Use of active peptides in the preparation of products for the prevention and treatment of ald

Abstract

This invention provides the application of active peptides in the preparation of products for the prevention and treatment of ALD. The amino acid sequence of the active peptides of this invention is shown in SEQ ID NO:1. These peptides not only activate ADH and ALDH enzymes to promote ethanol metabolism, but also significantly scavenge DPPH and ABTS free radicals, exerting antioxidant effects, thereby effectively preventing and / or treating ALD. Therefore, they are highly suitable for the preparation of products for the prevention and treatment of ALD.

Description

Application of bioactive peptides in the preparation of products for the prevention and treatment of ALD Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology. More specifically, it relates to the application of bioactive peptides in the preparation of products for the prevention and treatment of ALD. Background Technology

[0002] Alcoholic liver disease (ALD) is a common liver disease, mainly resulting from the interaction of multiple factors, including inflammatory responses, oxidative stress, intestinal endotoxins, inflammatory mediators, and nutritional imbalances induced directly or indirectly during the metabolism of ethanol and its derivatives. Clinical symptoms of ALD are nonspecific, ranging from asymptomatic to include right upper quadrant abdominal distension and pain, loss of appetite, fatigue, weight loss, and jaundice. As the condition worsens, neuropsychiatric symptoms, spider angiomas, and palmar erythema may also appear.

[0003] Bioactive peptides have received considerable attention in the treatment of diseases such as diabetes, tumors, thrombosis, and hypertension due to their advantages such as safety, non-toxicity, and suitability for targeted therapy. However, the existing bioactive peptide resources available for the prevention and treatment of ALD are still relatively scarce. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing an active peptide with an amino acid sequence as shown in SEQ ID NO:1, thereby enriching the resource library of active peptides with ALD prevention and treatment effects and providing more raw material options for products that prevent and treat ALD.

[0005] The primary objective of this invention is to provide an active peptide.

[0006] A second objective of this invention is to provide a biomaterial related to an active peptide.

[0007] A third objective of this invention is to provide the application of the above-mentioned active peptides or related biomaterials in the preparation of products for the prevention and treatment of ALD.

[0008] The fourth objective of this invention is to provide a product.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention provides an active peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0011] This bioactive peptide not only activates ADH and ALDH enzymes to promote ethanol metabolism, but also significantly scavenge DPPH and ABTS free radicals, exerting an antioxidant effect, thereby effectively preventing and / or treating ALD. It is highly suitable for preparing products for the prevention and treatment of ALD. Therefore, this invention also provides the above-mentioned bioactive peptide, related biomaterials containing the above-mentioned bioactive peptide, and their application in the preparation of products for the prevention and treatment of ALD. The related biomaterials are nucleic acid molecules capable of expressing the bioactive peptide, or recombinant bacteria, recombinant cells, recombinant plasmids, or expression cassettes containing the nucleic acid molecules.

[0012] Preferably, the prevention and treatment of ALD involves promoting ethanol metabolism.

[0013] More preferably, the promotion of ethanol metabolism is the activation of ADH enzyme.

[0014] More preferably, the promotion of ethanol metabolism involves activating the ALDH enzyme.

[0015] Preferably, the prevention and treatment of ALD is an antioxidant.

[0016] More preferably, the antioxidant is the scavenging of DPPH free radicals.

[0017] More preferably, the antioxidant is the scavenger of ABTS free radicals.

[0018] Based on this, the present invention also provides a product comprising the above-mentioned active peptide or the above-mentioned related biological material, wherein the related biological material is a nucleic acid molecule capable of expressing the active peptide, or a recombinant bacterium, recombinant cell, recombinant plasmid or expression cassette containing the nucleic acid molecule.

[0019] Preferably, the product is a drug.

[0020] More preferably, the dosage form of the drug is one of tablets, powder inhalers, aerosols, injections, granules, or powders.

[0021] More preferably, the drug also contains excipients.

