Multifunctional bioactive peptide based on highland barley wine lees protein and preparation method thereof

Abstract

The application belongs to the technical field of biotechnology and particularly relates to a multifunctional bioactive peptide based on highland barley wine lees protein and a preparation method thereof, wherein the amino acid sequence of the bioactive peptide is shown as SEQ ID NO:1. The bioactive peptide has small molecular weight, is easy to absorb, can inhibit tyrosinase activity and efficiently scavenge free radicals, and has the advantages of non-toxicity, non-carcinogenicity, high hydrophilicity, long half-life and the like through bioinformatics analysis prediction, and can be applied to the development of drugs for treating skin pigmentation and oxidative stress related diseases or daily chemicals for whitening, anti-aging, antioxidant and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a multifunctional bioactive peptide based on highland barley wine lees protein and its preparation method. Background Technology

[0002] Barley is an important highland specialty grain, and its distiller's grains, a byproduct of barley wine brewing, are rich in nutrients such as protein, dietary fiber, vitamins, and minerals. Barley distiller's grains protein, as a high-quality plant protein source, has excellent nutritional value and development potential. However, currently, the utilization efficiency of barley distiller's grains protein is low, with most being treated as feed or fertilizer, failing to fully realize its potential bioactive value.

[0003] Small molecule peptides can be obtained by processing highland barley lees protein through enzymatic hydrolysis and other methods. Some of these peptides may have specific biological activities, such as antioxidant and blood pressure lowering functions. However, there is relatively little research on characteristic peptides with clear functions in the enzymatic hydrolysis products of highland barley lees protein.

[0004] Therefore, the screening of barley lees protein peptides with multiple biological activities and clear efficacy is of great significance for the application of barley lees protein products in the development of pharmaceuticals or daily chemical products. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, this invention provides a multifunctional bioactive peptide based on highland barley lees protein and its preparation method. The bioactive peptide of this invention has a small molecular weight, is easily absorbed, can inhibit tyrosinase activity and efficiently scavenge free radicals, and, according to bioinformatics analysis, has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and a long half-life. It can be applied to the development of drugs for the treatment of skin pigmentation and oxidative stress-related diseases, or to daily chemical products for whitening, anti-aging, and antioxidant purposes, showing broad application prospects.

[0006] This invention is based on the inventor's discoveries and understanding of the following problems:

[0007] In the research process of bioactive peptides from highland barley lees protein, the inventors screened a bioactive peptide, DHWDIM, which has the activity of inhibiting tyrosinase, through a large number of virtual screening operations and experimental verifications. The amino acid sequence of this bioactive peptide is: aspartic acid-histidine-tryptophan-asparagine-isoleucine-methionine (Asp-His-Trp-Asn-Ile-Met, DHWDIM). The inventors further prepared this bioactive peptide using the Fmoc solid-phase synthesis method and verified through experiments that it has the biological activities of simultaneously inhibiting tyrosinase activity and efficiently scavenging free radicals.

[0008] In a first aspect, the present invention provides a bioactive peptide. According to embodiments of the present invention, the amino acid sequence of the bioactive peptide is shown in SEQ ID NO: 1. The bioactive peptide of the present invention has a small molecular weight, is easily absorbed, can inhibit tyrosinase activity and efficiently scavenge free radicals, and, according to bioinformatics analysis, has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and a long half-life. It can be applied to the development of drugs for the treatment of skin pigmentation and oxidative stress-related diseases, or to daily chemical products for whitening, anti-aging, and antioxidant purposes, showing broad application prospects.

[0009] In a second aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the bioactive peptide described in the first aspect. According to an embodiment of the present invention, the bioactive peptide encoded by the nucleic acid molecule has the advantages of small molecular weight, easy absorption, ability to inhibit tyrosinase activity and efficiently scavenge free radicals, and, as predicted by bioinformatics analysis, non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. The nucleic acid molecule enables the efficient expression of this bioactive peptide, facilitates its large-scale industrial production, and provides convenience for the development and application of drugs for the treatment of skin pigmentation and oxidative stress-related diseases, as well as whitening, anti-aging, and antioxidant daily chemical products.

[0010] In a third aspect, the present invention provides a construct comprising the nucleic acid molecule described in the second aspect. According to embodiments of the present invention, the construct enables the nucleic acid molecule to be efficiently expressed in host cells, thereby achieving stable and efficient production of the aforementioned bioactive peptide.

