Alcohol effect dispelling and liver protecting nonapeptide and preparation method thereof
By preparing and purifying the nonapeptide NNYNLPILR, ethanol dehydrogenase is activated, and the problem of slow ethanol metabolism is solved, achieving the effect of understanding the liver protection of wine.
Patent Information
- Application Number
- CN202510263587.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art is difficult to effectively activate ethanol dehydrogenase, resulting in slow alcohol metabolism and increased liver burden, and is unable to effectively prevent or treat alcoholic liver disease.
Using biological enzymatic lysis technology and molecular docking technology, a kind of nonapeptide (NNYNLPILR) was prepared from hemp seed protein, and purified by solid phase synthesis and high-performance liquid chromatography to obtain a polypeptide with the activation ability of ethanol dehydrogenase, which is used to activate ethanol dehydrogenase to accelerate alcohol metabolism.
Activate ethanol dehydrogenase, improve alcohol metabolism rate, reduce the adverse effects of alcohol on the liver, and provide alcohol protection function.
Smart Images
Figure CN120289569A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to an anti-alcoholic and liver-protecting nonapeptide and a preparation method thereof. Background Art
[0002] The history of human alcohol consumption is long. Since ancient times, as a special food, alcohol has played an important role in daily life and social interactions. However, excessive alcohol consumption can not only lead to alcohol dependence but also increase the risk of people getting sick, such as alcoholic fatty liver, liver cirrhosis, alcoholic hepatitis, digestive system diseases, etc.
[0003] Alcohol dehydrogenase (ADH) is the rate-limiting enzyme for normal human metabolism of alcohol, catalyzing the dehydrogenation of alcohol to produce acetaldehyde. Acetaldehyde is further catalyzed by aldehyde dehydrogenase (ALDH) to produce acetic acid harmless to humans, and finally decomposed into water and carbon dioxide and excreted from the body. Activating the activity of ADH can not only accelerate alcohol metabolism and the excretion of alcohol and metabolic intermediates from the body but also reduce its adverse effects on the liver.
[0004] Bioactive peptides exhibit various biological activities due to different amino acid compositions and sequences, and are considered natural efficacy factors for regulating body metabolism and promoting human health. Bioactive peptides have the advantages of small molecular weight, easy absorption, wide sources of food proteins, and good safety, and are widely favored. Therefore, finding a safe and effective liver-protecting active peptide to prevent or treat ALD has become a research and attention hotspot. Hemp seeds contain 20% - 25% protein and are high-quality raw materials for preparing bioactive peptides. Summary of the Invention
[0005] The purpose of the present invention is to provide a bioactive peptide that activates the activity of ADH, thereby accelerating alcohol metabolism and the excretion of alcohol and metabolic intermediates from the body, and reducing its adverse effects on the liver. The present invention uses hemp seed protein as a raw material and prepares a nonapeptide with the ability to activate alcohol dehydrogenase by using bioenzymolysis technology, chromatography technology, bioinformatics, molecular docking, and polypeptide coupling technology. This nonapeptide has the ability to activate alcohol dehydrogenase and can be used for the development of anti-alcoholic and liver-protecting functional foods, and is applied in the fields of biopharmaceuticals and functional foods.
[0006] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:
[0007] An anti-alcoholic and liver-protecting nonapeptide, the abbreviation of the nonapeptide is NNYNLPILR, the molecular weight is 1116.30, and the sequence is: Asn-Asn-Tyr-Asn-Leu-Pro-Ile-Leu-Arg.
[0008] A method for preparing the above-mentioned nine-peptide for relieving hangover and protecting the liver, the method comprising: selecting a high-molecular resin, connecting the hydroxyl group of Arg to the resin according to the characteristics of the amino acid sequence Asn-Asn-Tyr-Asn-Leu-Pro-Ile-Leu-Arg, then performing a condensation reaction between the amino group of Leu and the carboxyl group of Arg, after treatment, adding Ile, reacting the amino group of Ile with the carboxyl group of Leu, adding amino acids sequentially from right to left, and after adding the last Asn amino acid, removing the resin to obtain the target polypeptide.
[0009] Further, high performance liquid chromatography is used to purify the target polypeptide. The chromatographic column is NanoChromcoreTM120 C18 (4.6*250mm*5um), mobile phase A: 0.1% trifluoroacetic acid in 100% water; mobile phase B: 0.1% trifluoroacetic acid in 100% acetonitrile; within 20 min, the mobile phase B rises from 23% to 43%, the flow rate is 1.0 mL / min, the detection wavelength is 214 nm; quick-freeze with liquid nitrogen and freeze-dry to obtain the final product, and the required purity is above 98%.