[0022] Furthermore, the excipients include antioxidants (used to prevent the drug from deteriorating due to oxidation during storage or use, thus extending the drug's shelf life), vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and / or cocoa butter, used as solvents or carriers to help the drug components dissolve or disperse better), alginic acid (to maintain the physical stability of the drug and prevent drug particles from settling or agglomerating), isotonic salt solutions (used to adjust the osmotic pressure of the drug to meet the physiological requirements of the human body, thereby reducing irritation and adverse reactions to the human body), and emulsifiers (such as Tween and / or polyoxyethylene castor oil). Oils (e.g., used to reduce interfacial tension, promote emulsification of the dispersed phase, and maintain emulsion stability); wetting agents (e.g., sodium lauryl sulfate, used to help drug components disperse and dissolve better); colorants (used to improve the appearance and color of drugs, making them easier to identify and distinguish); flavoring agents (used to improve the taste of drugs and improve patient medication compliance); stabilizers (increase the physical stability of drugs and prevent them from deteriorating or degrading during storage or transportation); lubricants (e.g., talc, used to reduce drug friction); and phosphate buffers (used to adjust the pH of drugs to meet their requirements). The ingredients include: (1) those that meet human physiological requirements, thereby reducing irritation and adverse reactions to the human body; (2) polyols (such as propylene glycol, glycerin, sorbitol, mannitol, and / or polyethylene glycol, used to improve drug stability, prevent dehydration or deterioration during storage, and also to improve the taste and solubility of the drug); (3) sugars (such as lactose, glucose, and / or sucrose, used to help drug components disperse and dissolve better, while providing necessary energy support); and (4) starches (such as corn starch and / or potato starch, used to help tablets disintegrate rapidly after administration and release drug components, thereby improving drug bioavailability). The excipients may be selected from one or more of the following: cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, and / or methyl cellulose, used to improve drug compressibility and formability, and also to control drug release rate); and binders (such as gelatin, used to help drug components bind together better). The type of excipient can be selected based on the need to improve drug stability, activity, and / or bioavailability.

[0023] The present invention has the following beneficial effects:

[0024] The active peptides of this invention, whose amino acid sequences are shown in SEQ ID NO:1, can not only activate ADH and ALDH enzymes and promote ethanol metabolism, but also significantly scavenge DPPH and ABTS free radicals, exerting antioxidant effects, thereby effectively preventing and / or treating ALD. They are very suitable for preparing products for the prevention and treatment of ALD, and also enrich the resource library of active peptides with ALD prevention and treatment effects. Attached Figure Description

[0025] Figure 1 shows the mass spectrometry identification results of the bioactive peptides.

[0026] Figure 2 shows the purity identification results of the active peptide.

[0027] Figure 3 shows the ADH activation rate results of the active peptides.

[0028] Figure 4 shows the ALDH enzyme activity results after treatment with the active peptide.

[0029] Figure 5 shows the DPPH free radical scavenging rate results of the active peptide.

[0030] Figure 6 shows the ABTS free radical scavenging rate of the active peptide. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0032] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0033] Example 1: Synthesis, Mass Spectrometry Identification, and Purity Identification of Active Peptides

[0034] The bioactive peptide (amino acid sequence as shown in SEQ ID NO:1: WGAPGP) was synthesized with the assistance of Nanjing Peptide Valley Biotechnology Co., Ltd., and the bioactive peptide was identified by mass spectrometry and purity.

[0035] (1) Mass spectrometry identification

[0036] The molecular weight of the bioactive peptides was identified using ESI-MS mass spectrometry.

[0037] The identification conditions for ESI-MS mass spectrometry are as follows:

[0038] Mobile phase: A is water (containing 0.1% (v / v) formic acid), B is acetonitrile (containing 0.1% (v / v) formic acid);

[0039] Flow rate: 0.2 mL / min;

[0040] Running time: 1 min;

[0041] Positive ion mode;

[0042] Scan range: 0–2000 Da.

