Bird's nest peptide copolymer capable of effectively resisting fatigue and regulating emotion and application of bird's nest peptide copolymer

Abstract

The invention relates to a cubilose peptide copolymer capable of effectively resisting fatigue and regulating emotion and application thereof.The cubilose peptide copolymer is obtained through copolymerization of raw materials including cubilose peptide and oak extract, has excellent stability, remarkably improves the environment factor influence resistance of cubilose peptide and has good application prospects. The cubilose peptide copolymer has excellent digestive juice enzymolysis resistance, antioxidant activity and ATP content increasing efficacy activity in the application process, and the cubilose peptide copolymer provided by the invention has excellent nerve regulating activities such as fatigue resistance, vigor improvement, emotion regulation and concentration improvement.

Description

Technical Field

[0001] This application relates to the field of bird's nest peptide technology, and in particular to an effective anti-fatigue and mood-regulating bird's nest peptide copolymer and its application. Background Technology

[0002] Bird's nest has been hailed as a precious tonic and health-preserving product since ancient times, rich in nutrients, especially proteins, polysaccharides, and amino acids, which possess certain health benefits. In recent years, research has found that bird's nest peptides extracted from bird's nest exhibit superior biological activity, including antioxidant, anti-aging, and immunomodulatory activities. However, due to the unique structure of bird's nest peptides, they are easily affected by the environment. For example, in the human digestive tract, they are degraded into smaller peptides or amino acids by digestive enzymes such as pepsin and trypsin, leading to loss of activity. Furthermore, they are easily oxidized and degraded, and their efficacy fluctuates significantly due to factors such as temperature and humidity, making it difficult to guarantee consistent quality. Therefore, improving the stability of bird's nest peptides in the digestive tract and reducing their degradation by digestive enzymes are crucial to ensuring that bird's nest peptides exert their superior biological activity. Summary of the Invention

[0003] Based on this, this application provides an effective anti-fatigue and mood-regulating bird's nest peptide copolymer that improves the stability of bird's nest peptides, reduces their degradation by digestive enzymes, and ensures the stable performance of their bioactivity. This copolymer can then be used in bird's nest peptide complexes and bird's nest products to achieve good bioactive functions in the complexes and products.

[0004] In one aspect, this application provides an effective anti-fatigue and mood-regulating bird's nest peptide copolymer, which is obtained by copolymerizing raw materials containing bird's nest peptides and oak extract;

[0005] The oak extract is prepared by extracting the heartwood of oak with water, and the active ingredients of the oak extract include phenolic compounds.

[0006] The copolymerization process includes: mixing the raw materials with a solvent and ultrasonically treating them to prepare the effective anti-fatigue and mood-regulating bird's nest peptide copolymer.

[0007] In some embodiments, the mass ratio of the bird's nest peptide to the oak extract is 1:(0.02-6).

[0008] In some embodiments, the frequency of the ultrasound is 20 kHz to 80 kHz; and / or the power of the ultrasound is 100 W to 800 W; and / or the temperature of the ultrasound is 25°C to 60°C; and / or the duration of the ultrasound is 0.5 h to 2.0 h.

[0009] In some embodiments, the method further includes a step of freeze-drying the ultrasonically treated material.

[0010] Secondly, this application also provides an effective anti-fatigue and mood-regulating bird's nest peptide complex, wherein the active components of the effective anti-fatigue and mood-regulating bird's nest peptide complex include a first active component and a second active component;

[0011] The first active component includes the effective anti-fatigue and mood-regulating bird's nest peptide copolymer provided in the first aspect;

[0012] The second active ingredient includes one or more of larch extract, rosemary extract, chicken extract, and coenzyme Q10.

[0013] In some embodiments, the mass ratio of the first active component to the second active component is 1:(0.5-2).

[0014] In some embodiments, the larch extract is an extract of the larch cambium and xylem, and the larch cambium and xylem extract includes piezorin.

[0015] In some embodiments, the rosemary extract includes rosmarinic acid, and the rosemary extract is extracted from rosemary raw material.

[0016] In some embodiments, the chicken extract includes imidazole dipeptide, and the chicken extract is extracted from chicken raw materials.

[0017] In some embodiments, the coenzyme Q10 includes reduced coenzyme Q10.

[0018] Thirdly, this application also provides a bird's nest peptide product, which includes the effective anti-fatigue and mood-regulating bird's nest peptide copolymer provided in the first aspect and / or the effective anti-fatigue and mood-regulating bird's nest peptide complex provided in the second aspect.

[0019] Compared with traditional technologies, the beneficial effects of the technical solution of this application include: This application uses raw materials containing bird's nest peptides and oak extracts to copolymerize under ultrasonic treatment to form bird's nest peptide copolymers, which significantly improves the stability of bird's nest peptides, has excellent resistance to digestive enzyme hydrolysis, reduces their degradation in the body, and improves their activity in the body. Moreover, the bird's nest peptide copolymers exhibit excellent antioxidant, ATP-enhancing, and neuroinflammatory-improving biological activities. When used in bird's nest peptide compound products, they can enhance vitality and combat fatigue, and have excellent neuromodulation effects, showing good application prospects in multiple fields. Detailed Implementation

[0020] To facilitate understanding of the present invention, preferred embodiments are provided below to provide a more complete description of the invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to enable a thorough and complete understanding of the disclosure of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0022] As used herein, "optional," "optional," and "optional" refer to either "with" or "without" parallel options. If multiple "optional" entries appear in a technical solution, each "optional" entry is independent unless otherwise specified and there are no contradictions or mutual constraints. The term "and / or" as used herein includes any and all combinations of one or more related listed items. Unless otherwise specified, "multiple," "multiple," etc., as used herein refer to a quantity greater than 2 or equal to 2; for example, "one or more" indicates one, two, or more than two. In open-ended technical features or solutions described herein using words such as "containing," "including," and "comprising," unless otherwise specified, additional members beyond the listed members are not excluded. This can be considered as providing both a closed-ended feature or solution consisting of the listed members and an open-ended feature or solution that includes additional members beyond the listed members.

