptide composition.

[0011] The third object of the present invention is to provide the application of the traditional Chinese medicine protein peptide composition in cosmetics.

[0012] The above objects of the present invention are achieved by the following technical solutions:

[0013] The present invention first provides a traditional Chinese medicine protein peptide composition, which is composed of polygonatum sibiricum protein peptide and odoratum officinale protein peptide with a mass ratio of 1-3:1; the preparation methods of the polygonatum sibiricum protein peptide and the odoratum officinale protein peptide are as follows: first, use a composite enzyme to enzymatically hydrolyze to obtain crude protein, and then the crude protein is enzymatically hydrolyzed twice with a composite alkaline protein and pepsin to obtain small molecular peptides with a molecular weight below 10KDa. The composite enzyme is α-mannosidase, β-galactosidase, α-glucosidase and β-glucosidase, and the composite alkaline protein is trypsin and papain.

[0014] The present invention proposes a traditional Chinese medicine protein peptide composition with a scientific ratio, and polygonatum sibiricum protein peptide and odoratum officinale protein peptide are obtained by three enzymatic hydrolysis methods. Specifically, for the first enzymatic hydrolysis, the main purpose is to hydrolyze glycoprotein and decompose glycoprotein into sugar and protein. The main purpose of the second enzymatic hydrolysis is to decompose macromolecular proteins into smaller peptide segments. The third enzymatic hydrolysis is to further decompose medium-sized peptide segments into small molecular peptides on the basis of the second enzymatic hydrolysis. The protein peptides obtained by this preparation process have a relatively high purity. The content range of polygonatum sibiricum protein peptide is between 83.6% and 87.3%, and the content range of odoratum officinale protein peptide is between 85.5% and 88.1%. Further combining polygonatum sibiricum protein peptide and odoratum officinale protein peptide in a specific ratio, it is found that the combination of the two protein peptides has a synergistic effect, significantly enhancing multiple skin care effects such as whitening, anti-aging and moisturizing. Specifically, at the same dosage, the traditional Chinese medicine protein peptide composition with a specific ratio of polygonatum sibiricum protein peptide and odoratum officinale protein peptide has higher whitening, anti-aging and moisturizing effects than single protein peptides.

[0015] Furthermore, the preparation method of the polygonatum sibiricum protein peptide and the odoratum officinale protein peptide includes the following steps:

[0016] S1. Hydrothermally extract the traditional Chinese medicine raw materials to obtain a traditional Chinese medicine extract;

[0017] S2. Use a composite enzyme to perform primary enzymatic hydrolysis on the traditional Chinese medicine extract to obtain traditional Chinese medicine crude protein; the composite enzyme is α-mannosidase, β-galactosidase, α-glucosidase and β-glucosidase;

[0018] S3. Use a composite alkaline protease to perform secondary enzymatic hydrolysis on the traditional Chinese medicine crude protein solution to obtain a secondary enzymatic hydrolysate; the composite alkaline protease is trypsin and papain;

[0019] S4. Use pepsin to perform a third enzymatic hydrolysis on the second enzymatic hydrolysate to obtain a third enzymatic hydrolysate;

[0020] S5. Ultrafilter the third enzymatic hydrolysate using a membrane with a molecular weight cut-off of 10 KDa and perform post-treatment to obtain the traditional Chinese medicine protein peptide;

[0021] The traditional Chinese medicine raw material is polygonatum odoratum or polygonatum sibiricum.

[0022] Therefore, by optimizing the preparation process, the present invention prepares protein peptides by means of three enzymatic hydrolyses, avoiding the destruction of components under extreme conditions. The operation is simple, mild and environmentally friendly, which helps to maintain the biological activity of the protein peptides and improves the purity of the protein peptides. The obtained protein peptides are safe, non-irritating and have a relatively high purity.

[0023] Further, step S1 is to crush the traditional Chinese medicine raw material, immerse the polygonatum sibiricum powder in distilled water according to the mass ratio of material to liquid of 1:13 - 17, and decoct for 2 - 2.5 hours to obtain a traditional Chinese medicine extract.

[0024] Preferably, step S1 is to crush the traditional Chinese medicine raw material, immerse the polygonatum sibiricum powder in distilled water according to the mass ratio of material to liquid of 1:15, and decoct for 2 hours to obtain a traditional Chinese medicine extract.

[0025] Further, step S2 is to adjust the pH value of the extract to 6.0 - 8.0, add a complex enzyme, perform enzymatic hydrolysis at 50 °C for 3 - 7 hours to obtain a first enzymatic hydrolysate, and inactivate the enzyme; centrifuge the first enzymatic hydrolysate to take the supernatant, and perform alcohol precipitation to take the precipitate to obtain the traditional Chinese medicine crude protein.

[0026] Preferably, the mass ratio of α-mannosidase, β-galactosidase, α-glucosidase and β-glucosidase in the complex enzyme is 0.8 - 1.2:0.8 - 1.2:0.8 - 1.2:0.8 - 1.2.

[0027] Preferably, the mass ratio of α-mannosidase, β-galactosidase, α-glucosidase and β-glucosidase in the complex enzyme is 1:1:1:1.

[0028] Further, step S3 is to mix the traditional Chinese medicine crude protein and distilled water according to the mass ratio of 1:13 - 17, stir evenly, heat up to 45 - 50 °C, add a complex alkaline protease, adjust the pH value to 7.0 - 9.0, and stir and enzymatically hydrolyze for 2.0 - 6.0 hours to obtain a second enzymatic hydrolysate.

[0029] Preferably, step S3 is to mix the traditional Chinese medicine crude protein and distilled water according to the mass ratio of 1:15, stir evenly, heat up to 50 °C, add a complex alkaline protease, adjust the pH value to 7.0 - 9.0, and stir and enzymatically hydrolyze for 2.0 - 6.0 hours to obtain a second enzymatic hydrolysate.

[0030] Preferably, the mass ratio of trypsin to papain in the composite alkaline protease is 0.8 - 1.2:0.8 - 1.2.

[0031] Preferably, the mass ratio of trypsin to papain in the composite alkaline protease is 1:1.

[0032] Further, step S4 is to add pepsin to the second enzymatic hydrolysate, adjust the pH to 2.0 - 3.5, perform the third enzymatic hydrolysis for 2.0 - 6.0 hours, inactivate the enzyme, and obtain the third enzymatic hydrolysate.

[0033] Further, when the traditional Chinese medicine raw material is polygonatum sibiricum, the dosage of the composite enzyme in step S2 is 1 - 3% of the mass of the traditional Chinese medicine extraction solution; the dosage of the composite alkaline protease in step S3 is 1 - 5% of the mass of the traditional Chinese medicine crude protein solution; the dosage of pepsin in step S4 is 1 - 5% of the mass of the second enzymatic hydrolysate.

[0034] Further, when the traditional Chinese medicine raw material is odoratum, the dosage of the composite enzyme in step S2 is 1.5 - 3.5% of the mass of the traditional Chinese medicine extraction solution; the dosage of the composite alkaline protease in step S3 is 1.5 - 5.5% of the mass of the traditional Chinese medicine crude protein solution; the dosage of pepsin in step S4 is 1.5 - 5.5% of the mass of the second enzymatic hydrolysate.

[0035] The present invention provides the application of the traditional Chinese medicine protein peptide composition in the preparation of whitening, anti - aging and moisturizing cosmetics.

[0036] Further, the cosmetics are aqueous solutions, lotions, creams or serums.

[0037] The traditional Chinese medicine protein peptide composition is not only suitable for cosmetics in various dosage forms such as aqueous solutions, lotions, creams, serums, etc., but also provides a new, multi - functional and non - irritating raw material choice for the cosmetics industry, fully meeting the urgent needs of consumers for natural, safe and environmentally friendly cosmetics.

[0038] The present invention provides a cosmetic containing the above - mentioned traditional Chinese medicine protein peptide composition.

[0039] Further, the mass percentage content of the traditional Chinese medicine protein peptide composition in the cosmetics is 0.1 - 5%.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention provides a traditional Chinese medicine protein peptide composition, which is composed of polygonatum sibiricum protein peptide and odoratum officinale protein peptide. Both the polygonatum sibiricum protein peptide and the odoratum officinale protein peptide are protein peptides with a molecular weight below 10 KDa obtained by triple enzymatic hydrolysis. The traditional Chinese medicine protein peptide composition is obtained by mixing two raw materials in an appropriate ratio. This traditional Chinese medicine protein peptide composition not only has various skin care effects such as whitening, anti-aging, and moisturizing, but also the two protein peptide raw material components act synergistically, and the effect is significantly better than using only one of them. The present invention also provides a preparation method for the above-mentioned polygonatum sibiricum protein peptide and odoratum officinale protein peptide. Through multiple enzymatic hydrolysis techniques, the preparation process is optimized to achieve a mild and environmentally friendly production process, maximizing the preservation of the biological activity of the traditional Chinese medicine protein peptide, while improving the purity of the protein peptide to ensure no irritation. The preparation method is simple to operate, with mild and environmentally friendly conditions and is easy to produce. Compared with most chemically synthesized raw materials used in cosmetics, this traditional Chinese medicine protein peptide composition has the advantages of no irritation to the skin, being mild, safe, and having no toxic side effects. It is applied to cosmetics in different dosage forms such as aqueous solutions, lotions, creams, and essences, with a wide range of uses, providing a multifunctional, non-irritating, natural, and mild raw material choice for the cosmetics industry, meeting the urgent needs of consumers for safe, efficient, and environmentally friendly cosmetics. Detailed implementation mode

[0042] Specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0044] α-Mannosidase (product number: M923745-1mg; ≥15 units / mg protein (biuret)), β-Galactosidase (product number: S10066-1KU; purity / concentration: BR, 250-600 u / mg protein), α-Glucosidase (product number: S10050-1ku; purity level: 50 units / mg protein yeast), β-Glucosidase (product number: G824159-10mg; purity / concentration: 10-30 U / mg, source: almond), Trypsin (product number: S10034-100g; purity / concentration: BR; 1:250), Papain (product number: P815680-500g; purity level: ≥3 units / mg), Pepsin (product number: 9001-75-6; specification: >3000 U / mg).