[0011] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, it comprises the nucleic acid molecule described in the second aspect or the construct described in the third aspect. According to embodiments of the invention, the recombinant cell can efficiently express the aforementioned bioactive peptide under suitable conditions. This bioactive peptide has a small molecular weight, is easily absorbed, can inhibit tyrosinase activity, and efficiently scavenge free radicals. Furthermore, bioinformatics analysis predicts that it has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and a long half-life. The recombinant cell enables the efficient expression of this bioactive peptide, facilitating its large-scale industrial production.

[0012] In a fifth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, it comprises one or more of the following: the bioactive peptide described in the first aspect, the nucleic acid molecule described in the second aspect, the construct described in the third aspect, and the recombinant cell described in the fourth aspect.

[0013] Those skilled in the art will understand that the features and advantages described above for bioactive peptides, nucleic acid molecules, constructs or recombinant cells also apply to this pharmaceutical composition, and will not be repeated here.

[0014] According to embodiments of the present invention, the above-described pharmaceutical composition may further have the following additional technical features:

[0015] According to embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0016] In a sixth aspect, the present invention provides a daily chemical product. According to embodiments of the present invention, it comprises: the bioactive peptides described in the first aspect. The daily chemical product according to embodiments of the present invention has whitening, antioxidant, and other effects.

[0017] According to embodiments of the present invention, the above-mentioned daily chemical products may also have the following additional technical features:

[0018] According to an embodiment of the present invention, the daily chemical product further includes excipients acceptable in daily chemical products.

[0019] According to embodiments of the present invention, the daily chemical products include one or more of creams, lotions, aqueous solutions, gels, oils, wax-based products, and sheet products.

[0020] In a seventh aspect, the present invention provides an antioxidant article. According to embodiments of the invention, it comprises: the bioactive peptide described in the first aspect. The antioxidant article according to embodiments of the invention contains the aforementioned bioactive peptide capable of reducing free radical levels, the bioactive peptide having antioxidant effects and applicable to antioxidant applications.

[0021] In an eighth aspect of the invention, the invention proposes the use of the bioactive peptides of the first aspect, the nucleic acid molecules of the second aspect, the constructs of the third aspect, or the recombinant cells of the fourth aspect in the preparation of pharmaceuticals, daily chemical products, or antioxidant products, wherein the pharmaceuticals have at least one of the following uses: prevention and / or treatment of skin pigmentation; prevention and / or treatment of oxidative stress; the daily chemical products have at least one of the following uses: whitening; anti-aging; antioxidant; and the antioxidant products are used for antioxidant purposes.

[0022] Those skilled in the art will understand that the features and advantages described above for bioactive peptides, nucleic acid molecules, constructs or recombinant cells also apply to this application, and will not be repeated here.

[0023] In a ninth aspect of the invention, a method for inhibiting tyrosinase activity in a sample is provided. According to an embodiment of the invention, the method includes contacting the sample with the bioactive peptide described in the first aspect. The method according to an embodiment of the invention inhibits the level of tyrosinase activity in the sample by co-culturing the sample with the bioactive peptide of the invention.

[0024] In a tenth aspect of the invention, a tyrosinase inhibitor is provided. According to embodiments of the invention, it comprises the bioactive peptide described in the first aspect. The inhibitor according to embodiments of the invention can specifically inhibit tyrosinase activity through the action of the bioactive peptide described in the first aspect.

[0025] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0026] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0027] Figure 1 This is a chromatogram of the HPLC detection results of the pure bioactive peptide DHWDIM sample in Example 1 of the present invention;

[0028] Figure 2 This is a graph showing the MS detection results of the pure bioactive peptide DHWDIM sample in Example 1 of this invention.

[0029] Figure 3 This is a schematic diagram simulating the docking of the bioactive polypeptide DHWDIM with tyrosinase molecules in Example 2 of the present invention;

[0030] Figure 4 This is a graph showing the evaluation results of the whitening and antioxidant functions of the bioactive polypeptide DHWDIM in Example 3 of the present invention. Detailed Implementation

[0031] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0032] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0033] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0034] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0035] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0036] Terms and Definitions

[0037] In this paper, the term "free radical scavenging rate" is one of the important indicators for measuring antioxidant capacity. Specifically, it refers to the change in absorbance of the free radical solution before and after the addition of the scavenging agent in a free radical scavenging experiment. In the free radical scavenging rate determination experiment of the present invention, the scavenging agent is the bioactive polypeptide DHWDIM of the present invention.

