Glue traditional Chinese medicine protein peptide chelate with antioxidant and tyrosinase inhibitory activity and preparation method thereof

Abstract

The invention provides a glue traditional Chinese medicine protein peptide chelate with antioxidant and tyrosinase inhibitory activity and a preparation method thereof. The chelate is formed by chelation of glue traditional Chinese medicine protein peptide, zinc ions and selenium ions, the mass ratio of the zinc ions to the glue traditional Chinese medicine protein peptide is 0.1%-5%, and the mass ratio of the selenium ions to the glue traditional Chinese medicine protein peptide is 0.01%-0.5%. The gelatin traditional Chinese medicine protein peptide is derived from enzymolysis products of donkey-hide gelatin, oxhide gelatin, tortoise-shell glue or deer-horn glue. The preparation method is simple and comprises the following steps: dissolving the glue traditional Chinese medicine protein peptide in water or a buffer solution, adding a zinc salt and selenium salt solution, carrying out constant-temperature stirring reaction at 25-40 DEG C, and carrying out impurity removal, concentration and drying on a reaction product. Compared with the prior art, the chelate has the advantages that the ABTS free radical scavenging rate and tyrosinase inhibition activity are remarkably improved, the chelate is easily absorbed by a human body and can be widely applied to the field of other external preparations such as food, and a new thought is provided for development of skin whitening, anti-oxidation and anti-aging products.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a traditional Chinese medicine glue protein peptide chelate with antioxidant and tyrosinase inhibitory activities and a preparation method thereof. Background Art

[0002] In recent years, due to the influence of adverse factors such as air pollution, widespread use of electronic products, radiation, staying up late, etc., people's endocrine system will have various problems, manifested as pigmentation, dullness, etc. on the skin, and people's demand for skin whitening has increased significantly. Antioxidation and inhibition of tyrosinase activity are considered to be simple, direct and effective ways to achieve skin whitening. However, the whitening effects of most current whitening products are always unsatisfactory, and there are mainly the following problems: (1) The products cannot be absorbed by the skin;

[0003] (2) The selected whitening raw material components are single; (3) The selected combination of whitening raw materials is unreasonable, the synergistic effect is poor, and the maximum efficacy of each cannot be exerted when used in combination. The above problems seriously limit the application and effect of whitening products.

[0004] Collagen is mainly widely present in connective tissues such as muscles, skin, and bones of mammals, supporting the integrity of the morphology and structure of the skin and various tissues and organs, and playing an important role in protecting the body and supporting organs. Traditional Chinese medicine glues such as donkey-hide gelatin, yellow gelatin, tortoise shell glue, deer horn glue, etc. are made by boiling animal skins, horns or shells under high temperature and high pressure conditions for a long time, and are rich in collagen. Traditional Chinese medicine glue protein peptides, as the hydrolysis products of collagen in traditional Chinese medicine glues, retain many biological activity characteristics of collagen, have good biocompatibility, and are more easily absorbed by the human body.

[0005] Zinc is one of the important trace elements required by the human body and is widely involved in regulating physiological processes such as cell proliferation, protein synthesis, immunity, and endocrine. Zinc chelated collagen peptide is a complex formed by binding zinc ions to collagen peptides. This binding can enhance the biological activities of collagen peptides, including antioxidant and tyrosinase inhibitory activities. The State Intellectual Property Office publicly disclosed an invention patent "A Preparation Method and Application of Pearl Peptide Chelated Zinc" (Application No.: CN202410298242.7) on March 15, 2024, and found that the peptide-zinc chelate has a significant improvement in scavenging free radicals and inhibiting tyrosinase activity compared with the original pearl peptide, but the tyrosinase inhibition rate of a single zinc chelated polypeptide is less than 40%, and if zinc elements are ingested in excess, skin rashes, itching and other allergic phenomena will occur, so controlling the intake of zinc elements is crucial for the skin.