[0045] Example 1

[0046] (1) Preparation of polygonatum sibiricum protein peptide

[0047] S1. Clean the polygonatum officinale herbs, dry them, crush them, sieve them through a 120-mesh sieve, soak the polygonatum powder in distilled water according to the mass ratio of material to liquid of 1:15, stir evenly, and decoct at 90 °C for 2 hours to obtain the polygonatum extract.

[0048] S2. Adjust the pH value of the extract to 6.0, add a composite enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase, and the mass ratio of the four is 1:1:1:1) equivalent to 1.0% of the mass of the mixed solution, enzymatically hydrolyze at 50 °C for 3.0 hours to obtain the first enzymolysis solution, heat at 90 °C for 5 minutes to inactivate the enzyme and terminate the reaction. Centrifuge the first enzymolysis solution at 6000 rpm for 20 minutes and take the supernatant. Adjust the temperature to 20 °C, add ethanol equivalent to 70% of its volume to the supernatant, stir evenly, and place the mixed solution in a refrigerator at 4 °C for 24 hours. Centrifuge at 3500 rpm for 10 minutes to obtain the precipitate, which is the crude polygonatum protein.

[0049] S3. Mix the crude polygonatum protein and distilled water according to the mass ratio of 1:15, stir evenly, heat up to 50 °C, add a composite alkaline protease (a mixture of trypsin and papain, with a mass ratio of 1:1) equivalent to 1.0% of the mass of the protein solution, adjust the pH value to 7.0, and stir and enzymatically hydrolyze for 2.0 hours to obtain the second enzymolysis solution.

[0050] S4. After the enzymatic hydrolysis is completed, add pepsin equivalent to 1.0% of the mass of the solution to the second enzymolysis solution, adjust the pH to 2.0, and perform the third enzymatic hydrolysis for 2.0 hours. Heat the third enzymolysis solution to 90 °C and inactivate the enzyme for 15 minutes to terminate the reaction.

[0051] S5. Centrifuge and purify the inactivated enzymolysis solution with a centrifuge at a speed of 15000 r / min to obtain the centrifugate. Filter the centrifugate through an ultrafiltration membrane with a molecular weight cut-off of 10 KDa, concentrate the filtrate, and freeze-dry to obtain polygonatum protein peptide.

[0052] (2) Preparation of polygonatum odoratum protein peptide

[0053] Refer to the above method for preparing polygonatum protein peptide, with the differences being: in S2, add a composite enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase, and the mass ratio of the four is 1:1:1:1) equivalent to 1.5% of the mass of the mixed solution; in S3, add a composite alkaline protease (a mixture of trypsin and papain, with a mass ratio of 1:1) equivalent to 1.5% of the mass of the protein solution; in S4, add pepsin equivalent to 1.5% of the mass of the solution.

[0054] (3) Preparation of traditional Chinese medicine protein peptide composition

[0055] Mix the frozen powders of polygonatum sibiricum protein peptide and odoratum protein peptide evenly according to the weight ratio of 1:1 to obtain the traditional Chinese medicine protein peptide composition.

[0056] Example 2

[0057] (1) Preparation of polygonatum sibiricum protein peptide

[0058] S1. Wash the polygonatum sibiricum medicinal materials, dry them, crush them and pass through a 120-mesh sieve. Immerse the polygonatum sibiricum powder in distilled water according to the material-liquid mass ratio of 1:15, stir evenly, and decoct at 90 °C for 2 hours to obtain the polygonatum sibiricum extract.

[0059] S2. Adjust the pH value of the extract to 6.5, add a composite enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase, and the mass ratio of the four is 1:1:1:1) equivalent to 1.5% of the mass of the mixed solution, and enzymatically hydrolyze at a temperature of 50 °C for 4.0 hours to obtain the first enzymatic hydrolysate. Heat at 90 °C for 5 minutes to inactivate the enzyme and terminate the reaction. Centrifuge the first enzymatic hydrolysate at a speed of 6000 rpm for 20 minutes and take the supernatant. Adjust the temperature to 20 °C, add 70% of its volume of ethanol to the supernatant, stir evenly, and place the mixed solution in a refrigerator at 4 °C for 24 hours. Centrifuge at a speed of 3500 rpm for 10 minutes to obtain the precipitate, which is the crude polygonatum sibiricum protein.

[0060] S3. Mix the crude polygonatum sibiricum protein and distilled water according to the mass ratio of 1:15, stir evenly, heat up to 50 °C, add a composite alkaline protease (a mixture of trypsin and papain, and the mass ratio is 1:1) equivalent to 2.0% of the mass of the protein solution, adjust the pH value to 7.5, and stir and enzymatically hydrolyze for 3.0 hours to obtain the second enzymatic hydrolysate.

[0061] S4. After the enzymatic hydrolysis is completed, add pepsin equivalent to 2.0% of the mass of the solution to the second enzymatic hydrolysate, adjust the pH to 2.5, and perform the third enzymatic hydrolysis for 3.0 hours. Heat the third enzymatic hydrolysate to 90 °C and inactivate the enzyme for 15 minutes to terminate the reaction.

[0062] S5. Centrifuge and purify the inactivated enzymatic hydrolysate with a centrifuge at a speed of 15000 r / min to obtain the centrifugate. Filter the centrifugate through an ultrafiltration membrane with a molecular weight cut-off of 10 KDa, concentrate the filtrate, and freeze-dry it to obtain the polygonatum sibiricum protein peptide.

[0063] (2) Preparation of odoratum protein peptide [[ID=**26]]

[0064] Referring to the above-mentioned method for preparing yellow protamine peptide, the difference is that: in S2, a composite enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase, the mass ratio of the four is 1:1:1:1) equivalent to 2.0% of the mass of the mixed solution is added; in S3, a composite alkaline protease (a mixture of trypsin and papain, the mass ratio is 1:1) equivalent to 2.5% of the mass of the protein solution is added; in S4, pepsin equivalent to 2.5% of the mass of the solution is added to the second enzymatic hydrolysis solution.

[0065] (3) Preparation of Chinese medicine protein peptide compositions

[0066] The frozen powders of Polygonatum protein peptide and Polygonatum protein peptide are evenly mixed in a weight ratio of 1.5:1 to obtain a traditional Chinese medicine protein peptide composition.

[0067] Example 3

[0068] (1) Preparation of Polygonatum peptide

[0069] S1. Wash and dry the Polygonatum sibiricum, grind it, and pass it through a 120-mesh sieve. Soak the Polygonatum sibiricum powder in distilled water at a material-to-liquid ratio of 1:15, stir evenly, and boil at 90°C for 2 hours to obtain the Polygonatum sibiricum extract.

[0070] S2. The pH value of the extract was adjusted to 7.0, and a composite enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase, with a mass ratio of 1:1:1:1) equivalent to 2.0% of the mass of the mixed solution was added thereto. The solution was hydrolyzed at a temperature of 50°C for 5.0 hours to obtain the first hydrolyzate, which was heated at 90°C for 5 minutes to kill the enzyme and terminate the reaction. The first hydrolyzate was centrifuged at 6000rpm for 20 minutes, and the supernatant was taken. The temperature was adjusted to 20°C, 70% of the volume of ethanol was added to the supernatant, stirred evenly, and the mixed solution was placed under refrigeration at 4°C for 24 hours. The solution was centrifuged at 3500rpm for 10 minutes to obtain the precipitate, and the crude protein of Polygonatum sibiricum was obtained.

[0071] S3. Mix the crude protein of Polygonatum sibiricum with distilled water in a mass ratio of 1:15, stir evenly, raise the temperature to 50°C, add a composite alkaline protease (a mixture of trypsin and papain, mass ratio of 1:1) equivalent to 3.0% of the protein solution mass, adjust the pH value to 8.0, stir and hydrolyze for 4.0 hours to obtain the second enzymatic hydrolyzate.

[0072] S4. After the enzymatic hydrolysis is completed, pepsin equivalent to 3.0% of the solution mass is added to the second enzymatic hydrolysis solution, the pH is adjusted to 2.8, and the third enzymatic hydrolysis is performed for 4.0 hours. The third enzymatic hydrolysis solution is heated to 90°C and the enzyme is inactivated for 15 minutes to terminate the reaction.