[0038] In this article, the term "tyrosinase" refers to a copper-containing oxidoreductase, which is the rate-limiting enzyme in melanin synthesis. It is widely present in the human body and participates in the conversion of tyrosine into melanin. By inhibiting tyrosinase activity, melanin production can be reduced, thereby improving skin tone, whitening the skin, and is used to treat pigmentation-related diseases and delay skin aging.

[0039] Bioactive peptides

[0040] This invention proposes a bioactive peptide. According to embodiments of the invention, the amino acid sequence of the bioactive peptide is shown in SEQ ID NO: 1. The bioactive peptide of this invention has a small molecular weight, is easily absorbed, can inhibit tyrosinase activity, and efficiently scavenge free radicals. Furthermore, bioinformatics analysis predicts that it has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and a long half-life. It can be applied to the development of drugs for treating skin pigmentation and oxidative stress-related diseases, or to daily chemical products for whitening, anti-aging, and antioxidant purposes, showing broad application prospects.

[0041] According to an embodiment of the present invention, the amino acid sequence of the bioactive peptide is shown in SEQ ID NO: 1.

[0042] DHWDIM (SEQ ID NO: 1).

[0043] It should be noted that the amino acid sequences mentioned in this invention are all shown in the order from N-terminus to C-terminus.

[0044] For example, the bioactive peptides of the present invention have the ability to inhibit tyrosinase activity, thereby enabling them to be further used in the development of drugs for the treatment of diseases such as skin pigmentation or daily chemical products for whitening and anti-aging; the bioactive peptides of the present invention also have the ability to efficiently scavenge free radicals, thereby enabling them to be further used in the development of drugs for the treatment of oxidative stress-related diseases or daily chemical products for antioxidant purposes.

[0045] Nucleic acid molecules

[0046] This invention proposes a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the aforementioned bioactive peptide. According to an embodiment of the invention, the aforementioned bioactive peptide encoded by the nucleic acid molecule has the advantages of small molecular weight, easy absorption, ability to inhibit tyrosinase activity and efficiently scavenge free radicals, and bioinformatics analysis predicts that it is non-toxic, non-carcinogenic, highly hydrophilic, and has a long half-life. The nucleic acid molecule enables the efficient expression of this bioactive peptide, facilitates its large-scale industrial production, and provides convenience for the development and application of drugs for the treatment of skin pigmentation and oxidative stress-related diseases, as well as whitening, anti-aging, and antioxidant daily chemical products.

[0047] It should be noted that those skilled in the art should understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands; although in most cases only one strand is given, the other complementary strand is actually disclosed as well; in addition, the nucleic acid molecule sequences in this invention include DNA or RNA forms, and disclosing one of them means that the other is also disclosed.

[0048] Construct

[0049] This invention proposes a construct. According to embodiments of the invention, it includes the aforementioned nucleic acid molecule. According to embodiments of the invention, the construct enables the nucleic acid molecule to be efficiently expressed in host cells, thereby achieving stable and efficient production of the aforementioned bioactive peptide.

[0050] Exemplarily, the construct may be a vector; it should be noted that, for the purposes of this document, a vector refers to a nucleic acid molecule capable of self-replication within a suitable host, which transfers the inserted nucleic acid molecule to host cells and / or between host cells; the vector may include vectors primarily used for inserting DNA or RNA into cells, vectors primarily used for replicating DNA or RNA, and expression vectors primarily used for transcription and / or translation of DNA or RNA; the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell; by culturing a suitable host cell containing the vector, the vector can produce the aforementioned bioactive peptides, the vector including viral vectors, plasmids, bacteriophages, etc.

[0051] Recombinant cells

[0052] This invention proposes a recombinant cell. According to embodiments of the invention, it includes the aforementioned nucleic acid molecule or the aforementioned construct. According to embodiments of the invention, the recombinant cell can efficiently express the aforementioned bioactive peptide under suitable conditions. This bioactive peptide has a small molecular weight, is easily absorbed, can inhibit tyrosinase activity and efficiently scavenge free radicals, and, according to bioinformatics analysis, has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and a long half-life. The recombinant cell method enables the efficient expression of this bioactive peptide, facilitating its large-scale industrial production.

[0053] Pharmaceutical Composition

[0054] This invention provides a pharmaceutical composition. According to embodiments of the invention, it comprises one or more of the aforementioned bioactive peptides, nucleic acid molecules, constructs, and recombinant cells.

[0055] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0056] According to embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. Thus, the addition of excipients allows the pharmaceutical composition to better exert the efficacy of the bioactive peptides.