[0023] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0024] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0025] Bird's nest peptides are essentially small molecular fragments resulting from the hydrolysis of bird's nest protein. Their core components are short peptides, sialic acid, and sugar chains, and they contain various essential amino acids. Compared to bird's nest itself, bird's nest peptides have better bioactivity and are more easily absorbed by the body. However, due to their unique structure, they are easily affected by environmental factors such as temperature, humidity, enzymes, and microorganisms during use, leading to a decrease or even inactivation of their efficacy. Therefore, improving the stability of bird's nest peptides is crucial to ensuring their full bioactivity.

[0026] In one aspect, this application provides an effective anti-fatigue and mood-regulating bird's nest peptide copolymer, obtained by copolymerizing raw materials containing bird's nest peptide and oak extract; the oak extract is prepared by extracting the heartwood of oak with water, and the active ingredients of the oak extract include phenolic compounds.

[0027] The copolymerization process includes mixing bird's nest peptides, the oak extract, and a solvent, followed by ultrasonic treatment to obtain the final product.

[0028] This application utilizes a covalent cross-linking reaction between bird's nest peptides and oak extracts to form a bird's nest peptide-oak extract copolymer, which significantly improves the stability of bird's nest peptides and ensures that their bioactivity is fully utilized. The copolymer structure increases the steric hindrance of the bird's nest peptide molecular structure, reducing the degradation of the bird's nest peptide molecular chain by enzymes and microorganisms in the environment, thereby improving its resistance to enzymatic degradation. For example, it can prevent digestive enzymes in the digestive tract from reaching the cleavage site, thus enhancing the bird's nest peptide's resistance to digestion. The copolymer structure can also prevent the active ingredients in oak extract (such as ellagic acid) from being digested, increasing their concentration in the large intestine, where they are metabolized by the gut microbiota into smaller, more active metabolites such as urolithin. These metabolites can be absorbed into the bloodstream by the large intestine, exerting a systemic health effect. At the same time, the introduction of oak extract into the bird's nest peptide molecular structure forms a protective effect on the bird's nest peptide molecular structure. For example, under the influence of the external environment (light, heat, air, humidity, etc.), the oak extract portion in the copolymer structure can form a protective layer, on the one hand is isolating part of the external environment from attacking the bird's nest peptide molecular structure, and on the other hand, the oak extract portion can react with the external environment first, such as undergoing oxidation, thereby protecting the bird's nest peptide.

[0029] In some embodiments, the mass ratio of the bird's nest peptide to the oak extract is 1:(0.02-6). Within this mass ratio range, the bird's nest peptide and the oak extract can form a copolymer with good stability, thereby improving its good stability while ensuring its original efficacy. Further, the mass ratio of the bird's nest peptide to the oak extract is 1:(0.01-6).

[0030] In some embodiments, the molecular weight of the bird's nest peptide is ≤3 kDa.

[0031] In some embodiments, the active ingredients of the oak extract include phenolic compounds, which are copolymerized with bird's nest peptides. Exemplarily, the phenolic compounds include one or more of ellagic acid, ellagitannins, gallic acid, chlorogenic acid, and protocatechuic acid.

[0032] This application utilizes a copolymer formed from bird's nest peptides and oak extract. This increased steric hindrance makes it difficult for enzymes to reach their target sites, thus reducing the digestibility of the bird's nest peptides, protecting their intact structure, and enhancing their stability and activity. Simultaneously, oxidized proteins, upon reaching the colon, negatively regulate the gut microbiota and impair colonic health. This application addresses this by using oak extract in the copolymer to reduce the production of oxidized proteins during digestion. Therefore, the bird's nest peptide copolymer of this application possesses diverse and excellent functional activities, including enhanced antioxidant capacity, increased ATP levels, and reduced neuroinflammation, further enhancing its role in regulating neuroactive activity. This results in superior anti-fatigue, energy-boosting, mood-regulating, and concentration-enhancing neuroactive activity-regulating functions.

[0033] This application employs physical methods to copolymerize bird's nest peptides with oak extract through covalent bonds, including CO, CC, CN, and CS covalent bonds.

[0034] Ultrasonic treatment generates cavitation. When cavitation bubbles collapse, water molecules are broken down, producing hydroxyl radicals (•OH) and hydrogen radicals (•H). These highly reactive free radicals attack the active hydrogens (such as phenolic hydroxyl groups, thiol groups, and hydrogen on the α-carbon atom) of phenolic compounds in bird's nest peptides and oak extracts, forming protein free radicals and phenolic free radicals, respectively. These activated free radicals collide with each other to form stable covalent bonds, thus producing bird's nest peptide-oak extract copolymers.

[0035] Furthermore, the cavitation process, accompanied by intense shock waves and microjets, significantly increases the local concentration and collision frequency of phenolic compounds in bird's nest peptides and oak extracts, creating more opportunities for the reaction. On the other hand, the mechanical vibration and thermal effects of ultrasound disrupt the hydrogen bonds and hydrophobic interactions of bird's nest peptides, causing partial unfolding and exposing internal hydrophobic groups and reactive sites, increasing the probability and quantity of copolymer formation. Thus, the above treatment method greatly enhances the production of bird's nest peptide-oak extract copolymers, thereby improving the stability of bird's nest peptides and increasing their resistance to digestion.