[0073] S5. Centrifuge the enzyme-hydrolyzed solution after inactivating the enzyme at a rotational speed of 15,000 r / min for purification to obtain a centrifugate. Filter the centrifugate through an ultrafiltration membrane with a molecular weight cut-off of 10 KDa, concentrate the filtrate, and perform freeze-drying to obtain polygonatum protein peptide.

[0074] (2) Preparation of odoratum protein peptide

[0075] Referring to the above method for preparing polygonatum protein peptide, the differences are as follows: In S2, add a complex enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase, with a ratio of 1:1:1:1 for the four) equivalent to 2.5% of the mass of the mixed solution; in S3, add a complex alkaline protease (a mixture of trypsin and papain with a mass ratio of 1:1) equivalent to 3.5% of the mass of the protein solution; in S4, add pepsin equivalent to 3.5% of the mass of the solution.

[0076] (3) Preparation of traditional Chinese medicine protein peptide composition

[0077] Mix the freeze-dried powders of polygonatum protein peptide and odoratum protein peptide evenly according to a weight ratio of 2:1 to obtain the traditional Chinese medicine protein peptide composition.

[0078] Example 4

[0079] (1) Preparation of polygonatum protein peptide

[0080] S1. Clean and dry the polygonatum medicinal materials, crush them and pass through a 120-mesh sieve. Immerse the polygonatum powder in distilled water according to a material-liquid mass ratio of 1:15, stir evenly, and decoct at 90°C for 2 hours to obtain a polygonatum extract.

[0081] S2. Adjust the pH value of the extract to 7.5, add a complex enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase, with a mass ratio of 1:1:1:1 for the four) equivalent to 2.5% of the mass of the mixed solution, enzymatically hydrolyze at a temperature of 50°C for 6.0 hours to obtain a first enzyme-hydrolyzed solution, heat at 90°C for 5 minutes to inactivate the enzyme and terminate the reaction. Centrifuge the first enzyme-hydrolyzed solution at a rotational speed of 6,000 rpm for 20 minutes, and take the supernatant. Adjust the temperature to 20°C, add ethanol with a volume of 70% of the supernatant, stir evenly, and place the mixed solution in a refrigerated condition at 4°C for 24 hours. Centrifuge at a rotational speed of 3,500 rpm for 10 minutes to take the precipitate, and obtain polygonatum crude protein.

[0082] S3. Mix the polygonatum crude protein and distilled water according to a mass ratio of 1:15, stir evenly, raise the temperature to 50°C, add a complex alkaline protease (a mixture of trypsin and papain with a mass ratio of 1:1) equivalent to 4.0% of the mass of the protein solution, adjust the pH value to 8.5, and stir and enzymatically hydrolyze for 5.0 hours to obtain a second enzyme-hydrolyzed solution.

[0083] S4. After the enzymatic hydrolysis is completed, pepsin equivalent to 4.0% of the solution mass is added to the second enzymatic hydrolysis solution, the pH is adjusted to 3.0, and the third enzymatic hydrolysis is performed for 5.0 hours. The third enzymatic hydrolysis solution is heated to 90°C and the enzyme is inactivated for 15 minutes to terminate the reaction.

[0084] S5. The enzyme-inactivated hydrolyzate is centrifuged at 15,000 rpm to obtain a centrifuge. The centrifuge is filtered through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The filtrate is concentrated and freeze-dried to obtain polyanthocyanin peptide.

[0085] (2) Preparation of Polygonatum protein peptide

[0086] Referring to the above-mentioned method for preparing yellow protamine peptide, the difference is that: S2 is added with a composite enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase, the ratio of the four is 1:1:1:1) equivalent to 3.0% of the mass of the mixed solution; S3 is added with a composite alkaline protease (a mixture of trypsin and papain, the mass ratio is 1:1) equivalent to 4.5% of the mass of the protein solution; S4 is added with pepsin equivalent to 4.5% of the mass of the solution.

[0087] (3) Preparation of Chinese medicine protein peptide compositions

[0088] The frozen powders of Polygonatum protein peptide and Polygonatum protein peptide are evenly mixed in a weight ratio of 2.5:1 to obtain a traditional Chinese medicine protein peptide composition.

[0089] Example 5

[0090] (1) Preparation of Polygonatum peptide

[0091] S1. Wash and dry the Polygonatum sibiricum, grind it, and pass it through a 120-mesh sieve. Soak the Polygonatum sibiricum powder in distilled water at a material-to-liquid ratio of 1:15, stir evenly, and boil at 90°C for 2 hours to obtain the Polygonatum sibiricum extract.

[0092] S2. The pH value of the extract was adjusted to 8.0, and a composite enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase, with a mass ratio of 1:1:1:1) equivalent to 3.0% of the mass of the mixed solution was added thereto. The solution was enzymolyzed at a temperature of 50°C for 7.0 hours to obtain the first enzymolysis solution, which was heated at 90°C for 5 minutes to kill the enzyme and terminate the reaction. The first enzymolysis solution was centrifuged at 6000rpm for 20 minutes, and the supernatant was taken. The temperature was adjusted to 20°C, 70% of the volume of ethanol was added to the supernatant, stirred evenly, and the mixed solution was placed under refrigeration at 4°C for 24 hours. The crude protein of Polygonatum sibiricum was obtained by centrifugation at 3500rpm for 10 minutes.

[0093] S3. Mix the crude polygonatum protein with distilled water at a mass ratio of 1:15, stir evenly, heat up to 50 °C, add a compound alkaline protease (a mixture of trypsin and papain, with a mass ratio of 1:1) equivalent to 5.0% of the mass of the protein solution, adjust the pH value to 9.0, and stir for enzymatic hydrolysis for 6.0 hours to obtain the second enzymatic hydrolysate.

[0094] S4. After the enzymatic hydrolysis is completed, add pepsin equivalent to 5.0% of the mass of the solution to the second enzymatic hydrolysate, adjust the pH to 3.5, and perform the third enzymatic hydrolysis for 6.0 hours. Heat the third enzymatic hydrolysate to 90 °C to inactivate the enzyme for 15 minutes to terminate the reaction.

[0095] S5. Centrifuge and purify the enzyme-inactivated enzymatic hydrolysate with a centrifuge at a rotational speed of 15000 r / min to obtain the centrifugate. Filter the centrifugate through an ultrafiltration membrane with a molecular weight cut-off of 10 KDa, concentrate the filtrate, and freeze-dry it to obtain polygonatum protein peptide.

[0096] (2) Preparation of polygonatum odoratum protein peptide

[0097] Refer to the above method for preparing polygonatum protein peptide, with the differences being: in S2, add a compound enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase, with a ratio of 1:1:1:1 for the four) equivalent to 3.5% of the mass of the mixed solution; in S3, add a compound alkaline protease (a mixture of trypsin and papain, with a mass ratio of 1:1) equivalent to 5.5% of the mass of the protein solution; in S4, add pepsin equivalent to 5.5% of the mass of the solution.

[0098] (3) Preparation of traditional Chinese medicine protein peptide composition

[0099] Mix the frozen powders of polygonatum protein peptide and polygonatum odoratum protein peptide evenly according to a weight ratio of 3:1 to obtain the traditional Chinese medicine protein peptide composition.

[0100] Comparative Example 1

[0101] The difference between Comparative Example 1 and Example 3 is that in the preparation process of polygonatum protein peptide and polygonatum odoratum protein peptide, only use a compound enzyme (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase) and a compound alkaline protease for two enzymatic hydrolyses, and do not use pepsin for the third enzymatic hydrolysis, that is, omit step S4, and the other preparation conditions and the dosage ratio of polygonatum protein peptide and polygonatum odoratum protein peptide in the traditional Chinese medicine protein peptide composition are the same as those in Example 3.

[0102] Comparative Example 2

[0103] The difference between Comparative Example 2 and Example 3 is that in the preparation process of polygonatum protein peptide and odoratum protein peptide, only the complex enzymes (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase) and pepsin are used for two enzymatic hydrolyses, and the complex alkaline protease is not used for enzymatic hydrolysis, that is, step S3 is omitted, and the other preparation conditions and the dosage ratio of polygonatum protein peptide and odoratum protein peptide in the traditional Chinese medicine protein peptide composition are the same as those in Example 3.

[0104] Comparative Example 3

[0105] The difference between Comparative Example 3 and Example 3 is that in the preparation process of polygonatum protein peptide and odoratum protein peptide, only the complex alkaline protease and pepsin are used for two enzymatic hydrolyses, and the complex enzymes (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase) are not used for enzymatic hydrolysis, that is, step S2 is omitted, and the other preparation conditions and the dosage ratio of polygonatum protein peptide and odoratum protein peptide in the traditional Chinese medicine protein peptide composition are the same as those in Example 3.

[0106] Comparative Example 4

[0107] The difference between Comparative Example 4 and Example 3 is that in the preparation process of polygonatum protein peptide and odoratum protein peptide, only the complex enzymes (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase) are used for enzymatic hydrolysis, and the complex alkaline protease and pepsin are not used for enzymatic hydrolysis, that is, steps S3 and S4 are omitted, and the other preparation conditions and the dosage ratio of polygonatum protein peptide and odoratum protein peptide in the traditional Chinese medicine protein peptide composition are the same as those in Example 3.