[0043] The results of mass spectrometry identification are shown in Figure 1. It can be seen that the quasi-molecular ion peak (M+H)+ of the active peptide in this embodiment is 584.53, which carries one charge and is consistent with its theoretical molecular weight value. This indicates that the active peptide synthesized in this embodiment is the active peptide with the amino acid sequence shown in SEQ ID NO:1.

[0044] (2) Purity identification

[0045] The purity of the bioactive peptides was determined using high performance liquid chromatography (HPLC).

[0046] The identification conditions for high-performance liquid chromatography (HPLC) are as follows:

[0047] Column: Kromasil C18 (4.6 × 150 mm, 5 μm);

[0048] Mobile phase: A is acetonitrile (containing 0.1% (v / v) trifluoroacetic acid), B is water (containing 0.1% (v / v) trifluoroacetic acid);

[0049] Elution gradient: 0→0.01 min, 5% (v / v) A; 0.01→25 min, 50% (v / v) A; 25→30 min, 90% (v / v) A;

[0050] Flow rate: 1 mL / min;

[0051] Detection wavelength: 214 nm;

[0052] Sample loading volume: 20 μL.

[0053] The results of purity identification are shown in Figure 2. It can be seen that in the active peptide synthesized in this embodiment, the main peak accounts for the majority, with only a few impurity peaks appearing, and the purity is as high as 98.26%.

[0054] Example 2 ADH enzyme activation assay

[0055] (1) Solution preparation

[0056] Preparation of ADH enzyme solution: Dissolve ADH enzyme (360 U / mg) in ultrapure water to obtain a 0.2 U / mL ADH enzyme solution, and store it at -20 ℃ for later use.

[0057] Preparation of ADH enzyme working solution: Prepared according to the instructions of the alcohol dehydrogenase (ADH) kit.

[0058] Preparation of active peptide solutions: The active peptides obtained in Example 1 were dissolved in ultrapure water to obtain active peptide solutions of 0, 0.01, 0.1, 1, 2 and 4 mg / mL, respectively, which were then stored at -20 ℃ for later use.

[0059] (2) Measurement method

[0060] Control group: In a 96-well plate, 150 μL of ADH enzyme working solution was mixed with 50 μL of ultrapure water and incubated at 37 °C for 5 min. Then, 50 μL of ADH enzyme solution was added to initiate the reaction. Immediately after plating, the absorbance was measured using a VIRIOSKAN LUX microplate reader at a wavelength of 340 nm. Scans were performed every 15 seconds for 10 min, for a total of 41 scans. The reaction kinetic curve was fitted, and the first derivative of the curve at 0 min was taken as the initial reaction rate V0.

[0061] Sample group: In a 96-well plate, 150 μL of ADH enzyme working solution was mixed with 50 μL of active peptide solution and incubated at 37 ℃ for 5 min. Then, 50 μL of ADH enzyme solution was added to initiate the reaction. Immediately after application, the absorbance was measured using a VIRIOSKAN LUX microplate reader at a wavelength of 340 nm. Scans were performed every 15 s for 10 min, for a total of 41 scans. The reaction kinetic curve was fitted, and the first derivative of the curve at 0 min was determined as the initial reaction rate V1.

[0062] The ADH activation rate of the active peptide solution was calculated using the formula "ADH activation rate (%) = [(V1-V0) / V0] × 100%".

[0063] (3) Measurement results

[0064] As shown in Figure 3, the ADH activation rate of the active peptide solution of 0.01–4 mg / mL reached (12.34% ± 6.42%) to (41.70% ± 19.95%), indicating that the active peptide of the present invention can effectively activate ADH enzyme and promote the metabolism of ethanol to acetaldehyde, thereby effectively preventing and / or treating ALD, and is very suitable for preparing products for the prevention and treatment of ALD.

[0065] Example 3 ALDH enzyme activation assay

[0066] (1) Solution preparation

[0067] Preparation of ALDH enzyme solution: Dissolve ALDH enzyme (15 U / mg) in ultrapure water to obtain 0.2 U / mL ALDH enzyme solution, and store at -20 ℃ for later use.