[0057] In this article, the term "pharmaceuticalally acceptable" applies to humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), and "pharmaceuticalally acceptable excipient" is a substance with a reasonable benefit / risk ratio.

[0058] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0059] Those skilled in the art will understand that the features and advantages described above for bioactive peptides, nucleic acid molecules, constructs or recombinant cells also apply to this pharmaceutical composition, and will not be repeated here.

[0060] Daily chemical products

[0061] This invention proposes a daily chemical product. According to embodiments of the invention, it includes the aforementioned bioactive peptides. The daily chemical product according to embodiments of the invention has whitening, antioxidant, and other effects.

[0062] According to embodiments of the present invention, the daily chemical product further includes excipients acceptable to daily chemical products. Thus, the addition of excipients allows the daily chemical product to better exert the efficacy of bioactive peptides and become more acceptable to consumers.

[0063] According to embodiments of the present invention, the daily chemical products include one or more of the following: creams, lotions, aqueous solutions, gels, oils, wax-based products, and sheet masks. The daily chemical products according to embodiments of the present invention, while fully realizing their efficacy, can meet the usage habits of different consumers and provide them with more choices.

[0064] For example, the daily chemical products may include, but are not limited to: gels, serums, moisturizers, face creams, toners, shower gels, shampoos, face masks, etc.

[0065] Antioxidant products

[0066] This invention provides an antioxidant product. According to embodiments of the invention, it comprises the aforementioned bioactive peptide. The antioxidant product according to embodiments of the invention contains the aforementioned bioactive peptide capable of reducing free radical levels, and this bioactive peptide has antioxidant effects and can be used for antioxidant applications.

[0067] Applications in the preparation of pharmaceuticals, daily chemical products, or antioxidant products

[0068] This invention proposes the use of the aforementioned bioactive peptides, nucleic acid molecules, constructs, or recombinant cells in the preparation of pharmaceuticals, daily chemical products, or antioxidant products. The pharmaceuticals have at least one of the following uses: prevention and / or treatment of skin pigmentation; the daily chemical products have at least one of the following uses: whitening; anti-aging; antioxidant; and the antioxidant products are used for antioxidant purposes.

[0069] Those skilled in the art will understand that the features and advantages described above for bioactive peptides, nucleic acid molecules, constructs or recombinant cells also apply to this application, and will not be repeated here.

[0070] Methods to inhibit tyrosinase activity in samples

[0071] This invention provides a method for inhibiting tyrosinase activity in a sample. According to embodiments of the invention, the method includes contacting the sample with the aforementioned bioactive peptide. The method according to embodiments of the invention inhibits tyrosinase activity levels in the sample by co-culturing the sample with the bioactive peptide of the invention.

[0072] Tyrosinase inhibitors

[0073] This invention provides a tyrosinase inhibitor. According to embodiments of the invention, it comprises the aforementioned bioactive peptide. The inhibitor according to embodiments of the invention can specifically inhibit tyrosinase activity through the action of the aforementioned bioactive peptide.

[0074] The amino acid sequences involved in this invention are detailed in Table 1.

[0075] Table 1. Amino acid sequences involved in this invention.

[0076] SEQ ID NO: sequence 1 DHWDIM 2 MDEWQW 3 EPFPWQPQ 4 SPVPMPQ 5 SDGTWWE 6 ILPPG

[0077] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0078] Example 1: Isolation and preparation of bioactive polypeptide DHWDIM

[0079] 1. Extraction of highland barley wine lees protein by alcohol-alkali method

[0080] Barley lees powder (purchased from Tibet Qizheng Tibetan Medicine Co., Ltd.) and an alcohol-alkali mixture [V(95% ethanol):V(0.5mol / L NaOH) = 1:2] were thoroughly mixed at a solid-liquid ratio of 1:40 and stirred for 4 hours. After centrifugation, the supernatant was collected, and the pH of the supernatant was adjusted to 4.7 using 1mol / L HCl. After adjustment, the solution was allowed to stand for 30 minutes, and then centrifuged at 8000×g for 20 minutes. The precipitate was collected, which was the crude protein precipitate. The crude barley lees protein precipitate was washed three times with distilled water and then dissolved in distilled water to obtain a barley lees protein solution. The pH of the barley lees protein solution was then adjusted to 7.0 using 0.5mol / L NaOH solution, and then freeze-dried to obtain barley lees protein, which was stored at -20℃ for later use.