[0108] Comparative Example 5

[0109] The difference between Comparative Example 5 and Example 3 is that in the preparation process of polygonatum protein peptide and odoratum protein peptide, only the complex alkaline protease is used for enzymatic hydrolysis, and the complex enzymes (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase) and pepsin are not used for enzymatic hydrolysis, that is, steps S2 and S4 are omitted, and the other preparation conditions and the dosage ratio of polygonatum protein peptide and odoratum protein peptide in the traditional Chinese medicine protein peptide composition are the same as those in Example 3.

[0110] Comparative Example 6

[0111] The difference between Comparative Example 6 and Example 3 is that in the preparation process of polygonatum protein peptide and odoratum protein peptide, only pepsin is used for enzymatic hydrolysis, and the complex enzymes (α-mannosidase, β-galactosidase, α-glucosidase, β-glucosidase) and the complex alkaline protease are not used for enzymatic hydrolysis, that is, steps S2 and S3 are omitted, and the other preparation conditions and the dosage ratio of polygonatum protein peptide and odoratum protein peptide in the traditional Chinese medicine protein peptide composition are the same as those in Example 3.

[0112] Comparative Example 7

[0113] The difference between Comparative Example 7 and Example 3 lies in that the dosage ratio of raw materials in the traditional Chinese medicine protein peptide composition is different. The dosage ratio of polygonatum sibiricum protein peptide to odoratum officinale protein peptide is 1:5, but the total amount is the same as that in Example 3, and the preparation process is the same as that in Example 3.

[0114] Comparative Example 8

[0115] The difference between Comparative Example 8 and Example 3 lies in that the dosage ratio of raw materials in the traditional Chinese medicine protein peptide composition is different. The dosage ratio of polygonatum sibiricum protein peptide to odoratum officinale protein peptide is 1:3, but the total amount is the same as that in Example 3, and the preparation process is the same as that in Example 3.

[0116] Comparative Example 9

[0117] The difference between Comparative Example 9 and Example 3 lies in that the dosage ratio of raw materials in the traditional Chinese medicine protein peptide composition is different. The dosage ratio of polygonatum sibiricum protein peptide to odoratum officinale protein peptide is 5:1, but the total amount is the same as that in Example 3, and the preparation process is the same as that in Example 3.

[0118] Comparative Example 10

[0119] The difference between Comparative Example 10 and Example 3 lies in that the dosage ratio of raw materials in the traditional Chinese medicine protein peptide composition is different. The dosage ratio of polygonatum sibiricum protein peptide to odoratum officinale protein peptide is 7:1, but the total amount is the same as that in Example 3, and the preparation process is the same as that in Example 3.

[0120] Comparative Example 11

[0121] The difference between Comparative Example 11 and Example 3 lies in that the traditional Chinese medicine protein peptide composition only contains polygonatum sibiricum protein peptide and does not add odoratum officinale protein peptide, but the total amount is the same as that in Example 3, and the preparation process of polygonatum sibiricum protein peptide is the same as that in Example 3.

[0122] Comparative Example 12

[0123] The difference between Comparative Example 12 and Example 3 lies in that the traditional Chinese medicine protein peptide composition only contains odoratum officinale protein peptide and does not add polygonatum sibiricum protein peptide, but the total amount is the same as that in Example 3, and the preparation process of odoratum officinale protein peptide is the same as that in Example 3.

[0124] Application Example 1 An aqueous solution containing a traditional Chinese medicine protein peptide composition and its preparation method

[0125] Raw materials (in parts by mass): 0.1 part of traditional Chinese medicine protein peptide composition, 3 parts of propylene glycol, 0.1 part of sodium hyaluronate, 3 parts of ethanol, 3 parts of glycerol, 0.1 part of EDTA-2Na, an appropriate amount of citric acid to adjust the pH range (from 5.5 to 6.5), 0.4 part of p-hydroxyacetophenone, 0.4 part of phenoxyethanol, 0.02 part of essence, and deionized water is added to 100 parts.

[0126] Preparation steps:

[0127] Accurately weigh all raw materials to ensure that the proportion of each raw material meets the formula requirements.

[0128] Add an appropriate amount of deionized water to the mixing tank. Slowly add the traditional Chinese medicine protein peptide composition, sodium hyaluronate, and glycerol to the water, and continuously stir until completely dissolved to obtain the main solution.

[0129] Take another clean container, add propylene glycol, ethanol, and essence, and stir until the essence is completely dissolved to obtain the essence solution.

[0130] Slowly add the prepared essence solution to the main solution, and stir until the two solutions are completely mixed evenly without separation.

[0131] Weigh an appropriate amount of citric acid and slowly add it to the mixing tank. While adding, use a pH meter to detect the pH value until the required pH range (5.5 - 6.5) is reached.

[0132] Add EDTA - 2Na to the mixing tank and stir until dissolved.

[0133] Add p - hydroxyacetophenone and phenoxyethanol to the mixing tank and continuously stir evenly. Finally, add deionized water to 100 parts, and conduct final stirring and adjustment to ensure that all components of the formula are fully mixed evenly, thus obtaining the aqueous solution containing the traditional Chinese medicine protein peptide composition.

[0134] Application Example 2 An emulsion containing a traditional Chinese medicine protein peptide composition and its preparation method

[0135] Raw materials (parts by mass): 1 part of traditional Chinese medicine protein peptide composition, 2 parts of glycerol, 0.1 part of sodium hyaluronate, 0.1 part of allantoin, 0.02 part of essence, 10 parts of sweet almond oil, 8 parts of jojoba oil, 2 parts of beeswax, 0.5 part of vitamin E, 5 parts of Span 80, 0.5 part of carbomer, 0.5 part of phenoxyethanol, appropriate amount of citric acid, and deionized water added to 100 parts.

[0136] Preparation steps:

[0137] Weigh raw materials: Accurately weigh all raw materials to ensure that the proportion of each raw material meets the formula requirements.

[0138] Preparation of oil phase: Mix sweet almond oil, jojoba oil, beeswax, and vitamin E in a container and heat to about 70 - 75°C until completely melted and mixed evenly.

[0139] Preparation of water phase: In a separate container, heat an appropriate amount of deionized water to about 70 - 75°C. Then, sequentially add glycerol, sodium hyaluronate, allantoin, and carbomer, and stir while adding until completely dissolved.

[0140] Emulsification: When the aqueous phase is still warm, slowly add Span 80 while vigorously stirring to achieve full emulsification. Slowly pour the preheated oil phase into the aqueous phase while quickly stirring with a stirrer to form a preliminary emulsion. Use a homogenizer to homogenize the preliminarily formed emulsion to improve the stability and fineness of the emulsion.

[0141] Cooling and adjustment: Cool the homogenized emulsion to room temperature and stir appropriately to prevent stratification. When the emulsion is cooled to near room temperature, add essence and phenoxyethanol and stir evenly. Add an appropriate amount of citric acid to adjust the pH value to 5.5 - 6.5.

[0142] Quality inspection: Conduct quality inspections on the emulsion, such as appearance, odor, stability, etc., to ensure that the product meets the standard requirements, thus obtaining the emulsion containing the traditional Chinese medicine protein peptide composition.

[0143] Application Example 3 A cream containing a traditional Chinese medicine protein peptide composition and its preparation method

[0144] Raw materials (parts by mass): 3 parts of traditional Chinese medicine protein peptide composition, 5 parts of trehalose, 0.3 part of sodium hyaluronate, 0.1 part of allantoin, 12 parts of rosehip oil, 10 parts of sweet almond oil, 2 parts of candelilla wax, 2.5 parts of cetearyl alcohol emulsifier, 0.4 part of vitamin E, 2 parts of Polysorbate 80, 0.3 part of phenoxyethanol, appropriate amount of citric acid, and deionized water is added to 100 parts

[0145] Preparation steps:

[0146] Weigh raw materials: Accurately weigh all raw materials to ensure that the proportion of each raw material meets the formula requirements.

[0147] Oil phase preparation: Mix rosehip oil, sweet almond oil, candelilla wax, vitamin E, and cetearyl alcohol emulsifier in a container and heat to 85°C until all solid components are completely melted and mixed evenly.

[0148] Aqueous phase preparation: In another container, add an appropriate amount of deionized water and heat to 85°C. Sequentially add trehalose, sodium hyaluronate, and allantoin while stirring until completely dissolved.

[0149] Emulsification: When the aqueous phase is still warm, add Polysorbate 80 to the aqueous phase while stirring to ensure complete dissolution. Slowly pour the preheated oil phase into the aqueous phase while quickly stirring with an electric stirrer to form a preliminary cream. Use a homogenizer to homogenize the preliminarily formed cream to improve the stability and fineness of the cream.

[0150] Cooling adjustment: Cool the emulsified cream to room temperature. During this process, stir appropriately to prevent stratification. When the cream is cooled to near room temperature, add phenoxyethanol and stir evenly. Check the pH value of the cream and add an appropriate amount of citric acid to adjust the pH value to 5.5 - 6.5.