[0036] Compared to alkaline methods, free radical methods, and enzymatic methods, this application utilizes a physical method to ensure the activity and efficacy of bird's nest peptides, reduce the introduction of impurities, and is cost-effective and practical. For example, the alkaline method requires a high pH environment, which may lead to the hydrolysis of some acetylamino groups of N-acetylneuraminic acid in the bird's nest peptides; the free radical method requires the introduction of ascorbic acid and hydrogen peroxide into the system, which may result in a small amount of unreacted hydrogen peroxide residue; while the enzymatic method has high specificity and mild reaction conditions, the preparation process is complex and costly. Therefore, this application employs a purely physical method to promote the covalent cross-linking reaction, which is efficient, safe, environmentally friendly, and easy to implement for industrial production. The bird's nest peptide copolymer preparation method provided by this application is simple, easy to operate, and highly effective, offering new ideas and methods for the development of bird's nest peptides and related products.

[0037] In some embodiments, the solvent includes water, such as purified water. As a non-limiting example, the bird's nest peptides and oak extracts may be dissolved separately in water before mixing.

[0038] In some embodiments, the frequency of the ultrasound is 20 kHz to 80 kHz, including but not limited to 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, or any combination thereof and values ​​within that range. Further, the frequency of the ultrasound is 30 kHz to 50 kHz.

[0039] In some embodiments, the power of the ultrasound is 100 W-800 W, including but not limited to 100 W, 200 W, 300 W, 400 W, 500 W, 600 W, 700 W, 800 W or any combination thereof and values ​​within that range. Further, the power of the ultrasound is 300 W-600 W.

[0040] In some embodiments, the temperature of the ultrasound is 25°C-60°C, including but not limited to 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, or any combination thereof and values ​​within that range. Further, the temperature of the ultrasound is 30°C-50°C.

[0041] In some embodiments, the ultrasound duration is 0.5 h to 2.0 h, including but not limited to 0.5 h, 1 h, 1.5 h, 2.0 h or any of the foregoing ranges and values ​​within that range.

[0042] To a certain extent, excessively high ultrasonic power and frequency can lead to excessive energy input, resulting in protein denaturation and aggregation, as well as the decomposition of phenolic compounds, reducing the binding capacity of phenolic compounds to proteins.

[0043] In some embodiments, the ultrasonically treated material is further subjected to freeze-drying, which involves concentrating and freeze-drying the ultrasonically treated solution to form a powdered bird's nest peptide copolymer.

[0044] Secondly, this application also provides an effective anti-fatigue and mood-regulating bird's nest peptide complex. The active components of the effective anti-fatigue and mood-regulating bird's nest peptide complex include a first active component and a second active component. The first active component includes the effective anti-fatigue and mood-regulating bird's nest peptide copolymer provided in the first aspect; the second active component includes one or more of larch extract, rosemary extract, chicken extract, and coenzyme Q10. The bird's nest peptide complex provided by this application has significant efficacy in regulating nerve function.

[0045] In some embodiments, the mass ratio of the first active component to the second active component is 1:(0.5-2), including but not limited to 1:0.5, 1:1, 1:1.5, 1:2 or any of the foregoing ranges and values ​​within those ranges.

[0046] In some embodiments, the second active component includes at least two of larch extract, rosemary extract, chicken extract, and coenzyme Q10. Exemplarily, the second active component includes larch extract and rosemary extract, with a mass ratio of larch extract to rosemary extract of 1:(0.5-2); Exemplarily, the second active component includes rosemary extract and chicken extract, with a mass ratio of rosemary extract to chicken extract of 1:(0.7-1.5); Exemplarily, the second active component includes larch extract, rosemary extract, and chicken extract, with a mass ratio of larch extract, rosemary extract, and chicken extract of 1:(0.5-2):(0.35-3); Exemplarily, the second active component includes larch extract, rosemary extract, chicken extract, and coenzyme Q10, with a mass ratio of larch extract, rosemary extract, chicken extract, and coenzyme Q10 of 1:(0.5-2):(0.35-3):(0.5-1.2).

[0047] In some embodiments, the larch extract is an extract of the cambium and xylem of larch, and the active ingredient in the extract of the cambium and xylem of larch includes piezorin.

[0048] In some embodiments, the rosemary extract is extracted from rosemary raw material, and the active ingredient of the rosemary extract includes rosmarinic acid.

[0049] In some embodiments, the chicken extract is extracted from chicken meat, and the active ingredient in the chicken extract includes imidazole dipeptide.

[0050] In some embodiments, the coenzyme Q10 includes reduced coenzyme Q10.

[0051] Thirdly, this application also provides a bird's nest peptide product, which includes the effective anti-fatigue and mood-regulating bird's nest peptide copolymer provided in the first aspect and / or the bird's nest peptide complex provided in the second aspect.

[0052] In some embodiments, excipients may also be added to the bird's nest peptide complex and bird's nest peptide products, including one or more of the following: excipients, thickeners, anti-caking agents, antioxidants, leavening agents, emulsifiers, coating agents, preservatives, color-protecting agents, colorants, acidity regulators, enzyme preparations, flavor enhancers, moisture-retaining agents, nutritional fortifiers, and stabilizers. As non-limiting examples, these include one or more of the following: starch, starch hydrolysates, hydroxypropyl methylcellulose, magnesium stearate, particulate silica, titanium dioxide, edible rice oil, edible soybean oil, edible rapeseed oil, edible safflower oil, gelatin, glycerol, and glycerol fatty acid esters.