[0006] Selenium is a very important trace element for the human body, and it plays the roles of antioxidant and immune enhancement in the human body. The World Health Organization (WHO) stipulates that the minimum selenium requirement for the human body is 17 μg / d, and the physiological selenium requirement for the human body is 40 μg / d. However, chronic poisoning will occur when the average daily selenium intake is higher than 2.4 mg for a long time. Inorganic selenium must be combined with other organic substances in the intestine before it can be absorbed by the body, while organic selenium is absorbed by the intestinal wall of the body in the form of active transport, so the absorption efficiency is higher than that of inorganic selenium. Compared with inorganic selenium, the selenium in selenium-chelated collagen peptide has higher bioavailability, thus more effectively exerting its antioxidant effect. The State Intellectual Property Office has published a patent for invention "A Preparation Method and Application of a Whey Protein Peptide-Selenium Chelate with Antioxidant Activity" (Application No.: CN201810802848.4). The prepared whey protein peptide-selenium chelate has high antioxidant activity, has a unique chelation mechanism and transport mechanism, is safe and non-toxic, is easily absorbed, and can supplement amino acids and selenium at the same time. However, its antioxidant pathway is relatively single, and there is a lack of synergistic effect between elements, which affects the exertion of activity. Summary of the Invention

[0007] To solve the deficiencies of the prior art, the present invention has developed a traditional Chinese medicine glue protein peptide chelate with antioxidant and tyrosinase inhibitory activities. On the basis of ensuring easy absorption by the human body, the maximum whitening effect is achieved by giving full play to the synergistic effect of the components.

[0008] A traditional Chinese medicine glue protein peptide chelate with antioxidant and tyrosinase inhibitory activities, which is chelated and formed by the following components:

[0009] Traditional Chinese medicine glue protein peptide;

[0010] Zinc ion;

[0011] Selenium ion;

[0012] Among them, the mass ratio of the zinc ion to the traditional Chinese medicine glue protein peptide is 0.1% - 5%, and the mass ratio of the selenium ion to the traditional Chinese medicine glue protein peptide is 0.01% - 0.5%.

[0013] Further, the traditional Chinese medicine glue protein peptide is selected from one or more of the enzymatic hydrolysis products of donkey-hide gelatin, yellow gelatin, tortoise shell glue or deer antler glue.

[0014] Further, the chelate is in the form of solid powder or solution and has uniform dispersibility.

[0015] Further, a method for preparing a traditional Chinese medicine glue protein peptide chelate includes the following steps:

[0016] 1) Dissolve the traditional Chinese medicine glue protein peptide in water or buffer solution to obtain a protein peptide solution;

[0017] 2) Add a zinc salt solution and a selenium salt solution to the protein peptide solution, and stir and react at 25°C to 40°C for 10 to 60 minutes to chelate zinc ions and selenium ions with the protein peptide of the glue-like traditional Chinese medicine.

[0018] 3) Remove impurities, concentrate, and dry the mixed solution after the reaction to obtain a chelate of the protein peptide of the glue-like traditional Chinese medicine.

[0019] Further, the zinc salt in step 2) is selected from zinc sulfate, zinc acetate or zinc chloride, and the selenium salt is selected from sodium selenite or sodium selenate.

[0020] Further, the pH value of the reaction system in step 2) is controlled within the range of 5 to 8, and the reaction is carried out under constant temperature stirring conditions.

[0021] Further, a composition containing a chelate of the protein peptide of the glue-like traditional Chinese medicine, the composition includes:

[0022] The chelate of the protein peptide of the glue-like traditional Chinese medicine;

[0023] A carrier or excipient;

[0024] Wherein, the mass fraction of the chelate of the protein peptide of the glue-like traditional Chinese medicine in the composition is 0.1% to 10%.

[0025] Further, the composition exists in the form of an external use product and further contains one or more of a humectant, a thickener, a preservative or a fragrance.

[0026] Further, the composition exists in an edible form and further contains one or more of proteins, carbohydrates, vitamins or minerals.