[0151] Quality inspection: Conduct quality inspections on the cream, such as appearance, odor, stability, etc., to ensure that the product meets the standard requirements, thus obtaining the cream containing the traditional Chinese medicine protein peptide composition.

[0152] Application Example 4 An essence containing a traditional Chinese medicine protein peptide composition and its preparation method

[0153] Raw materials (by mass parts): 5 parts of traditional Chinese medicine protein peptide composition, 0.5 part of sodium hyaluronate, 5 parts of glycerol, 5 parts of butanediol, 3 parts of 1,3 - propanediol, 2 parts of trehalose, 0.1 part of allantoin, 0.05 part of disodium EDTA, 0.5 part of phenoxyethanol, 0.3 part of carbomer, appropriate amount of triethanolamine, and deionized water is added to 100 parts.

[0154] Preparation steps: Add carbomer to an appropriate amount of deionized water, stir evenly to make it fully swell, and heat to about 70°C to promote dissolution. In a clean and sterilized container, add deionized water (reserving the part for dissolving carbomer), and then add the traditional Chinese medicine protein peptide composition, sodium hyaluronate, glycerol, butanediol, 1,3 - propanediol, trehalose, allantoin, and disodium EDTA in sequence. Heat to about 75°C and stir while heating to ensure that all components are completely dissolved. When the temperature of the aqueous phase drops to about 70°C, slowly add the pre - prepared carbomer solution while stirring to avoid generating a large amount of foam. Continue stirring until the carbomer is completely dissolved to form a uniform viscous solution. Slowly adjust the pH value of the solution to between about 6.0 - 6.5 using triethanolamine. When the solution temperature drops to near room temperature, add phenoxyethanol and stir evenly. Use a homogenizer to homogenize the solution to improve the uniformity and stability of the solution. Conduct quality inspections on the essence, including indicators such as appearance, odor, pH value, viscosity, etc., to ensure that the product meets the standard requirements, thus obtaining the essence containing the traditional Chinese medicine protein peptide composition.

[0155] Test Example 1 Determination of the contents of polygonatum protein peptide and odoratum protein peptide

[0156] Determine the contents of polygonatum protein peptide and odoratum protein peptide involved in Examples 1 - 5 and Comparative Examples 1 - 6 to evaluate the advantages and disadvantages of the protein peptide extraction and purification method of the present invention. The method and results are as follows:

[0157] 1. Method basis

[0158] According to the provisions of the "National Food Safety Standard Plant Protein Peptides for Food Processing" (GB 31611-2023), the "Kjeldahl Nitrogen Determination Method" in the "National Food Safety Standard Determination of Protein in Food" (GB 5009.5-2016) was used to determine the content of protein peptides.

[0159] 2. Principle of Assay: Protein peptides are decomposed under catalytic heating conditions, and the generated ammonia combines with sulfuric acid to form ammonium sulfate. Alkalinization and distillation free the ammonia, which is then absorbed with boric acid and titrated with a standard sulfuric acid or hydrochloric acid solution. The nitrogen content is calculated based on the acid consumption and multiplied by the conversion factor (6.25) to obtain the protein peptide content.

[0160] 3. Materials and Reagents

[0161] The Chinese medicine protein peptide compositions were prepared according to the methods of Examples 1 to 5 and Comparative Examples 1 to 6, and dried and stored separately for future use.

[0162] Copper sulfate (CuSO4·5H2O), potassium sulfate (K2SO4), sulfuric acid (H2SO4), boric acid (H3BO3), methyl red indicator (C 15 H 15 N3O2), bromocresol green indicator (C 21 H 14 Br4O5S), methylene blue indicator (C 16 H 18 CIN3S·3H2O), sodium hydroxide (NaOH), and 95% ethanol (C2H5OH) were all analytically pure.

[0163] Boric acid solution (20 g / L): Weigh 20 g of boric acid, dissolve it in water and dilute to 1000 mL.

[0164] Sodium hydroxide solution (400 g / L): Weigh 40 g of sodium hydroxide and dissolve it in water, then cool and dilute to 100 mL.

[0165] Sulfuric acid standard titration solution [c(1 / 2H2SO4)]: 0.0500mol / L.

[0166] Methyl red ethanol solution (1 g / L): Weigh 0.1 g of methyl red, dissolve it in 95% ethanol, and dilute to 100 mL with 95% ethanol.

[0167] Methylene blue ethanol solution (1 g / L): Weigh 0.1 g of methylene blue, dissolve it in 95% ethanol, and dilute to 100 mL with 95% ethanol.

[0168] Bromocresol green ethanol solution (1 g / L): Weigh 0.1 g of bromocresol green, dissolve it in 95% ethanol, and dilute to 100 mL with 95% ethanol.

[0169] Mixed indicator solution: Mix 2 parts of methyl red ethanol solution with 1 part of methylene blue ethanol solution immediately before use.

[0170] 4. Analysis steps

[0171] Sample treatment: Weigh 0.2 g of the well-mixed solid sample accurately to 0.001 g, transfer it into a dry 100 mL Kjeldahl flask, add 0.4 g of copper sulfate, 6 g of potassium sulfate and 20 mL of sulfuric acid. After gently shaking, place a small funnel at the mouth of the flask. Incline the flask at a 45° angle on an asbestos net with small holes. Heat carefully. Wait until all the contents are carbonized and the foam completely stops, then increase the heating intensity and keep the liquid in the flask slightly boiling. Continue heating until the liquid turns blue-green and clear and transparent, and then continue heating for another 0.5 h - 1 h. Remove and let it cool. Carefully add 20 mL of water. After cooling, transfer it into a 100 mL volumetric flask, wash the Kjeldahl flask with a small amount of water, and pour the washing liquid into the volumetric flask. Then add water to the scale and mix well for standby. At the same time, conduct a reagent blank test.

[0172] Determination: Assemble the Kjeldahl distillation device. Fill the steam generator with water to 2 / 3 of its capacity, add several glass beads, add a few drops of methyl red ethanol solution and several milliliters of sulfuric acid to keep the water acidic. Heat the water in the steam generator to boiling and maintain boiling.

[0173] Add 10.0 mL of boric acid solution and 1 - 2 drops of the mixed indicator into the receiving flask, and insert the lower end of the condenser into the liquid surface. According to the nitrogen content in the sample, accurately pipette 2.0 mL of the sample treatment solution into the reaction chamber from the small glass cup, wash the small glass cup with 10 mL of water and let it flow into the reaction chamber. Then tightly plug the rod-shaped glass stopper. Pour 10.0 mL of sodium hydroxide solution into the small glass cup, lift the glass stopper to let it slowly flow into the reaction chamber, immediately cover the glass stopper tightly and seal it with water. Clamp the screw clamp and start distillation. After distilling for 10 min, move the distillate receiving flask so that the liquid surface is away from the lower end of the condenser, and then distill for another 1 min. Then rinse the outside of the lower end of the condenser with a small amount of water and remove the distillate receiving flask. Titrate with the sulfuric acid standard titration solution to the end point as soon as possible, that is, the solution color changes to grayish blue. Conduct a reagent blank at the same time.

[0174] The protein content in the sample is calculated by the following formula:

[0175]

[0176] Where:

[0177] X—the protein content in the sample, unit: grams per hundred grams (g / 100 g);

[0178] V1—the volume of the sulfuric acid or hydrochloric acid standard titration solution consumed by the test solution, unit: milliliters (mL);

[0179] V2—the volume of the sulfuric acid or hydrochloric acid standard titration solution consumed by the reagent blank, unit: milliliters (mL);

[0180] c—the concentration of the sulfuric acid standard titration solution, in moles per liter (mol / L);

[0181] 0.0140—the mass of nitrogen equivalent to 1.0 mL of the sulfuric acid standard titration solution, in grams (g);

[0182] m—the mass of the sample, in grams (g);

[0183] V3—the volume of the digested solution taken, in milliliters (mL);

[0184] 6.25—the coefficient for converting nitrogen to protein peptide;

[0185] 100—the conversion coefficient.

[0186] Table 1 Protein peptide content of examples and comparative examples

[0187]

[0188]

[0189] By analyzing the determination results (Table 1) of the content of polygonatum protein peptide and odoratum protein peptide involved in Examples 1-5 and Comparative Examples 1-6, the following conclusions can be drawn:

[0190] (1) The protein peptide extraction and purification method of the examples has high effectiveness and superiority. According to the determination results, the content range of polygonatum protein peptide in the examples is between 84.2% and 87.3%, and the content range of odoratum protein peptide is between 85.5% and 88.1%, which are significantly higher than those in the comparative examples (the content range of polygonatum protein peptide in the comparative examples is between 15.4% and 69.1%, and the content range of odoratum protein peptide is between 16.5% and 72.6%). This significant difference indicates that the protein peptide extraction and purification method adopted in the present invention has high effectiveness and superiority. Further analysis found that the main difference in the extraction and purification process between the examples and the comparative examples lies in the number of enzymatic hydrolysis. The examples adopted three enzymatic hydrolyses, while the comparative examples adopted one or two enzymatic hydrolyses. This shows that the number of enzymatic hydrolysis is an important factor affecting the protein peptide content, and by increasing the number of enzymatic hydrolysis, protein peptide can be extracted and purified more effectively.