[0053] In some embodiments, the bird's nest peptide copolymer and / or bird's nest peptide complex may also be filled, stored, or packaged in capsule form, and the form of bird's nest peptide products includes capsules. The capsules may be hard capsules and / or soft capsules, such as capsule filling: filling the capsules with powder of the bird's nest peptide copolymer and / or bird's nest peptide complex to prepare soft capsules or hard capsules.

[0054] Capsule filling: Filling bird's nest peptide copolymer and / or bird's nest peptide complex powder into capsules to prepare soft capsules or hard capsules.

[0055] Specifically, if made into hard capsules, use pharmaceutical-grade lock-type gelatin hollow capsules, with each capsule containing 400 mg to 600 mg of powder. Before use, place the capsules in a suitable temperature and humidity environment to achieve a moisture content of 12-15%. Use a capsule filling machine to complete the following operations: separate the capsule cap and body; arrange the capsule bodies and vacuum-hold them; quantitatively fill the powder into the capsule bodies; cap the capsules and lock them in place.

[0056] Specifically, if soft capsules are to be made, gelatin, plasticizer (such as glycerin), and water are mixed at a mass ratio of 1:(0.3-0.5):(0.7-1.0) and dissolved at 60℃-70℃. During this process, the mixture is degassed and filtered to remove impurities under a vacuum of -0.08 MPa to -0.09 MPa to obtain a clear gel solution. Next, the oil solution is prepared by using vegetable oil (such as rice oil, soybean oil, rapeseed oil, or safflower oil) or glycerol fatty acid esters as solvents. Bird's nest peptide copolymer and / or bird's nest peptide complex powder are added and stirred at 100 rpm-300 rpm until completely dissolved at 40℃-60℃. The mixture is then filtered through a 0.45 μm microporous membrane. The gel solution is then formed into a uniform sheet and fed into a capsule press along with the oil solution. The capsules are pressed at a mold temperature of 20℃-25℃. Finally, the pressed wet capsules are dried and shaped, first at 18℃-22℃. The mixture is initially shaped in a °C environment, then transferred to a drying chamber at 25°C-30°C and 35%-45% relative humidity to remove excess moisture, ultimately yielding qualified oil solution soft capsules.

[0057] Polishing inspection: Remove dust from the surface of the capsules to make them clean and shiny, and remove any capsules with abnormal appearance under light.

[0058] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0059] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0060] The specific embodiments of this application involve the following reagents and raw materials, including their types and sources:

[0061] Oak extract (containing phenolic compounds) is prepared by water extraction from the heartwood of oak. Specifically, the heartwood of oak is pulverized to 40-60 mesh, and repeatedly extracted with hot water at 70℃-90℃. The extract is filtered to remove insoluble substances, and then subjected to 5 kDa ultrafiltration to remove macromolecular impurities. Finally, it is dried to obtain the oak extract.

[0062] Larch cambium and xylem extract (Firtatidylcholine); rosemary extract (rosmarinic acid); chicken extract (imidazolium dipeptide).

[0063] Preparation of bird's nest peptides: Bird's nest is stewed, homogenized, subjected to conventional enzymatic hydrolysis (such as alkaline protease, neutral protease, flavor protease, trypsin, etc.), filtered, and dried to obtain bird's nest peptides. Specifically, bird's nest powder is placed in boiling water and stewed for 2 hours, followed by homogenization at 25-30 MPa for 20-30 minutes. After cooling to room temperature, alkaline protease or a complex enzyme is added to hydrolyze the stewed bird's nest peptides. During this process, the pH value is controlled between 7.0 and 9.0, and the temperature is maintained at 50℃-60℃. After hydrolysis for 3-4 hours, the bird's nest hydrolysate is obtained by filtration. The bird's nest hydrolysate is then concentrated and spray-dried to obtain bird's nest peptide powder. The inlet air temperature for spray drying is 170℃, and the outlet air temperature is greater than 90℃.

[0064] Examples 1-3

[0065] This embodiment provides a type of bird's nest peptide copolymer, as detailed below:

[0066] Table 1: Raw material composition of active ingredients

[0067]

[0068] Bird's nest peptide and oak extract were dissolved separately in purified water to form aqueous solutions of bird's nest peptide and oak extract as shown in Table 1. Then, the aqueous solutions of bird's nest peptide and oak extract were mixed in equal volumes to form a mixture.

[0069] The above mixture was subjected to ultrasonic treatment at a frequency of 45 kHz, a power of 500 W, and a temperature of 45 ℃ for 1 h to form a bird's nest peptide copolymer solution. This solution was then concentrated and freeze-dried to form three groups of bird's nest peptide copolymers in powder form.

[0070] Examples 4-7

[0071] This embodiment provides a type of bird's nest peptide copolymer and its preparation method, as detailed below:

[0072] Bird's nest peptide and oak extract were dissolved separately in purified water to form an aqueous solution of bird's nest peptide with a concentration of 1.4 mg / mL and an aqueous solution of oak extract with a concentration of 1.5 mg / mL. Then, the aqueous solution of bird's nest peptide and the aqueous solution of oak extract were mixed in equal volumes to form a mixture.

[0073] The above mixture was subjected to ultrasonic treatment with ultrasonic treatment parameters as shown in Table 2 to form a bird's nest peptide copolymer solution. The solution was then concentrated and freeze-dried to form four groups of bird's nest peptide copolymers in powder form.