[0027] The present invention aims to overcome the defects in the prior art such as low absorption rate of whitening products, single active ingredient and insufficient synergistic effect, and provides a chelate of the protein peptide of the glue-like traditional Chinese medicine that is easily absorbed by the human body and has excellent antioxidant and tyrosinase inhibitory activities and its preparation method. The chelate of the protein peptide of the glue-like traditional Chinese medicine in the present invention chelates zinc ions and selenium ions on the protein peptide of the glue-like traditional Chinese medicine respectively to form a stable and multi-active composite structure.

[0028] Specifically, the protein peptide of the glue-like traditional Chinese medicine used in the present invention can be selected from the enzymatic hydrolysis products of donkey-hide gelatin, yellow gelatin, tortoise shell gelatin or deer antler gelatin. While retaining the nutritional value and activity of the traditional glue-like traditional Chinese medicine, such protein peptides are more easily absorbed by the human body after enzymatic hydrolysis treatment. Further, by chelating zinc ions and selenium ions with the protein peptide respectively, the antioxidant performance and tyrosinase inhibitory effect of the chelate are significantly improved. Zinc element participates in the synthesis of various proteins and immune regulation in the body, while selenium element plays an important role in antioxidant and immune regulation. The chelate formed by the two with the protein peptide of the glue-like traditional Chinese medicine can synergistically enhance the effect within the safe dose range.

[0029] In the present invention, by controlling the mass ratio of zinc ions to the protein peptide of traditional Chinese medicine with glue-like property to be about 0.1% - 5%, and the mass ratio of selenium ions to the protein peptide of traditional Chinese medicine with glue-like property to be about 0.01% - 0.5%, and preferably carrying out the constant-temperature stirring reaction under the condition of 25°C - 40°C, the sufficient chelation of zinc ions and selenium ions can be achieved. The preparation process flow of the present invention is simple: after dissolving the protein peptide of traditional Chinese medicine with glue-like property, zinc salt and selenium salt are added in sequence for chelation reaction, and then through impurity removal, concentration and drying, a stable chelate of the protein peptide of traditional Chinese medicine with glue-like property is obtained. This preparation method is easy to operate and is suitable for large-scale production. The obtained chelate has higher stability and bioavailability.

[0030] Compared with the existing single zinc-chelated or selenium-chelated polypeptides, the chelate of the present invention has a greater improvement in both the ABTS free radical scavenging rate and the tyrosinase inhibition rate, and can be used to improve skin whitening and antioxidant activities, and is applied to fields such as food, health products, cosmetics or topical preparations. The related products prepared by using the chelate of the protein peptide of traditional Chinese medicine with glue-like property described in the present invention can not only provide necessary nutritional supplements for the body, but also reduce pigmentation caused by exogenous free radicals and tyrosinase, and have a positive improvement effect on skin dullness, age spots and early aging.

[0031] In summary, the beneficial effects of the present invention mainly include:

[0032] Using the protein peptide of traditional Chinese medicine with glue-like property as the main matrix, it is more easily absorbed by the human body and has high safety;

[0033] Through the synergistic chelation of zinc ions and selenium ions, the antioxidant and tyrosinase inhibition activities are significantly enhanced;

[0034] The preparation process is simple and easy to operate, and is suitable for large-scale production under industrial conditions;

[0035] The chelate has good stability and can be widely applied to multiple fields such as food, health products, cosmetics or topical preparations

[0036] The beneficial effects of the present invention: On the basis of ensuring easy absorption by the human body, the present invention maximizes the whitening effect by giving full play to the synergistic effect of components, and the main raw material used is the protein peptide of traditional Chinese medicine with glue-like property, which is easily absorbed by the human body. The extraction process is simple and will not cause adverse effects on the human body, nor will it cause addiction and drug dependence, etc., and has high safety, better antioxidant property and tyrosinase inhibition activity. The complex of the present invention creatively applies selenium element to the direction of tyrosinase inhibition, providing new ideas for the development of whitening products.