[0081] 2. Alkaline protease hydrolysis of barley wine lees protein

[0082] The barley lees protein obtained in step 1 was mixed with 35 mM sodium phosphate buffer (pH 6.2) at a mass-to-volume ratio of 1:20. The pH of the solution was then adjusted to 7.0 using 1 mol / L NaOH. 5% alkaline protease (w / w, purchased from Sigma) was added, and the mixture was stirred to obtain the barley lees protein hydrolysate. This hydrolysate was then placed in a water bath and shaken for 2 hours at 50°C. After hydrolysis, the barley lees protein hydrolysate was boiled in a water bath for 10 minutes to inactivate any residual alkaline protease. After cooling to room temperature, the solution was centrifuged at 10000×g for 15 minutes at 4°C, and the supernatant was collected to obtain the barley lees protein hydrolysate solution for later use.

[0083] 3. Isolation, purification, and sequence identification of highland barley distillers' grains protein hydrolysate

[0084] The 12 mL of barley distillers' grains protein hydrolysate solution obtained in step 2 was transferred to a 3 kDa ultrafiltration tube and centrifuged at 5000 × g for 30 min at 4 °C to obtain a barley distillers' grains protein hydrolysate sample with a molecular weight <3 kDa. The obtained sample was then desalted using a C18 desalting column to obtain a desalted sample with a molecular weight <3 kDa. LC-MS / MS analysis was performed on this sample using an online nanojet ion source. The system used was a Q-Exactive Plus mass spectrometer (ThermoFisher Scientific, MA, USA) with a tandem EASY-nanoLC 1200. The specific LC-MS / MS analysis steps are as follows:

[0085] 1 μL of desalted barley lees protein hydrolysate sample with a molecular weight <3kDa fraction was loaded (analytical column: Acclaim PepMap C18, 75μm×25cm). The sample was separated by a gradient at a rate of 60 min. The column flow rate was 300 nL / min, the column temperature was 40℃, and the electrospray voltage was 2kV. The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was 80% ACN solution containing 0.1% formic acid. The gradient started from 2% of the B phase, increased non-linearly to 35% in 47 min, increased to 100% in 1 min, and was maintained for 12 min.

[0086] The Q-Exactive Plus mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters are set as follows:

[0087] (1)MS

[0088] Scan range (m / z): 200~2000; Resolution: 70000; AGC target: 3e6; Maximum injection time: 50ms;

[0089] (2) HCD-MS / MS

[0090] Resolution: 17500; AGC target: 1e5; Maximum injection time: 45ms; Collision energy: 28; Dynamic exclusion time: 30s.

[0091] The obtained tandem mass spectra were then analyzed using PEAKS Studio version 10.6 (BioinformaticsSolutions Inc., Waterloo, Canada). The database used was uniprot-Hordeum_vulgare_subsp_vulgare (version 2023, 34528 entries), with the enzymatic digestion set to none. The search parameters were: fragment ion mass tolerance: 0.02 Da; parent ion mass tolerance: 10 ppm; variable modifications: Oxidation (M) 15.99; peptide card value: -10lgP ≥ 20. For peptides not found in the database, ALC (%) ≥ 80 was set to obtain the target peptide sequence.

[0092] Some target peptide sequences are shown in Table 2.

[0093] Table 2 Partial Target Peptide Sequences

[0094] Target peptide sequence Serial Number DHWDIM SEQ ID NO: 1 MDEWQW SEQ ID NO: 2 EPFPWQPQ SEQ ID NO: 3 SPVPMPQ SEQ ID NO: 4 SDGTWWE SEQ ID NO: 5 ILPPG SEQ ID NO: 6

[0095] 4. Virtual screening of bioactive peptides based on bioinformatics

[0096] Non-toxic, non-allergenic, non-carcinogenic, and highly bioactive are the basic requirements for screening bioactive peptides. Small molecule peptides with 2-10 amino acid residues and a molecular weight below 1000 Da can avoid gastrointestinal digestion, overcoming the drawback of proteins being destroyed by digestive enzymes and thus unable to be taken orally. Therefore, based on bioinformatics technology, the inventors preliminarily screened novel bioactive peptides that meet the development requirements based on the criteria of non-toxic, non-allergenic, non-carcinogenic, highly bioactive, easily absorbed by the human intestine, and stable in vitro existence. The specific steps are as follows:

[0097] First, the PeptideRanker online system (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.Php) was used to predict the bioactivity of the target peptide sequence obtained in step 3. Then, ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) was used to predict the water solubility and toxicity of the target peptide sequence obtained in step 3. Next, Expasy (https: / / web.expasy.org / protparam / ) was used to analyze the isoelectric point and half-life of the target peptide sequence obtained in step 3. Finally, admetSAR (http: / / lmmd.ecust.edu.cn / admetSAR1 / predict / ) was used to predict the human intestinal absorption and carcinogenicity of the target peptide sequence obtained in step 3.