[0074] Table 2: Ultrasonic processing parameters

[0075]

[0076] Comparative Example 1

[0077] The difference from Example 1 is that the same concentration of bird's nest peptide aqueous solution and oak extract aqueous solution are simply mixed, then concentrated and freeze-dried. The remaining composition and preparation method are the same as in Example 1.

[0078] Experimental Example 1: Determination of Free Amino Content in Bird's Nest Peptide Copolymer

[0079] The free amino group content in samples was determined using the o-phthalaldehyde (OPA) method. The OPA reagent was prepared as follows: 40.0 mg OPA was dissolved in 1.0 mL of methanol, mixed with 25.0 mL of sodium borate buffer (0.1 M, pH 9.8), 100 μL of β-mercaptoethanol, and 2.5 mL of 20% (w / v) sodium dodecyl sulfate (SDS) solution, and then diluted to 50.0 mL with distilled water. The sample protein concentration was adjusted to 0.2 mg / mL. Then, 200 μL of the sample solution was mixed with 4.0 mL of OPA reagent, reacted at 35 °C for 2 min, and the absorbance at 340 nm was measured. Using the OPA reagent and distilled water mixture as a blank, a standard curve was plotted using L-leucine standard solution to calculate the free amino group content in the sample.

[0080] Table 3: Results of determination of free amino content in bird's nest peptide copolymer

[0081]

[0082] Free amino groups can serve as reaction sites for covalent cross-linking between proteins and phenolic compounds. Therefore, a decrease in the free amino group content indicates the occurrence of covalent cross-linking, i.e., the formation of copolymers. The greater the decrease in free amino group content, the higher the degree of covalent cross-linking. It is evident that the free amino group content in Comparative Example 1 was not significantly different from that of the individual bird's nest peptide sample under the same protein content, indicating that the simply mixed bird's nest peptide aqueous solution and oak extract in Comparative Example 1 did not undergo covalent cross-linking, and no covalent bonds were formed between the components.

[0083] Experimental Example 2: Cytotoxicity Test of Bird's Nest Peptide Copolymer

[0084] The samples prepared in Examples 1-7 and Comparative Example 1 were subjected to cytotoxicity tests. The specific test methods were as follows:

[0085] HaCaT and BV2 cells in logarithmic growth phase were seeded in 96-well plates at a density of 1 × 10⁶ cells / well. 4Cells were cultured per well using the appropriate culture medium until they adhered (approximately 24 hours). The culture medium was then discarded, and the cells were divided into a control group and a sample group. The control group was cultured in serum-free medium after discarding the culture medium, while the sample group was cultured in serum-free medium containing the sample. After 24 hours of incubation, the culture medium was discarded, and the cells were replaced with 10% CCK-8 solution for another 2 hours. The absorbance (A) was measured at 450 nm. Six parallel experiments were conducted, and cell viability was used to assess the cytotoxicity of the samples. The specific calculation formula is shown in equation (I):

[0086] (I)

[0087] Table 4: Effect of bird's nest peptide copolymer on cell viability

[0088]

[0089] Therefore, the bird's nest peptide copolymer, bird's nest peptide, and oak extract prepared in this application have no obvious cytotoxicity and are safe.

[0090] Experimental Example 3: Test of the anti-digestive enzyme activity of bird's nest peptide copolymer

[0091] The samples prepared in Examples 1-7 and Comparative Example 1, along with the bird's nest peptide, were subjected to an anti-digestion enzymatic hydrolysis activity test. The specific test methods are as follows:

[0092] Preparation of test samples: Weigh out the samples prepared in Examples 1-7 and Comparative Example 1, and 1g of bird's nest peptide, respectively, and add deionized water to a final volume of 40g to form 8 groups of test sample solutions.

[0093] (1) Preparation of digestion stock solution

[0094] Table 5: Preparation of Digestive Stock Solution

[0095]

[0096] (2) Preparation of digestion electrolyte

[0097] Table 6: Preparation of Digestion Electrolyte

[0098]

[0099] (3) Simulating oral digestion

[0100] The sample solution to be tested was mixed with 40 g of salivary digestion electrolyte and 0.2 mL of CaCl2(H2O)2 and incubated at 37 °C for 2 min to simulate oral digestion.

[0101] (4) Simulating gastric digestion

[0102] Add 80 mL of gastric digestion electrolyte to the oral digests, adjust the pH of the digestion solution to 3 with 6 M HCl solution, add 0.04 mL of CaCl2(H2O)2 and mix to dissolve pepsin in the gastric digestion electrolyte. The final pepsin concentration of each sample is 500 U / mL. Incubate at 37 ℃ for 2 h.

[0103] (5) Simulated intestinal digestion

[0104] Add 160 mL of intestinal digestion electrolyte to the gastric digest, adjust the pH of the digestion solution to 7 with 1 M NaOH solution, add 0.32 mL of CaCl2(H2O)2 and mix, dissolve trypsin in the intestinal digestion electrolyte, the final trypsin concentration of each sample is 25 U / mL, and incubate at 37 ℃ for 2 h.

[0105] (6) Enzyme inactivation

[0106] After digestion, the sample was placed in boiling water and heated for 7 minutes to inactivate the enzymes in the solution. After enzyme inactivation, the sample was cooled and the digestion product was filtered through medium-speed filter paper to obtain the digestion supernatant.