[0037] The complex of the present invention has good synergistic effect, can maximize the antioxidant property and tyrosinase inhibition activity, and achieve the maximization of whitening results. Description of the Drawings

[0038] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0039] Figure 1 ABTS scavenging rate of the 5% Zn colloidal traditional Chinese medicine protein peptide chelate of the present invention;

[0040] Figure 2 Schematic diagram of the ABTS scavenging rate of the 5% Zn / 0.1% Se colloidal traditional Chinese medicine protein peptide chelate of the present invention;

[0041] Figure 3 Schematic diagram of the ABTS scavenging rate of the 5% Zn / 0.2% Se colloidal traditional Chinese medicine protein peptide chelate of the present invention;

[0042] Figure 4 Schematic diagram of the tyrosinase inhibitory activity of the 5% Zn colloidal traditional Chinese medicine protein peptide chelate of the present invention;

[0043] Figure 5 Schematic diagram of the tyrosinase inhibitory activity of the 5% Zn / 0.1% Se colloidal traditional Chinese medicine protein peptide chelate of the present invention;

[0044] Figure 6 Schematic diagram of the tyrosinase inhibitory activity of the 5% Zn / 0.2% Se colloidal traditional Chinese medicine protein peptide chelate of the present invention. Detailed implementation manners

[0045] The present invention will be described in detail below in conjunction with the drawings and specific embodiments. At the same time, it should be noted here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments. For some well-known technologies, those skilled in the art can also adopt other alternative methods for implementation; moreover, the drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0046] It should be noted that in the specification, references to "an embodiment", "embodiment", "exemplary embodiment", "some embodiments", etc. indicate that the described embodiment may include specific features, structures, or characteristics, but not necessarily every embodiment includes such specific features, structures, or characteristics. Additionally, when combining an embodiment to describe a specific feature, structure, or characteristic, implementing such feature, structure, or characteristic in combination with other embodiments (whether explicitly described or not) should be within the knowledge of those skilled in the relevant art.

[0047] Generally, terms can be understood at least in part from their use in context. For example, depending at least in part on the context, the term "one or more" as used herein can be used to describe any feature, structure, or property in the singular sense, or can be used to describe a combination of features, structures, or properties in the plural sense. Additionally, the term "based on" can be understood as not necessarily intended to convey a set of exclusive factors, but rather can alternatively, depending at least in part on the context, allow for the existence of other factors that are not necessarily explicitly described.

[0048] The preparation steps of a traditional Chinese medicine protein peptide chelate of glue with antioxidant and tyrosinase inhibitory activities are as follows:

[0049] Step 1: Mix 7.4 mmol / L ABTS and 2.6 mmol / L potassium persulfate in equal volumes. After standing in the dark for 12 h, adjust the ABTS radical solution with phosphate buffer solution at pH 7.4 to obtain an absorbance of 0.700 ± 0.005 at 734 nm to complete the dilution of the ABTS solution.

[0050] Step 2: In a 96-well plate, add 175 μL of the diluted ABTS radical solution to 25 μL of the sample solution. After spotting the plate, react in the dark at 37 °C for 10 min, then set the wavelength to 734 nm and measure the absorbance value of the well plate using a full-wavelength microplate reader. Compared with the experimental group, in the control group, an equal volume of distilled water is used instead of the sample and added to the 96-well plate, and the other added solutions remain unchanged. Compared with the experimental group, in the blank group, an equal volume of buffer solution is used instead of the ABTS radical-containing solution and added to the 96-well plate, and the other added solutions remain unchanged. The calculation formula is as shown in Equation (1).

[0051]

[0052] Where A in Equation (1) C is the absorbance of the control group; A S is the absorbance of the sample; A b is the absorbance of the blank group.