[0191] (2) The protein peptide extraction and purification method of the embodiment has good stability and reproducibility. The data in the embodiment show that although there are certain fluctuations in the protein peptide content among the embodiments, overall, the fluctuation range is relatively small. The fluctuation range of the polygonatum sibiricum protein peptide content is between 84.2% and 87.3%, and the fluctuation range of the polygonatum odoratum protein peptide content is between 85.5% and 88.1%. This relatively small fluctuation range indicates that the extraction and purification method has good stability and reproducibility, and can maintain a relatively consistent protein peptide extraction effect in different batches.

[0192] In summary, the protein peptide extraction and purification method adopted by the present invention can not only effectively increase the contents of polygonatum sibiricum protein peptide and polygonatum odoratum protein peptide, but also has good stability and reproducibility.

[0193] Test Example 2 Skin Irritation Test

[0194] The skin irritation test was conducted on the traditional Chinese medicine protein peptide compositions involved in Examples 1 to 5. The method and results are as follows:

[0195] 1. Test method basis: It was carried out according to the "Skin Irritation Test Method" in the "Technical Specifications for Cosmetics Safety (2015 Edition)".

[0196] 2. Experimental principle: The test substance was applied once on the skin of the test animals, and the degree of local irritation of the animal skin was observed and scored at regular time intervals. Self-control was adopted to evaluate the irritation effect of the test substance on the skin.

[0197] 3. Test samples and animals: The test substance was a 1% aqueous solution of the traditional Chinese medicine protein peptide composition involved in Examples 1 to 5. The test animals were ordinary-grade New Zealand white rabbits purchased from the Guangdong Provincial Medical Experimental Animal Center.

[0198] 4. Experimental steps:

[0199] 4.1 Twenty-five white New Zealand rabbits were selected and randomly divided into 5 groups, with 5 rabbits in each group, namely Examples 1 to 5 groups.

[0200] 4.2 Approximately 24 hours before the test, the hair on both sides of the spine of the New Zealand rabbits was cut off, taking care not to damage the epidermis. The hair removal area was approximately 3 cm × 3 cm on the left and right sides respectively.

[0201] 4.3 Approximately 0.5 mL of the test substance was directly applied to the skin, then covered with two layers of gauze (2.5 cm × 2.5 cm) and one layer of glass paper, and then fixed with non-irritating adhesive tape and bandage. The skin on the other side was used as a control. The closed test was adopted, and the application time was 4 hours. After the test, the residual test substance was removed with warm water.

[0202] 4.4 Observe the skin reactions at the application sites at 1, 24, 48, and 72 hours after removing the test substance, and score the skin reactions according to Table 2. The comprehensive evaluation uses the average value of the integral of the test animals, and determines the skin irritation intensity according to the highest integral average value at each observation time point of 24, 48, and 72 hours according to Table 3.

[0203] Table 2 Skin Irritation Reaction Score

[0204]

[0205] Table 3 Classification of Skin Irritation Intensity

[0206]

[0207] 5. Results and Analysis

[0208] Within 1 - 72 hours after administering the traditional Chinese medicine protein peptide compositions of each example of the present invention, no erythema or edema was observed on the skin of New Zealand rabbits. According to the classification of skin irritation intensity in Table 2, it shows that the traditional Chinese medicine protein peptide compositions of each example of the present invention have no irritation to the skin.

[0209] Test Example 3 Whitening Efficacy Test

[0210] Perform in vitro whitening efficacy tests on Examples 1 - 5 and Comparative Examples 1 - 12.

[0211] 1. Method Basis

[0212] The test is carried out according to "T / GDCA 006—2021 Test Method for Inhibiting the Activity of Tyrosinase by Cosmetic Raw Materials (In Vitro Method)".

[0213] 2. Test Principle

[0214] The generation process of melanin is finely regulated by tyrosinase. Tyrosinase can catalyze L - tyrosine to become dopa, and further oxidize dopa to dopaquinone. This series of reactions ultimately contributes to the synthesis of melanin. Therefore, tyrosinase plays a crucial role in melanin production. It controls the melanin production process, and its activity level is directly related to the deposition of pigments in the skin. Inhibiting the activity of tyrosinase is of great significance for delaying the skin browning process and reducing melanin production, and is one of the effective ways to whiten the skin.

[0215] Test substances with the ability to inhibit tyrosinase activity can effectively slow down the rate of tyrosinase-catalyzed conversion of L-tyrosine to dopaquinone. By measuring the absorbance value of dopaquinone at a wavelength of 475 nm, the inhibitory effect of the test substance on tyrosinase activity can be quantitatively evaluated. To a certain extent, the ability of a substance to inhibit tyrosinase activity can be used as an indicator to measure its whitening potential. Specifically, the higher the degree of inhibition of tyrosinase activity, the more significant the whitening effect of the substance.

[0216] 3. Materials and Reagents

[0217] The traditional Chinese medicine protein peptide composition was prepared according to the methods of Examples 1-5 and Comparative Examples 1-12, and stored separately after drying for later use.

[0218] L-tyrosine was purchased from Macklin, with a purity of ≥99.0%, and was a biochemical reagent BR. Mushroom tyrosinase was purchased from Macklin, with a specification of 25 KU. Kojic acid was purchased from Macklin, with a purity of ≥99.0%, and was a biochemical reagent BR. The remaining reagents were all of analytical grade. Thermo scientific 1530 type full wavelength microplate reader.

[0219] 4. Experimental Procedures:

[0220] 4.1 Solution Preparation

[0221] Phosphate buffer solution (PBS), pH = 6.8, 0.1 mol / L: Weigh 1.361 g of potassium dihydrogen phosphate and add distilled water to a total volume of 100 mL to obtain Solution A. Weigh 1.78 g of disodium hydrogen phosphate and also add distilled water to 100 mL to obtain Solution B. Subsequently, mix Solution A and Solution B evenly in a 1:1 ratio to obtain the phosphate buffer solution (PBS).

[0222] L-tyrosine solution: Weigh 0.025 g of L-tyrosine, dissolve it with the above-prepared PBS buffer solution, and assist with ultrasonic treatment to promote dissolution, and finally make up the volume to 50 mL. This solution needs to be prepared and used immediately to ensure its freshness and effectiveness.

[0223] Tyrosinase solution: Use PBS buffer solution as the solvent to prepare mushroom tyrosinase into 500 U / mL. After preparation, it was aliquoted and stored at -20 °C, and attention should be paid to avoiding secondary freeze-thaw of the solution to ensure the activity of the enzyme.

[0224] Positive control kojic acid: Use PBS buffer to dilute kojic acid into a series of solutions with gradient mass concentrations. The specific concentrations are 0.080 g / L, 0.040 g / L, 0.020 g / L, 0.010 g / L, 0.008 g / L, 0.005 g / L, 0.002 g / L, and 0.001 g / L respectively.

[0225] Traditional Chinese medicine protein peptide composition solution: Prepare the traditional Chinese medicine protein peptide compositions in Examples 1 - 5 and Comparative Examples 1 - 12 into solutions with a concentration of 1.0 mg / mL using PBS buffer respectively, and set aside.

[0226] Use distilled water as the blank control.

[0227] 4.2 Experimental grouping

[0228] On a 96 - well microplate, the following four types of wells are set: solvent background well (labeled as Ta), solvent reaction well (labeled as Tb), sample background well (labeled as Tc), and sample reaction well (labeled as Td). Among them, the Ta group serves as the solvent background group, and its operation is not to add the substrate L - tyrosine solution and also not to add the sample solution; the Tb group serves as the solvent reaction group and needs to add the substrate L - tyrosine solution but not add the sample solution; the Tc group is set as the sample background group, and its operation is not to add the substrate L - tyrosine solution but to add the sample solution; while the Td group serves as the sample reaction group and needs to add both the substrate L - tyrosine solution and the sample solution. Each group of experiments is set with three replicate wells to ensure the accuracy and reliability of the data.

[0229] 4.3 Experimental steps

[0230] According to the reagent addition amounts provided in Table 4, add the L - tyrosine solution, sample solution / solvent, and PBS buffer to each well in sequence. After the addition, mix the contents of each well thoroughly and incubate them in a constant temperature environment at 37°C for 10 min. Subsequently, add 20 μL of tyrosinase solution to each well and continue to mix and react at 37°C, ensuring that the reaction time is 5 min ± 5 s. After the reaction ends, immediately place the microplate into the microplate reader and measure the absorbance values of different samples and the positive control at a wavelength of 475 nm. Three wells are set for measurement in each group, and the average value is taken. From adding the tyrosinase solution to the final measurement of absorbance, the operation time for all wells is kept consistent, which is 5 min ± 5 s.

[0231] Table 4 Reagent addition table for tyrosinase activity inhibition experiment

[0232]

[0233] Calculation of tyrosinase activity inhibition rate:

[0234] Inhibition rate = [1 - (Td - Tc) / (Tb - Ta)] × 100%

[0235] In the formula:

[0236] Td—Absorbance of the sample reaction well

[0237] Tc—Absorbance of the sample background well

[0238] Tb—Average absorbance of the solvent reaction wells

[0239] Ta—Average absorbance of the solvent background wells

[0240] The results are shown in Table 5.