[0098] The superior results of in vitro functional prediction of peptides by virtual screening are shown in Table 3.

[0099] Table 3. Optimal results of in vitro functional prediction for peptide virtual screening.

[0100]

[0101]

[0102] The results showed that six peptide sequences were obtained through screening. Among them, DHWDIM had a high PeptideRanker score (0.819857), low hydrophilicity (-0.17), and a short half-life (1.1 hours), indicating that it may have good biological activity and stability. Considering the activity score, toxicity, safety, and physicochemical properties of the peptide sequences obtained through screening, and since no relevant studies on DHWDIM were found in public databases, DHWDIM was selected for subsequent experiments.

[0103] 5. Artificial synthesis of bioactive peptide DHWDIM

[0104] The inventors prepared the bioactive peptide DHWDIM using the Fmoc solid-phase synthesis method. The specific steps are as follows:

[0105] (1) Solvent pretreatment

[0106] Before use, N,N-dimethylformamide (DMF) and methanol were soaked overnight using G3-pore molecular sieves to remove impurities and moisture from the solvents.

[0107] (2) Resin swelling

[0108] Weigh 2.0g of blank Wang resin into a clean, dry reaction tube, add 15mL of DMF, and activate at room temperature for 30min.

[0109] (3) Introduce the first amino acid

[0110] At room temperature, DMF from step 2 was removed by sand core filtration, and 1 mmol of 5-fold excess of the first amino acid at the C-terminus of the target sequence, 5-fold excess of 4-dimethylaminopyridine (DMAP), 5-fold excess of N,N-diisopropylcarbodiimide (DIC), and 60 mL of DMF were added as solvent. The reaction was carried out at room temperature for 3 h. After the reaction was completed, the mixture was washed 5 times with 6 mL of DMF each time. Then, 6 mL of pyridine and acetic anhydride in a 1:1 volume ratio was added, and the mixture was reacted for 30 min. After the reaction was completed, the mixture was washed 5 times with 6 mL of DMF each time.

[0111] (4) Removal of Fmoc protecting groups

[0112] Remove the solvent from step 3 by core filtration. Add 10 mL of 20% piperidine DMF solution to the above resin, stir for 10 min under N2 protection, and filter out the solution. Add another 10 mL of 20% piperidine DMF solution, stir under N2 protection for 5 min, and filter out the solution again. Repeat the above operation twice. Then wash with DMF 4 times and methanol 2 times, 6 mL each time.

[0113] (5) Detection of Fmoc protecting group removal effect by ninhydrin

[0114] Take out a small amount of resin treated in step 4, wash it three times with methanol, add one drop each of ninhydrin, KCN and phenol solution, heat at 110°C for 5 minutes. If it turns dark blue, it is a positive reaction, indicating that the Fmoc protecting group has been completely removed and the next reaction operation can be carried out. If it is colorless, it means that the protecting group has not been completely removed and the above step (4) Fmoc protecting group removal operation needs to be repeated.

[0115] (6) Introduction of the second amino acid and removal of the Fmoc protecting group

[0116] Weigh out 3 times the molar excess of the second amino acid at the C-terminus of the target sequence, 3 times the molar excess of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), and 3 times the molar excess of 1-hydroxybenzotriazole (HOBT) into the above reaction tube, and add an appropriate amount of DMF to completely dissolve them; then add 10 times the molar excess of N,N-diisopropylethylamine (DIPEA), and react at room temperature for 40 min. After the reaction is complete, wash 5 times with DMF, 6 mL each time. Take a small amount of the above-treated resin and test it with ninhydrin reagent. If it turns colorless, then add 10 mL of 20% piperidine DMF solution to remove the Fmoc protecting group, repeating twice, for 10 min and 5 min respectively. After that, wash 4 times with DMF and 2 times with methanol, 6 mL each time. Take a small amount of the above-treated resin again and test it with ninhydrin reagent. If the test is blue, the next reaction can proceed.

[0117] (7) Introducing amino acids again

[0118] Repeat the operation described in step (6) until the last amino acid at the N-terminus of the target sequence is synthesized. After removing the Fmoc protecting group, the solution is dried to obtain a resin containing the target bioactive peptide DHWDIM.