[0053] Step 3: Tyrosinase inhibitory activity test: Prepare the tyrosine solution, polyphenol oxidase solution required for the reaction, and the solvent is PBS buffer solution. Prepare the sample group solution and the control group solution. In the experimental group, add 60 μL of tyrosine, 60 μL of the composite peptide solution, and 80 μL of PBS; in the control group, PBS is used to replace the tyrosine solution; in the blank group, deionized water is used to replace the composite peptide solution; in the complete control group, deionized water is used to replace the composite peptide solution and PBS is used to replace the tyrosine solution. Store in the dark at 25 °C for 10 min, then add 20 μL of polyphenol oxidase and react in the dark at 25 °C for 20 min. Use a microplate reader to measure the absorbance value at 475 nm, which are respectively recorded as A s 、A c 、Ab , A 0 .

[0054] The tyrosinase inhibitory activity was calculated according to the following formula.

[0055]

[0056] The following are specific embodiments.

[0057] Example 1: Weigh a certain amount of gelatinous Chinese medicine protein peptide and zinc sulfate so that the Zn ion content is 5% of the mass of the gelatinous Chinese medicine protein peptide, add deionized water to prepare solutions of different concentrations, and then place them in a reactor with a stirring function for constant temperature reaction, the temperature is set to 25°C, the reaction time is set to 30min, and a gelatinous Chinese medicine protein peptide zinc chelate is prepared, recorded as 5% Zn gelatinous Chinese medicine protein peptide chelate.

[0058] Example 2: Weigh a certain amount of gelatinous Chinese medicine protein peptide, zinc sulfate and sodium selenite so that the Zn ion content is 5% of the mass of the gelatinous Chinese medicine protein peptide, and the Se ion content is 0.1% of the mass of the gelatinous Chinese medicine protein peptide. Add deionized water to prepare solutions of different concentrations, and then place them in a reactor with a stirring function for constant temperature reaction. The temperature is set to 25°C and the reaction time is set to 30 minutes to prepare a gelatinous Chinese medicine protein peptide zinc-selenium chelate, which is recorded as 5% Zn / 0.1% Se gelatinous Chinese medicine protein peptide chelate.

[0059] Example 3: Weigh a certain amount of gelatinous Chinese medicine protein peptide, zinc sulfate and sodium selenite so that the Zn ion content is 5% of the mass of the gelatinous Chinese medicine protein peptide, and the Se ion content is 0.2% of the mass of the gelatinous Chinese medicine protein peptide, add deionized water to prepare solutions of different concentrations, and then place them in a reactor with a stirring function for constant temperature reaction, the temperature is set to 25°C, and the reaction time is set to 30min, to prepare a gelatinous Chinese medicine protein peptide zinc-selenium chelate, recorded as 5% Zn / 0.2% Se gelatinous Chinese medicine protein peptide chelate.

[0060] The antioxidant and tyrosinase inhibitory activities of the composites prepared in Examples 1-3 are shown in the attached Figure 1-6 shown.

[0061] In the present invention, a traditional Chinese medicine protein peptide of glue type is chelated with zinc and selenium ions in a specific ratio, endowing it with better antioxidant and tyrosinase inhibitory activities than when used alone, thus achieving a better whitening effect. The maximum concentrations of zinc and selenium ions in the human body are 10 ppm and 3 ppm respectively. Exceeding these concentrations may pose a risk of poisoning. The ion concentrations used in the present invention are within the safe range. Meanwhile, after chelating zinc and selenium ions with the traditional Chinese medicine protein peptide of glue type, it is beneficial to the absorption of the ions. The experimental results show that after the traditional Chinese medicine protein peptide of glue type chelates zinc ions and selenium ions respectively, the antioxidant property of the chelate has been greatly improved compared with that of the traditional Chinese medicine protein peptide of glue type, and the improvement effect of the selenium chelate of the traditional Chinese medicine protein peptide of glue type is more obvious. Therefore, in the subsequent examples, only the addition amount of selenium ions was changed, and it was found that the 5% Zn / 0.1% Se chelate of the traditional Chinese medicine protein peptide of glue type has better antioxidant property, and with the increase of the selenium ion content, the antioxidant property has little improvement compared with the 5% Zn / 0.1% Se chelate of the traditional Chinese medicine protein peptide of glue type.