[0068] Preparation of ALDH enzyme inactivation solution: Dissolve ALDH enzyme (15 U / mg) in ultrapure water to obtain 0.2 U / mL ALDH enzyme solution. Inactivate the enzyme at 100 ℃ for 5 min, and then store at -20 ℃ for later use.

[0069] Preparation of ALDH enzyme working solution: Prepared according to the instructions of the acetaldehyde dehydrogenase (ALDH) kit.

[0070] Preparation of active peptide solutions: The active peptides obtained in Example 1 were dissolved in ultrapure water to obtain active peptide solutions of 0, 0.1, 1, 2 and 4 mg / mL, respectively, which were stored at -20 ℃ for later use.

[0071] (2) Construction of standard curve

[0072] The acetaldehyde dehydrogenase kit contains the following reagents: standard (powder, 1 vial), reagent 2 (2.5 mL, 1 vial), and reagent 3 (70 mL, 1 vial). Dissolve the standard in 1.41 mL of distilled water to obtain a 1 nmol / μL standard solution. Then dilute this solution with distilled water to prepare six concentration gradients (0, 0.1, 0.2, 0.3, 0.4, 0.5 nmol / μL). Take 20 μL of each concentration gradient, add 6.5 μL of reagent 2 and 174 μL of reagent 3, mix well, and incubate at 37 ℃ for 5 min. Read the absorbance at 450 nm. Plot a standard curve y = ax + b with standard concentration as the x-axis and absorbance as the y-axis.

[0073] (3) Measurement method

[0074] Control group: In a 96-well plate, 180 μL of ALDH enzyme working solution was mixed with 10 μL of active peptide solution, and then 10 μL of ALDH enzyme inactivation solution was added and mixed. After 30 s, the absorbance A1 was measured in a VIRIOSKAN LUX microplate reader at a wavelength of 450 nm. After 30 min, the absorbance A2 was measured again. The result was ΔA0 = A2 - A1.

[0075] Sample group: In a 96-well plate, 180 μL of ALDH enzyme working solution was mixed with 10 μL of active peptide solution, and then 10 μL of ALDH enzyme solution was added and mixed. After 30 s, the absorbance A3 was measured in a VIRIOSKAN LUX microplate reader at a wavelength of 450 nm. After 30 min, the absorbance A4 was measured again. The result was ΔA1 = A4 - A3.

[0076] The ALDH enzyme activity after treatment with the active peptide solution was calculated according to the formula “ALDH enzyme activity (nmol / min / mL) = (△Ab) / avt”, where △A = △A1 - △A0, v is the system volume (200 μL) and t is the reaction time (30 min).

[0077] (4) Measurement results

[0078] The results are shown in Figure 4. It can be seen that the ALDH enzyme activity reached (2.67±0.11) to (3.43±0.20) nmol / min / mL after treatment with 0.1–4 mg / mL of the active peptide solution. This indicates that the active peptide of the present invention can effectively activate ALDH enzyme, promote the conversion of acetaldehyde, a metabolite of ethanol, into acetic acid, and thus effectively prevent and / or treat ALD, making it highly suitable for preparing products for the prevention and treatment of ALD.

[0079] Example 4: DPPH Free Radical Scavenging Test

[0080] (1) Solution preparation

[0081] Preparation of DPPH solution: Dissolve 4 mg of DPPH (2,2-biphenyl-1-picrylhydrazyl) solid in 100 mL of anhydrous ethanol and store at 4 °C protected from light for later use.

[0082] Preparation of active peptide solutions: The active peptides obtained in Example 1 were dissolved in ultrapure water to obtain active peptide solutions with concentrations of 0.005, 0.01, 0.1, 0.5, and 1 mg / mL, respectively, which were then stored at -20 °C for later use.

[0083] (2) Measurement method

[0084] Blank group: In a 96-well plate, 150 μL of DPPH solution was mixed with 50 μL of ultrapure water, incubated in the dark for 40 min, and the absorbance A0 was measured in a microplate reader at a wavelength of 517 nm.