[0119] (8) Resin shedding and separation and detection of pure bioactive peptide DHWDIM

[0120] Cutting: Add 6 times the resin volume of cutting solution (95% trifluoroacetic acid: 2% triisopropylsilane: 2% ethylenedithiol: 1% H2O), shake on a shaker for 2 hours, filter the reaction solution to obtain a trifluoroacetic acid solution containing the target peptide, dry the lysis buffer with nitrogen, precipitate with diethyl ether and centrifuge, then wash the precipitate 3 times with anhydrous diethyl ether to obtain a white solid, dissolve it in pure water, and desalt and purify the crude peptide by HPLC. After freeze-drying, crystals are precipitated, which is the pure bioactive peptide DHWDIM, and stored at -20℃ for later use.

[0121] (9) Quality testing of pure bioactive peptide DHWDIM

[0122] Take a small amount of the pure bioactive peptide DHWDIM obtained in step (8), dissolve it in trifluoroacetic acid solution by ultrasound to obtain the test solution; place the test solution in a high performance liquid chromatograph for detection to perform the quality test of the pure bioactive peptide DHWDIM.

[0123] The HPLC parameters are set as follows:

[0124] Chromatographic column: ZORBAX SB-C18, 4.6×250mm, 5μm; Aqueous phase: 100% water plus 0.1% trifluoroacetic acid; Organic phase: 100% acetonitrile plus 0.1% trifluoroacetic acid; Flow rate: 1mL / min; Injection volume: 10μL; Detection wavelength: 220nm;

[0125] The MS parameter settings are as follows:

[0126] Ion source: Electrospray ionization source (ESI source); Atomizing gas flow rate: 1.5 L / min; CDL: -20.0 V; CDL temperature: 250 °C; Heating block temperature: 200 °C; Ion source voltage: +4.5 kV; Detector voltage: 1.5 kV; Mobile phase flow rate: 0.2 mL / min; Mobile phase ratio: 50% H2O / 50% ACN.

[0127] The HPLC results of the pure bioactive peptide DHWDIM sample are shown below. Figure 1 MS detection results of pure bioactive peptide DHWDIM sample are shown in [reference needed]. Figure 2 .

[0128] The results showed that the purity of the bioactive peptide DHWDIM obtained in step (8) was greater than 95% as determined by HPLC detection; and the polypeptide sequence of the bioactive peptide DHWDIM obtained in step (8) was the target sequence DHWDIM as determined by MS detection.

[0129] Example 2: Molecular docking and interaction force analysis of bioactive polypeptide DHWDIM

[0130] Using the bioactive peptide DHWDIM screened in Example 1 as a ligand and tyrosinase as a receptor, the inventors used molecular docking technology to clarify the interaction site and interaction force between the peptide DHWDIM and the active enzyme, thereby achieving theoretical verification of its skin whitening function.

[0131] The crystal structure of tyrosinase (PDB number: 2Y9X) was obtained from the RCSB protein database (http: / / www.rcsb.org / ). Then, ZDOCK was used to perform molecular docking between the bioactive peptide DHWDIM and tyrosinase. After screening for the optimal binding conformation, the binding force between the bioactive peptide DHWDIM and tyrosinase was analyzed by PDBePISA, and the interaction force was further analyzed in detail using LigPlus+. Finally, the binding between the ligand (bioactive peptide DHWDIM) and the receptor (tyrosinase) was visualized using PyMOL software.

[0132] Simulation of the docking of bioactive peptide DHWDIM with tyrosinase molecules Figure 3 .

[0133] The results showed that the molecular binding energy between the bioactive peptide DHWDIM and the amino acid residues of tyrosinase was -5.1 kcal / mol. The amino acid residues Asp1(C), His2(C), Trp3(C), Asp4(C), Ile5(C), and Met6(C) of the bioactive peptide DHWDIM were tightly bound to the key amino acid residues of tyrosinase (TYR) through various interactions. Specifically, Asp1(C) and His2(C) formed stable bindings with Val283(A) and Ser282(A) of tyrosinase through hydrogen bonding, respectively; Trp3(C) and Asp4(C) bound through... The bioactive peptide DHWDIM binds to tyrosinase residues such as Arg268(A), Phe264(A), and Val248(A) through strong hydrophobic interactions, enhancing the stability of the bioactive peptide DHWDIM-tyrosinase complex. Furthermore, the side chains of Met6(C) and Ile5(C) form strong hydrophobic interactions with tyrosinase residues Pro284(A) and His244(A), further solidifying the stability of their binding. Therefore, it is hypothesized that the bioactive peptide DHWDIM binds tightly to the active site residues of tyrosinase through the synergistic effect of hydrophobic interactions and hydrogen bonding, thereby inhibiting tyrosinase activity.