[0062] The 5% Zn / 0.1% Se chelate of the traditional Chinese medicine protein peptide of glue type has stronger tyrosinase inhibitory activity than zinc ions and selenium ions used alone, and with the increase of the selenium ion content, the tyrosinase inhibitory activity decreases instead. In summary, the 5% Zn / 0.1% Se chelate of the traditional Chinese medicine protein peptide of glue type has the optimal antioxidant and tyrosinase inhibitory activities. The main raw material used in the present invention is the traditional Chinese medicine protein peptide of glue type, which is easily absorbed by the human body. The extraction process is simple, does not cause adverse effects on the human body, and will not cause addiction and drug dependence, etc., with high safety. At the same time, the complex has a good synergistic effect, and the antioxidant and tyrosinase inhibitory activities are greatly improved compared with the single component, providing a new development idea for better realizing whitening.

[0063] The present invention covers any substitutions, modifications, equivalent methods and solutions made on the essence and scope of the present invention. In order to enable the public to have a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention, and those skilled in the art can fully understand the present invention without the description of these details. In addition, in order to avoid unnecessary confusion to the essence of the present invention, well-known methods, processes, procedures, components and circuits, etc. are not described in detail.

[0064] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A gelatinous Chinese medicine protein peptide chelate with antioxidant and tyrosinase inhibitory activities, characterized in that: The gelatinous Chinese medicine protein peptide chelate is formed by chelating the following components: Glue-based Chinese medicine protein peptides; Zinc ions; Selenium ion; The mass ratio of the zinc ions to the gelatinous Chinese medicine protein peptides is 0.1% to 5%, and the mass ratio of the selenium ions to the gelatinous Chinese medicine protein peptides is 0.01% to 0.5%.

2. The gelatinous Chinese medicine protein peptide chelate according to claim 1, characterized in that: The gelatinous Chinese medicine protein peptide is selected from one or more of the enzymatic hydrolysis products of donkey-hide gelatin, yellow gelatin, tortoise shell glue or deer antler glue.

3. The gelatinous Chinese medicine protein peptide chelate according to claim 1 or 2, characterized in that: The chelate is in the form of solid powder or solution and has uniform dispersion.

4. A method for preparing the gelatinous Chinese medicine protein peptide chelate according to any one of claims 1 to 3, comprising the following steps: 1) dissolving the gelatinous Chinese medicine protein peptide in water or buffer to obtain a protein peptide solution; 2) adding a zinc salt solution and a selenium salt solution to the protein peptide solution, stirring and reacting at 25° C. to 40° C. for 10 to 60 minutes to chelate the zinc ions and selenium ions with the gelatinous Chinese medicine protein peptide; 3) After the reaction is completed, the mixed solution is removed from impurities, concentrated, and dried to obtain a gelatinous Chinese medicine protein peptide chelate.

5. The method according to claim 4, characterized in that The zinc salt in step 2) is selected from zinc sulfate, zinc acetate or zinc chloride, and the selenium salt is selected from sodium selenite or sodium selenate.

6. The method according to claim 4 or 5, characterized in that The pH value of the reaction system in step 2) is controlled within the range of 5 to 8, and the reaction is carried out under constant temperature stirring conditions.

7. A composition containing the gelatinous Chinese medicine protein peptide chelate according to any one of claims 1 to 3, the composition comprising: Gum-based Chinese medicine protein peptide chelates; Carrier or excipient; Wherein, the mass fraction of the gelatinous traditional Chinese medicine protein peptide chelate in the composition is 0.1% to 10%.

8. The composition according to claim 7, characterized in that The composition is in the form of an external product and further comprises one or more of a moisturizer, a thickener, a preservative or a fragrance.

9. The composition according to any one of claims 7 or 8, characterized in that The composition is in an edible form and further comprises one or more of protein, carbohydrate, vitamin or mineral.

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