[0241] Table 5 Inhibition rate of tyrosinase activity

[0242]

[0243]

[0244] It can be seen from the data in Table 5 that the traditional Chinese medicine protein peptide compositions of Examples 1 to 5 have good inhibitory effects on tyrosinase activity, and their inhibition rates are between 59.89% and 65.47%, showing strong whitening effects. In contrast, the inhibition rates of Comparative Examples 1 to 12 are relatively low, ranging from 20.87% to 56.24%, indicating that there are significant differences in the inhibitory effects between different compositions.

[0245] The inhibition rate of the positive control group (kojic acid) is 72.09%, which is higher than that of all examples and comparative examples, indicating that the positive control has the strongest tyrosinase inhibitory effect. The inhibition rate of the blank control group is only 0.21%, showing the effectiveness of the experimental design and further verifying the reliability of the experimental results. Although kojic acid has the strongest inhibitory effect on tyrosinase activity and is a special ingredient in current whitening and freckle-removing cosmetics, excessive use of kojic acid will cause white spots and a sense of irritation on human skin. The traditional Chinese medicine protein peptide composition is mild and has a high inhibitory effect on tyrosinase activity, and is more suitable as an active ingredient for skin care cosmetics.

[0246] Further analysis found that due to the different number of enzymatic hydrolysis times in the extraction processes of Comparative Examples 1 to 6 from those of the examples, the protein peptide content obtained is relatively low, and at the same time, its tyrosinase activity inhibition rate is also low. This shows that there may be a certain correlation between the tyrosinase activity inhibition rate and the protein peptide content. In other words, the level of the protein peptide content may directly affect the inhibitory effect of the composition on tyrosinase.

[0247] In addition, by comparing the traditional Chinese medicine protein peptide compositions in Examples 1-5 and Comparative Examples 7-12, it can be found that different ratios of polygonatum sibiricum protein peptide and odoratum protein peptide have a significant impact on the whitening effect. When the ratio of polygonatum sibiricum protein peptide to odoratum protein peptide is within a certain range (polygonatum sibiricum protein peptide: odoratum protein peptide is 1-3:1), the whitening effect of the composition is more significant. However, in Comparative Example 11 and Comparative Example 12, only polygonatum sibiricum protein peptide or odoratum protein peptide is contained, and the inhibition rate of tyrosinase activity is relatively low. This indicates that there may be a synergistic effect between the two protein peptides, jointly enhancing the whitening effect of the composition. When only one of the protein peptides is contained under the same dosage, the whitening effect is reduced. In summary, when preparing the traditional Chinese medicine protein peptide composition, it is crucial to reasonably control the ratio of polygonatum sibiricum protein peptide to odoratum protein peptide.

[0248] Test Example 4 In Vitro Anti-Aging Efficacy Test

[0249] Anti-aging efficacy tests of Examples 1-5 and Comparative Examples 1-12.

[0250] 1. Method Basis

[0251] The test was carried out in accordance with the General Rules for Anti-Aging - Antioxidant Evaluation Methods (T / ZGKSL001-2020).

[0252] 2. Test Principle

[0253] Excessive production of free radicals is the main cause of natural skin aging and photoaging. With the increase of age, the activity of enzymes that naturally scavenge free radicals in the skin decreases, and excess free radicals will damage skin cells, leading to the appearance of aging symptoms. Therefore, scavenging free radicals has become an effective means to delay skin aging. Cosmetics (or raw materials) with the ability to scavenge free radicals can protect skin cells from damage by reducing the production of free radicals or scavenging existing free radicals, thereby delaying the skin aging process and reducing the generation of wrinkles. When evaluating the anti-aging efficacy of cosmetics, the scavenging ability of the sample on free radicals (such as DPPH free radicals, ABTS free radicals, superoxide anions, hydroxyl free radicals, etc.) can be determined through in vitro experiments, and its anti-aging effect can be objectively evaluated. This method has the advantages of simple operation and clear results, and is one of the commonly used in vitro methods for evaluating the anti-aging efficacy of cosmetics.

[0254] 3. Materials and Reagents

[0255] The traditional Chinese medicine protein peptide composition was prepared according to the methods of Examples 1 to 5 and Comparative Examples 1 to 12, and was respectively formulated into a 0.1% aqueous solution with distilled water as the test sample. Absolute ethanol (Guangzhou Chemical Reagent Factory); sodium dihydrogen phosphate dihydrate (Maclin); PBS phosphate buffer powder (White Shark, 1x); 1,1-diphenyl-2-picrylhydrazine (Solarbio Science & Technology Co., Ltd.); Trolox (Maclin); total antioxidant capacity detection kit ABTS method (Beyotime Biotechnology Co., Ltd.); Thermoscientific 1530 full-wavelength microplate reader.

[0256] Weigh 3 mg of 1,1-diphenyl-2-trinitrophenylhydrazine into a 25 mL volumetric flask, and make up the volume with 95% ethanol to prepare a 0.12 mg / mL DPPH ethanol solution.

[0257] The traditional Chinese medicine protein peptide composition was prepared according to the methods of Examples 1 to 5 and Comparative Examples 1 to 12, and was respectively formulated into a 1.0 mg / mL aqueous solution with distilled water as the test sample.

[0258] Accurately weigh 25.0 mg of Trolox reference substance, make up the volume to 25 mL with absolute ethanol, and refrigerate it in the refrigerator at low temperature in the dark as the positive control solution.

[0259] Preparation of PBS buffer solution (pH = 7.4, 0.01 mol / L): Weigh 8.0 g of NaCl, 0.2 g of KCl, 2.88 g of Na2HPO4·2H2O and 0.27 g of KH2PO4, dissolve them in about 800 mL of purified water, and stir with a magnetic stirrer until completely dissolved. Adjust the pH to 7.4 with 0.1 M HCl, and make up the volume to 1000 mL with distilled water.

[0260] 4 Experimental procedures

[0261] 4.1 Determination of DPPH free radical scavenging ability

[0262] In a 96-well plate, set up sample wells, sample background wells, blank wells and solvent background wells respectively. Add 30 μL of the test sample solution, 150 μL of distilled water and 60 μL of DPPH ethanol solution to the sample wells; replace the DPPH ethanol solution with 95% ethanol in the sample background wells. Add 180 μL of distilled water and 60 μL of DPPH ethanol solution to the blank wells; replace the DPPH ethanol solution with 95% ethanol in the solvent background wells. Mix well, store in the dark for 5 minutes, and measure the absorbance of each well at a wavelength of 517 nm. Use Trolox as the positive control.

[0263] The calculation formula for the DPPH free radical scavenging rate S1 is:

[0264] T = Absorbance of the sample well; T0 = Absorbance of the sample background well; C = Absorbance of the solvent well; C0 = Absorbance of the solvent background well

[0265] The measurement results are shown in Table 6

[0266] Table 6 DPPH radical scavenging rate

[0267]

[0268]

[0269] 4.2 Determination of ABTS radical scavenging ability

[0270] According to the kit instructions, mix the ABTS solution and the ABTS working stock solution in equal proportion and store in the dark for 16 hours. When in use, dilute the ABTS working stock solution with PBS buffer to obtain the ABTS working solution. It is required that after subtracting the corresponding PBS buffer blank control from the absorbance of the ABTS working solution, A 734 is 0.7 ± 0.05, and the corresponding A 405 is around 1.4

[0271] In a 96-well plate, set up sample wells, sample background wells, and blank wells respectively. Add 10 μL of the test sample solution and 200 μL of the ABTS working solution to the sample wells; replace the sample solution with the corresponding solvent in the sample background wells. Add 10 μL of the corresponding solvent and 200 μL of PBS buffer (80% ethanol) to the blank wells. Mix well and store in the dark for 4 minutes, and measure the absorbance at each wavelength A 734 of each well. Use Trolox as the positive control

[0272] The calculation formula for the ABTS radical scavenging rate S2 is as follows

[0273] P = Absorbance of the sample well; P0 = Absorbance of the sample background well; L = Absorbance of the solvent well

[0274] The measurement results are shown in Table 7

[0275] Table 7 ABTS radical scavenging rate

[0276]

[0277] As shown in Tables 6 and 7, the DPPH free radical scavenging rates of the traditional Chinese medicine protein peptide compositions of Examples 1 to 5 ranged from 80.68% to 83.75%, and the ABTS free radical scavenging rates ranged from 78.53% to 84.25%, demonstrating strong anti-aging capabilities. The DPPH free radical scavenging rates of the examples were similar to the 86.12% DPPH free radical scavenging rate of the positive control, while the ABTS free radical scavenging rates of the examples were much higher than the 68.48% ABTS free radical scavenging rate of the positive control.