[0134] The above results indicate that the bioactive polypeptide DHWDIM has a good affinity for tyrosinase, suggesting that it has a good whitening effect.

[0135] Example 3: Experimental verification of the whitening and antioxidant functions of the bioactive polypeptide DHWDIM

[0136] 1. Determination of the tyrosinase activity inhibition rate of the bioactive polypeptide DHWDIM

[0137] 50 μL of a pure 8 mg / mL solution of the bioactive peptide DHWDIM obtained in Example 1 (the blank control group was replaced with 0.2 mol / L pH 7.5 phosphate buffer) and 50 μL of tyrosinase solution were incubated at 37°C for 5 min. Then, 50 μL of L-tyrosine substrate solution was added. The absorbance value was recorded at 475 nm every 20 s using a microplate reader, and the change in absorbance value was recorded continuously for 10 min. The tyrosinase activity inhibition rate was calculated according to Equation 1.

[0138]

[0139] In Formula 1: A: Absorbance of the 0.2 mol / L pH 7.5 phosphate buffer group (blank control group); B: Absorbance of the bioactive peptide DHWDIM pure product reaction solution sample group (experimental group) obtained in Example 1.

[0140] 2. Determination of DPPH free radical scavenging rate of bioactive peptide DHWDIM

[0141] 50 μL of a 5 mg / mL pure solution of the bioactive peptide DHWDIM obtained in Example 1 (the blank control group was replaced with distilled water) and 50 μL of a 0.2 mmol / L DPPH solution (dissolved in methanol) were mixed and incubated at 37°C in the dark for 20 min. The absorbance was recorded at 517 nm using a microplate reader. The DPPH free radical scavenging rate was calculated according to Equation 2.

[0142]

[0143] In Formula 2: A: absorbance of the distilled water group (blank control group); B: absorbance of the bioactive peptide DHWDIM pure product reaction solution sample group (experimental group) obtained in Example 1.

[0144] The evaluation results of the whitening and antioxidant functions of the bioactive polypeptide DHWDIM are shown in the figure. Figure 4 .

[0145] The results showed that the tyrosinase activity inhibition rate of the bioactive peptide DHWDIM obtained in Example 1 was 45.04%, which can exert good whitening and other effects; the DPPH free radical scavenging rate of the bioactive peptide DHWDIM obtained in Example 1 was 46.44%, which can exert good whitening and antioxidant capabilities.

[0146] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0147] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A bioactive peptide, characterized in that, The amino acid sequence of the bioactive peptide is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bioactive peptide of claim 1.

3. A construct, characterized in that, Includes the nucleic acid molecule as described in claim 2.

4. A recombinant cell, characterized in that, Includes the nucleic acid molecule of claim 2 or the construct of claim 3.

5. A pharmaceutical composition, characterized in that, include: One or more of the bioactive peptide of claim 1, the nucleic acid molecule of claim 2, the construct of claim 3, and the recombinant cell of claim 4.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition further includes pharmaceutically acceptable excipients.

7. A daily chemical product, characterized in that, include: The bioactive peptide according to claim 1.

8. The daily chemical product according to claim 7, characterized in that, The daily chemical products further include acceptable excipients in the daily chemical products.

9. The daily chemical product according to claim 7, characterized in that, The daily chemical products include one or more of the following: creams, lotions, liquids, gels, oils, wax-based products, and sheet products.

10. An antioxidant product, characterized in that, include: The bioactive peptide according to claim 1.

11. The use of the bioactive peptide of claim 1, the nucleic acid molecule of claim 2, the construct of claim 3, or the recombinant cell of claim 4 in the preparation of pharmaceuticals or daily chemical products, wherein the pharmaceuticals have at least one of the following uses: Prevention and / or treatment of skin pigmentation; Prevention and / or treatment of oxidative stress; The daily chemical product has at least one of the following uses: Skin whitening; Antioxidant; Delaying skin aging through antioxidant and / or inhibition of tyrosinase activity.

12. A method for inhibiting the activity of tyrosinase in a sample, characterized in that, include: The sample is contacted with the bioactive peptide of claim 1, the method being for non-therapeutic purposes.

13. A tyrosinase inhibitor, characterized in that, include: The bioactive peptide according to claim 1.

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