[0107] (7) Determination of degree of hydrolysis

[0108] The free amino group content in samples was determined using the o-phthalaldehyde (OPA) method. The OPA reagent was prepared as follows: 40.0 mg OPA was dissolved in 1.0 mL of methanol, mixed with 25.0 mL of sodium borate buffer (0.1 M, pH 9.8), 100 μL of β-mercaptoethanol, and 2.5 mL of 20% (w / v) sodium dodecyl sulfate (SDS) solution, and then diluted to 50.0 mL with distilled water. The protein concentration of the sample was adjusted to 0.2 mg / mL, and then 200 μL of the mixture was mixed with 4.0 mL of the OPA reagent. The mixture was reacted at 35 °C for 2 min, and the absorbance at 340 nm was measured. Using the OPA reagent and distilled water mixture as a blank, a standard curve was plotted using L-leucine standard solution to calculate the free amino group content in the sample, and the degree of hydrolysis was calculated accordingly.

[0109] Table 7: Results of the test on the anti-digestive enzyme hydrolysis activity of bird's nest peptide copolymer

[0110]

[0111] During digestion, peptide bonds break and free amino groups are released. The degree of digestion is directly proportional to the increase in free amino groups. Therefore, the degree of hydrolysis can be reflected by the content of free amino groups in the sample. As shown in Table 7, compared with Comparative Example 1, the samples in Examples 1-7 showed significantly reduced degrees of hydrolysis and excellent resistance to digestive enzymes. This demonstrates that the combination of bird's nest peptide and oak extract significantly improves the digestibility of bird's nest peptide. However, the resistance to digestive enzymes in the bird's nest peptide sample alone did not differ significantly from that in Comparative Example 1. This indicates that no copolymer structure was formed in the sample of Comparative Example 1, thus failing to promote the digestibility of bird's nest peptide.

[0112] Experimental Example 4: Effect of Bird's Nest Peptide Copolymer on Cellular ATP Content

[0113] The cellular ATP content of the samples prepared in Examples 1-7 and Comparative Example 1, as well as the bird's nest peptide and oak extract, was tested using the following specific methods:

[0114] HaCaT cells in logarithmic growth phase were seeded into 6-well plates at a density of 5 × 10⁶ cells / well. 5 Cells per well were cultured until they adhered (approximately 24 hours). The culture medium was then discarded, and the cells were cultured in the following groups.

[0115] Blank group and model group: Add culture medium.

[0116] Sample group: Add culture medium to prepare sample solutions of the same concentration.

[0117] After the samples were added, the model group and sample group were irradiated under UV light for 15 min, and then the cells were transferred to an incubator for further incubation for 24 h. The cell control group was not treated. Subsequently, the culture medium was aspirated, and the cells were collected; centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the cells were washed with PBS, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 200 μL of lysis buffer was added to each well to lyse the cells. After lysis, the cells were centrifuged at 12000 rpm for 5 min at 4 ℃, and the supernatant was collected for subsequent assays.

[0118] Add 100 μL of ATP detection working solution to a 96-well plate and incubate at room temperature for 3-5 min to allow all background ATP to be consumed, thereby reducing the background level. Add 20 μL of sample to each well and measure the RLU value using a chemiluminescence analyzer. Perform three parallel experiments, with the blank group as 100% ATP content. Calculate the percentage of ATP content in the sample group relative to the blank group cells using formula (II):

[0119] (II)

[0120] The results obtained using the above testing method are shown in Table 8:

[0121] Table 8: Effects of bird's nest peptide copolymer on cellular ATP

[0122]

[0123] ATP directly provides energy for various life activities within cells, including synthesis, active transport, muscle contraction, and nerve impulse transmission. Ultraviolet (UV) radiation can penetrate the cell membrane and directly damage the structure of mitochondria, leading to reduced ATP production. Furthermore, UV irradiation can cause cells to produce excessive amounts of reactive oxygen species (ROS), resulting in mitochondrial damage.

[0124] As shown in Table 8, the ATP content in the model group cells was significantly reduced, indicating successful modeling. Compared to the model group, the ATP content in the example group was significantly increased, far exceeding that of Comparative Example 1 and the individual bird's nest peptide and oak extract raw materials. This indicates that the bird's nest peptide copolymer provided in this application can promote ATP production in cells and increase cellular energy, showing better results than the simple mixture group (Comparative Example 1). It also shows that this application achieves synergistic effects through the raw materials of bird's nest peptide and oak extract.

[0125] Experimental Example 5: Antioxidant Activity of Bird's Nest Peptide Copolymer

[0126] The samples prepared in Examples 1-7 and Comparative Example 1, as well as bird's nest peptides and oak extract, were subjected to antioxidant tests. The specific test methods were as follows:

[0127] HaCaT cells in logarithmic growth phase were seeded into 96-well plates at a density of 1 × 10⁶ cells / well. 4 Cells were cultured at 100 cells / well until adherence (approximately 24 hours). The culture medium was then discarded, and the cells were divided into a control group, a model group, and a sample group. After discarding the culture medium, serum-free medium was added to the control and model groups, while serum-free medium containing the sample solution (at the same concentration) was added to the sample groups. After 24 hours of culture, except for the control group, the remaining groups were cultured for another 4 hours with 300 μM hydrogen peroxide solution. After the culture was finished, the medium was discarded, and the cells were replaced with 10% CCK-8 solution for another 2 hours. The absorbance (A) was measured at 450 nm. Three parallel experiments were conducted, and antioxidant activity was assessed based on cell viability. The survival rate of the control group was 100%. The specific calculation formula is shown in equation (III).