[0278] In contrast, the DPPH free radical scavenging rate and ABTS free radical scavenging rate of the Chinese medicine protein peptide compositions of Comparative Examples 1 to 12 are significantly lower, wherein the DPPH free radical scavenging rate is between 38.21% and 72.29%, and the ABTS free radical scavenging rate is between 26.97% and 75.11%. This result shows that the polyanthocyanidin and yuzhu protein peptide in the Chinese medicine protein peptide composition can synergistically enhance the anti-aging effect, and their ratio and preparation process also have a significant effect on their anti-aging effect (free radical scavenging rate). Although the composition of the comparative example also shows a certain free radical scavenging ability, the overall effect is not as good as the composition of the embodiment.

[0279] In summary, the Chinese herbal protein peptide compositions of Examples 1 to 5 have strong in vitro anti-aging efficacy and outstanding antioxidant capacity, and are expected to provide an effective solution for delaying skin aging. While the comparative example compositions also have some anti-aging effects, their overall performance is inferior to that of the examples, further illustrating the importance of the raw material ratio and preparation process of Chinese herbal protein peptide compositions.

[0280] Test Example 5 Moisturizing Efficacy Test

[0281] Moisturizing efficacy test of Examples 1 to 5 and Comparative Examples 1 to 12.

[0282] 1. Method basis: The test was conducted in accordance with QBT4256-2011 Guidelines for Evaluation of Moisturizing Efficacy of Cosmetics.

[0283] 2. Materials and Reagents: Traditional Chinese medicine protein peptide compositions were prepared according to the methods of Examples 1 to 5 and Comparative Examples 1 to 12, and were prepared into 0.1% aqueous solutions with distilled water as test samples. The control sample was 1% glycerol, and the blank control was 100% distilled water.

[0284] 3. Experimental Methods: 95 healthy subjects aged 18 to 55 years were selected on a voluntary basis. They were required to have no history of serious illness, immunodeficiency, or allergies, had not used hormones or immunosuppressants in the past month, and had not participated in other experiments. The subjects were randomly divided into 19 groups of 5 people each, namely, Example Groups 1 to 5, Comparative Example Groups 1 to 12, a control group, and a blank group. During the experiment, the subjects did not apply any medications or cosmetics unrelated to the experiment.

[0285] Before the test, clean the inner forearms of both hands and wipe them with a dry tissue. Mark the measurement area on the inner forearms of both hands. Before the actual test, sit quietly in a standard room (test environment temperature: (22±1)°C, humidity: (50±5)%, real-time dynamic monitoring) for at least 30 minutes. Do not drink water during this time. Keep your forearms exposed and in the test position, and remain relaxed.

[0286] During the experiment, a 3×3cm area was marked on the inside of each arm. 2 The test area can be marked on the same arm, and multiple areas can be marked on the same arm, and the interval between each area is 1 cm. The test samples and control samples were randomly assigned to the left and right arms. The skin moisture content of the test area was measured and recorded using the Corneometer CM 825 skin moisture tester. The results were expressed as moisture measurement values (Moisture Measurement Value, MMV) in units of CU (Corneometer Units). Each area was measured in parallel 5 times, and the average value was taken as the final test value. First, the initial (blank) value of each test area was measured, and then the value was calculated according to (2.0±0.1) mg / cm 2 Using a latex fingertip, apply the test sample evenly to the designated area using a latex fingertip. One and two hours after application, measure the skin moisture content of the test and control areas. To ensure consistency, all measurements on the same volunteer were performed by the same person. The skin moisture content growth rate at two hours was calculated using the following formula and averaged. A higher skin moisture content growth rate indicates better moisturizing efficacy. The test results are shown in Table 8 below.

[0287] Moisture content growth rate = (MMVt-MMV0) / MMV0×100%

[0288] Where MMV0 is the skin MMV before use; MMVt is the skin MMV at time t after use.

[0289] Table 8 Skin moisture content and 2h moisture content growth rate

[0290]

[0291] As can be seen from the data in Table 8, after the traditional Chinese medicine protein peptide composition in the examples was applied to the skin of the subjects, the growth rate of skin moisture content within 2 hours was between 45.60% and 49.30%. This fully shows that the traditional Chinese medicine protein peptide composition in the examples has a strong moisturizing effect, which is similar to the moisturizing effect of the positive control group. In contrast, the moisturizing effects of the traditional Chinese medicine protein peptide compositions in Comparative Examples 1-12 were significantly weaker. The range of the 2-hour skin moisture content growth rate of these compositions was only 16.32% to 43.82%, far lower than that of the compositions in the examples. This confirms that there are significant differences in the moisturizing effects among different traditional Chinese medicine protein peptide compositions.

[0292] The relatively weak moisturizing ability of Comparative Examples 1-6 is related to the number of enzymatic hydrolysis times in their extraction processes. Since the number of enzymatic hydrolysis times in these two groups of comparative examples is different from that in the examples, the resulting protein peptide content is relatively low. At the same time, the 2-hour skin moisture content growth rate is also correspondingly low. This indicates that different numbers of enzymatic hydrolysis times and the resulting differences in protein peptide content may both have important effects on the moisturizing ability of protein peptides.

[0293] By comparing the data of the examples with those of Comparative Examples 11-12, it can be found that the moisturizing ability of the traditional Chinese medicine protein peptide composition also has a synergistic effect. Comparative Example 11 and Comparative Example 12 contained only polygonatum protein peptide or odoratum protein peptide respectively, and their 2-hour skin moisture content growth rates were relatively low. This shows that when polygonatum protein peptide and odoratum protein peptide are used together, their moisturizing effect is significantly better than that of using only one of the protein peptides alone.

[0294] By comparing the data of the examples with those of Comparative Examples 7-10, it can be seen that different ratios of polygonatum protein peptide and odoratum protein peptide in the traditional Chinese medicine protein peptide composition have a significant impact on the moisturizing ability. When the ratio of these two protein peptides is within a certain range (the ratio in the examples), the moisturizing effect of the traditional Chinese medicine protein peptide composition is relatively significant.

[0295] Therefore, this test example shows that under the preparation conditions and raw material dosage ratios described in the present invention, a traditional Chinese medicine protein peptide composition with a significant moisturizing effect can be obtained.

Claims

1. A traditional Chinese medicine protein peptide composition, characterized in that, It is composed of polygonatum protein peptide and polygonatum odoratum protein peptide with a mass ratio of 1-3:1; the preparation method of the polygonatum protein peptide and polygonatum odoratum protein peptide is to first use a composite enzyme to enzymatically hydrolyze to obtain crude protein, and then the crude protein is enzymatically hydrolyzed twice with a composite alkaline protease and pepsin to obtain small molecular peptides with a molecular weight below 10KDa. The composite enzyme is α-mannosidase, β-galactosidase, α-glucosidase and β-glucosidase, and the composite alkaline protease is trypsin and papain; the preparation method of the polygonatum protein peptide and polygonatum odoratum protein peptide includes the following steps: S1. Hydrothermally extract the traditional Chinese medicine raw material to obtain a traditional Chinese medicine extract; S2. Use a composite enzyme to perform primary enzymatic hydrolysis on the traditional Chinese medicine extract to obtain traditional Chinese medicine crude protein; the composite enzyme is α-mannosidase, β-galactosidase, α-glucosidase and β-glucosidase; S3. Use a composite alkaline protease to perform secondary enzymatic hydrolysis on the traditional Chinese medicine crude protein solution to obtain a secondary enzymatic hydrolysate; the composite alkaline protease is trypsin and papain; S4. Use pepsin to perform tertiary enzymatic hydrolysis on the secondary enzymatic hydrolysate to obtain a tertiary enzymatic hydrolysate; S5. Ultrafilter the tertiary enzymatic hydrolysate with a membrane having a cut-off molecular weight of 10KDa and perform post-treatment to obtain traditional Chinese medicine protein peptide; The traditional Chinese medicine raw material is polygonatum odoratum or polygonatum sibiricum; The mass ratio of α-mannosidase, β-galactosidase, α-glucosidase and β-glucosidase in the composite enzyme is (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2); The mass ratio of trypsin and papain in the composite alkaline protease is (0.8-1.2):(0.8-1.2); When the traditional Chinese medicine raw material is polygonatum sibiricum, the dosage of the composite enzyme in step S2 is 1-3% of the mass of the traditional Chinese medicine extract; the dosage of the composite alkaline protease in step S3 is 1-5% of the mass of the traditional Chinese medicine crude protein solution; the dosage of pepsin in step S4 is 1-5% of the mass of the secondary enzymatic hydrolysate; When the traditional Chinese medicine raw material is polygonatum odoratum, the dosage of the composite enzyme in step S2 is 1.5-3.5% of the mass of the traditional Chinese medicine extract; the dosage of the composite alkaline protease in step S3 is 1.5-5.5% of the mass of the traditional Chinese medicine crude protein solution; the dosage of pepsin in step S4 is 1.5-5.5% of the mass of the secondary enzymatic hydrolysate.

2. Use of the traditional Chinese medicine protein peptide composition according to claim 1 in the preparation of whitening, anti-aging and moisturizing cosmetics.

3. According to the use of claim 2, the cosmetics are aqueous solutions, emulsions, creams or serums.

4. A cosmetic, characterized in that, Contains the traditional Chinese medicine protein peptide composition according to claim 1.

5. The cosmetic according to claim 4, characterized in that, The mass percentage content of the traditional Chinese medicine protein peptide composition in the cosmetics is 0.1-5%.

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