[0085] Sample group: In a 96-well plate, 150 μL of DPPH solution and 50 μL of active peptide solution were mixed and incubated in the dark for 40 min. The absorbance A1 was then measured in a microplate reader at a wavelength of 517 nm.

[0086] Control group: In a 96-well plate, 150 μL of anhydrous ethanol and 50 μL of active peptide solution were mixed and incubated in the dark for 40 min. The absorbance A2 was then measured in a microplate reader at a wavelength of 517 nm.

[0087] Four parallel samples were set up in each group, and the DPPH free radical scavenging rate of the active peptide solution was calculated according to the formula "DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0]×100%".

[0088] (3) Measurement results

[0089] As shown in Figure 5, the DPPH free radical scavenging rate of the active peptide solution of 0.005–1 mg / mL reached (35.93% ± 4.84%) to (81.55% ± 1.04%), indicating that the active peptide of the present invention can significantly scavenge DPPH free radicals, exert antioxidant effects, and thus effectively prevent and / or treat ALD, making it very suitable for preparing products for the prevention and treatment of ALD.

[0090] Example 5: ABTS Free Radical Scavenging Test

[0091] (1) Solution preparation

[0092] Preparation of ABTS stock solution: Dissolve 0.096 g of ABTS (2,2-aminodiphenyl-1-picrylhydrazyl) solid in 25 mL of ultrapure water.

[0093] Preparation of potassium persulfate solution: Dissolve 0.017 g of potassium persulfate (K2S2O8) solid in 25 mL of ultrapure water.

[0094] Preparation of ABTS free radical working solution: Mix ABTS stock solution and potassium persulfate solution at a volume ratio of 1:1, and let stand in the dark until the system turns blue-green.

[0095] Preparation of active peptide solutions: The active peptides obtained in Example 1 were dissolved in ultrapure water to obtain active peptide solutions with concentrations of 0.005, 0.01, 0.1, 0.5, and 1 mg / mL, respectively, which were then stored at -20 °C for later use.

[0096] (2) Measurement method

[0097] Blank group: In a 96-well plate, 150 μL of ABTS free radical working solution was mixed with 50 μL of ultrapure water, incubated in the dark for 40 min, and the absorbance A0 was measured in a microplate reader at a wavelength of 734 nm.

[0098] Sample group: In a 96-well plate, 150 μL of ABTS free radical working solution and 50 μL of active peptide solution were mixed and incubated in the dark for 40 min. The absorbance A1 was then measured in a microplate reader at a wavelength of 734 nm.

[0099] Control group: In a 96-well plate, 150 μL of ultrapure water and 50 μL of active peptide solution were mixed and incubated in the dark for 40 min. The absorbance A2 was then measured in a microplate reader at a wavelength of 734 nm.

[0100] Four parallel samples were set up in each group, and the ABTS free radical scavenging rate of the active peptide solution was calculated according to the formula "ABTS free radical scavenging rate (%) = [1-(A1-A2) / A0]×100%".

[0101] (3) Measurement results

[0102] As shown in Figure 6, the ABTS free radical scavenging rate of the active peptide solution of 0.005–1 mg / mL reached (59.51% ± 2.88%) to (95.44% ± 2.58%), indicating that the active peptide of the present invention can significantly scavenge ABTS free radicals, exert antioxidant effects, and thus effectively prevent and / or treat ALD, making it very suitable for preparing products for the prevention and treatment of ALD.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An active peptide, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. A biomaterial related to an active peptide, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID NO:

1. The related biological material is a nucleic acid molecule that can express the active peptide, or a recombinant bacterium, recombinant cell, recombinant plasmid or expression cassette containing the nucleic acid molecule.

3. The use of the active peptide of claim 1 or the related biomaterial of claim 2 in the preparation of products for preventing and treating alcoholic liver injury.

4. A product characterized in that, It contains the active peptide of claim 1 or the related biomaterial of claim 2.

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