[0128] (III)

[0129] The results obtained using the above testing method are shown in Table 9:

[0130] Table 9: Antioxidant Activity of Bird's Nest Peptide Copolymer

[0131]

[0132] After treatment with hydrogen peroxide, the cell survival rate of the model group was significantly reduced. The cell survival rate of the example group was significantly higher than that of the bird's nest peptide and oak extract group and the model group, indicating that the example significantly repaired the oxidative damage caused by hydrogen peroxide. It was more effective than the simple mixture group (Comparative Example 1) and significantly higher than the antioxidant effect of bird's nest peptide and oak extract alone.

[0133] Experimental Example 6: Inhibitory Neuroinflammatory Activity of Bird's Nest Peptide Copolymer

[0134] The samples prepared in Examples 1-7 and Comparative Example 1, as well as the samples of bird's nest peptide and oak extract, were subjected to tests for inhibiting neuroinflammation. The specific test method was as follows:

[0135] BV2 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 5 × 10⁶ cells / well. 4 Cells were cultured at 100 cells / well until they adhered (approximately 24 hours). The culture medium was then discarded, and the cells were divided into a control group, a model group, and a sample group. After discarding the culture medium, serum-free medium was added to the control and model groups, while serum-free medium containing the sample solution (at the same concentration) was added to the sample groups. After 2 hours of further culture, except for the control group, the remaining groups were added to a lipopolysaccharide solution at a final concentration of 500 ng / mL and cultured for another 24 hours. After the culture was completed, the culture medium was discarded, and the NO concentration was measured using the supernatant. Three parallel experiments were conducted.

[0136] The results obtained using the above testing method are shown in Table 10:

[0137] Table 10: Inhibitory neuroinflammatory activity of bird's nest peptide copolymers

[0138]

[0139] After treatment with lipopolysaccharide, the NO content released by cells in the model group increased significantly. The NO released by cells in the example group was significantly lower than that in the bird's nest peptide and oak extract group and the model group. This indicates that the bird's nest peptide copolymer significantly reduced lipopolysaccharide-induced inflammatory damage and had the effects of improving neuroinflammation, regulating nerve activity, enhancing cognition, and maintaining the homeostasis of the central nervous system. Moreover, it was more effective than the simple mixture group (Comparative Example 1) and significantly higher than the inhibitory neuroinflammatory activity of bird's nest peptide and oak extract alone.

[0140] Example 8

[0141] The bird's nest peptide copolymer prepared in Example 1 was mixed with larch cambium and xylem extracts at a mass ratio of 1:1 to form a bird's nest peptide complex.

[0142] Example 9

[0143] The bird's nest peptide copolymer prepared in Example 1 was mixed with rosemary extract at a mass ratio of 1:1 to form a bird's nest peptide complex.

[0144] Example 10

[0145] The bird's nest peptide copolymer prepared in Example 1 was mixed with chicken extract at a mass ratio of 1:1 to form a bird's nest peptide complex.

[0146] Example 11

[0147] The bird's nest peptide copolymer prepared in Example 1 was mixed with reduced coenzyme Q10 at a mass ratio of 1:1 to form a bird's nest peptide complex.

[0148] Example 12

[0149] The bird's nest peptide copolymer prepared in Example 1 was mixed with larch cambium and xylem extracts and rosemary extract at a ratio of 1:0.5:0.5 to form a bird's nest peptide complex.

[0150] Example 13

[0151] The bird's nest peptide copolymer prepared in Example 1 was mixed with rosemary extract and chicken extract at a ratio of 1:0.5:0.5 to form a bird's nest peptide complex.

[0152] Example 14

[0153] The bird's nest peptide copolymer prepared in Example 1 was mixed with larch cambium and xylem extract, rosemary extract, and chicken extract in a ratio of 1:0.33:0.33:0.33 to form a bird's nest peptide complex.

[0154] Example 15

[0155] The bird's nest peptide copolymer prepared in Example 1 was mixed with larch cambium and xylem extract, rosemary extract, chicken extract, and coenzyme Q10 in a ratio of 1:0.25:0.25:0.25:0.25 to form a bird's nest peptide complex.

[0156] Experimental Example 6: Effect of Bird's Nest Peptide Complex on Anti-enzymatic Hydrolysis by Digestive Fluids

[0157] The samples from Examples 8-15 were subjected to an enzymatic hydrolysis test using the same method as in Example 3. The results are shown in Table 11.

[0158] Table 11: Effect of bird's nest peptide complex on degree of hydrolysis

[0159]

[0160] Compared to bird's nest peptide copolymers, the degree of hydrolysis is further reduced after bird's nest peptide copolymers form complexes with one or more of larch cambium and xylem extracts, rosemary extract, chicken extract, and reduced coenzyme Q10, indicating that the bird's nest peptide complex provided in this application can more effectively protect the bird's nest peptide structure from being destroyed during digestion.

[0161] Experimental Example 7: Effect of Bird's Nest Peptide Complex on Cellular ATP Content

[0162] Cellular ATP content was tested in samples from Examples 8-15, larch cambium and xylem extracts, rosemary extract, chicken extract, and reduced coenzyme Q10. The amount of ATP added to all samples was the same, and the specific testing method was consistent with that in Example 4. The results are shown in Table 12.

[0163] Table 12: Effects of bird's nest peptide complex on cellular ATP

[0164]

[0165] The results in Table 12 show that the complex formed by combining the bird's nest peptide copolymer with one or more of larch cambium and xylem extracts, rosemary extract, chicken extract, and reduced coenzyme Q10 can significantly increase the cellular ATP content level. Furthermore, the activity of each of these components—bird's nest peptide copolymer, larch cambium and xylem extract, rosemary extract, chicken extract, and reduced coenzyme Q10—is significantly higher than that of any single component. This indicates that the components in the complex prepared in this application have a synergistic effect in promoting ATP production in cells. The bird's nest peptide complex provided in this application can enhance cellular energy, significantly improve energy levels, and reduce fatigue.

[0166] Experimental Example 8: Antioxidant Activity of Bird's Nest Peptide Complex

[0167] The antioxidant activity of samples from Examples 8-15, larch cambium and xylem extracts, rosemary extract, chicken extract, and reduced coenzyme Q10 was tested. The amount of each sample added was the same, and the specific testing method was the same as in Example 5. The results are shown in Table 13.

[0168] Table 13: Antioxidant Activity of Bird's Nest Peptide Complex

[0169]

[0170] The results in Table 13 show that the complex formed by combining the bird's nest peptide copolymer with one or more of larch cambium and xylem extracts, rosemary extract, chicken extract, and reduced coenzyme Q10 can significantly enhance the antioxidant activity of cells. Compared with the individual activities of bird's nest peptide copolymer, larch cambium and xylem extract, rosemary extract, chicken extract, and reduced coenzyme Q10, the activity of these components is significantly greater. This indicates that the components in the complex prepared in this application synergistically achieve excellent antioxidant effects, reduce the degree of oxidative stress in cells, thereby enabling cells to perform normal physiological functions and enhance the efficacy and activity of bird's nest peptides.

[0171] Experimental Example 9: Inhibitory Neuroinflammatory Activity of Bird's Nest Peptide Complex

[0172] The samples from Examples 8-15, larch cambium and xylem extracts, rosemary extract, chicken extract, and reduced coenzyme Q10 were tested for inhibiting neuroinflammation. The amount of each sample added was the same, and the specific test method was the same as in Example 6. The results are shown in Table 14.

[0173] Table 14: Inhibitory neuroinflammatory activity of bird's nest peptide complex

[0174]

[0175] The results in Table 14 show that the complex formed by combining the bird's nest peptide copolymer with one or more of larch cambium and xylem extracts, rosemary extract, chicken extract, and reduced coenzyme Q10 has a significant effect on improving neuroinflammation. Compared with the individual bird's nest peptide copolymer, larch cambium and xylem extract, rosemary extract, chicken extract, and reduced coenzyme Q10, the activity of these components is significantly higher. This indicates that the components in the complex prepared in this application synergistically achieve excellent neuroinflammation-improving activity, reduce the degree of inflammation of nerve cells, regulate nerve activity, enhance cognition, and maintain the homeostasis of the central nervous system.

[0176] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0177] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A bird's nest peptide copolymer that effectively combats fatigue and regulates mood, characterized in that, It is obtained by copolymerization of raw materials containing bird's nest peptides and oak extract; The oak extract is prepared by extracting the heartwood of oak with water, and the active ingredients of the oak extract include phenolic compounds. The copolymerization process includes: mixing the raw materials with a solvent and ultrasonically treating them to prepare the effective anti-fatigue and mood-regulating bird's nest peptide copolymer.

2. The effective anti-fatigue and mood-regulating bird's nest peptide copolymer according to claim 1, characterized in that, The mass ratio of the bird's nest peptide to the oak extract is 1:(0.02-6).

3. The effective anti-fatigue and mood-regulating bird's nest peptide copolymer according to claim 1, characterized in that, The frequency of the ultrasound is 20 kHz-80 kHz; and / or the power of the ultrasound is 100 W-800 W; and / or the temperature of the ultrasound is 25 ℃-60 ℃; and / or the duration of the ultrasound is 0.5 h-2.0 h.

4. The effective anti-fatigue and mood-regulating bird's nest peptide copolymer according to any one of claims 1 to 3, characterized in that, It also includes the step of freeze-drying the ultrasonically treated material.

5. A bird's nest peptide complex that effectively combats fatigue and regulates mood, characterized in that, The active components of the bird's nest peptide complex include a first active component and a second active component; The first active component includes the effective anti-fatigue and mood-regulating bird's nest peptide copolymer as described in any one of claims 1 to 4; The second active ingredient includes one or more of larch extract, rosemary extract, chicken extract, and coenzyme Q10.

6. The effective anti-fatigue and mood-regulating bird's nest peptide complex according to claim 5, characterized in that, The mass ratio of the first active component to the second active component is 1:(0.5-2).

7. The effective anti-fatigue and mood-regulating bird's nest peptide complex according to claim 5 or 6, characterized in that, The larch extract is an extract of the cambium and xylem of larch, and the extract of the cambium and xylem of larch includes piezin.

8. The effective anti-fatigue and mood-regulating bird's nest peptide complex according to claim 5 or 6, characterized in that, The rosemary extract includes rosmarinic acid, and the rosemary extract is extracted from rosemary raw material.

9. The effective anti-fatigue and mood-regulating bird's nest peptide complex according to claim 5 or 6, characterized in that, The chicken extract includes imidazole dipeptide, and the chicken extract is extracted from chicken raw materials.

10. The effective anti-fatigue and mood-regulating bird's nest peptide complex according to claim 5 or 6, characterized in that, The coenzyme Q10 includes reduced coenzyme Q10.

11. Bird's nest peptide products, characterized in that, The bird's nest peptide products include the effective anti-fatigue and mood-regulating bird's nest peptide copolymers according to any one of claims 1 to 4 and / or the effective anti-fatigue and mood-regulating bird's nest peptide complexes according to any one of claims 5 to 10.

Images