[0010] Further, the vacuum degree of the freeze-drying is 50 Pa, the temperature is -80°C, and the time is 16 h.
[0011] The beneficial effects of the present invention compared with the prior art are as follows: The present invention uses hemp seed protein as a raw material, and through the combination of bioenzymolysis technology, bioinformatics and molecular docking technology, a nine-peptide with ethanol dehydrogenase activation activity is screened, and through solid-phase synthesis technology for polypeptide synthesis and in vitro activity determination, a polypeptide with hemp seed protein source having ethanol dehydrogenase activation activity is obtained, which can be used for the development of drugs for relieving hangover and protecting the liver. Description of the Drawings
[0012] Figure 1 It is a graph of the activation rate of each component of hemp seed peptide on ethanol dehydrogenase.
[0013] Figure 2 It is a molecular docking diagram of the synthetic polypeptide Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile and ethanol dehydrogenase.
[0014] Figure 3 It is an HPLC diagram of the synthetic polypeptide Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile.
[0015] Figure 4 It is an MS diagram of the synthetic polypeptide Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile.
[0016] Figure 5 Activation rate graph of synthetic polypeptide Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile with different concentrations on alcohol dehydrogenase. Specific implementation mode
[0017] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the protection scope of the present invention.
[0018] Example 1:
[0019] The ethanol dehydrogenase-activating nonapeptide synthesized by the present invention is abbreviated as NNYNLPILR, with a molecular weight of 1116.30 and the sequence: Asn-Asn-Tyr-Asn-Leu-Pro-Ile-Leu-Arg.
[0020] 1. Preparation of hemp seed liver-protecting peptide
[0021] Weigh 10 g of hemp seed protein powder, add 50 mL of deionized water, fully dissolve and stir evenly, adjust the pH of the enzymatic hydrolysis solution to 6.2, add 3.36% neutral protease, carry out enzymatic hydrolysis at 44 °C for 2.75 h, inactivate the enzyme at 95 °C for 15 min after enzymatic hydrolysis, centrifuge at 10000 r / min at 4 °C for 15 min, and take the supernatant (polypeptide solution) for vacuum freeze-drying for later use.
[0022] 2. Isolation and purification of hemp seed liver-protecting peptide
[0023] Put anhydrous ethanol in a 4 °C refrigerator overnight, and add it drop by drop to the prepared 40 mg / mL hemp seed peptide solution until the ethanol volume fraction reaches 20%. Then let the solution stand at 4 °C for 1 h, and centrifuge at 7500 g for 10 min. The precipitate part is denoted as E20. Add the supernatant part to cold anhydrous ethanol until the ethanol volume fraction reaches 40%, let it stand at 4 °C for 1 h, and the precipitate part obtained by centrifugation is denoted as E40. Add the supernatant part to cold anhydrous ethanol until the ethanol volume fraction reaches 60%, let it stand at 4 °C for 1 h, and the precipitate part obtained by centrifugation is denoted as E60, and the supernatant is denoted as SPT. All components are concentrated under reduced pressure at 55 °C to remove ethanol, and then freeze-dried, and the activation rate of alcohol dehydrogenase of each component is measured to screen the strongest active component ( Figure 1 ).
[0024] The activation rate of alcohol dehydrogenase was measured by the Warburg-Hoch method. First, 1.5 mL (0.1 M, pH 8.8) of sodium pyrophosphate buffer solution, 1.0 mL of 0.01 M NAD +Nicotinamide adenine dinucleotide (oxidized form)、0.5 mL (11.5%, v / v) ethanol and 0.1 mL of hydrolyzed hemp protein were mixed at 25 °C and reacted for 5 min. Then, 0.1 mL of 0.25 U / mL ADH was added to the reaction system. The absorbance was immediately measured at 340 nm for 10 min. The initial linear part of the reaction was plotted, and the slope was denoted as Ka. For the control group, 0.2 mL of distilled water was used instead of the sample, and the slope was denoted as Kc, which was calculated according to formula (1).
[0025]
[0026] 3. Virtual Screening and Molecular Docking of Hemp Seed Liver-Protecting Peptide Sequences
[0027] For the most active component SPT obtained by separation and purification, LC-MS / MS mass spectrometry was used to analyze and identify the polypeptide sequence. Dock 6.9 was used for virtual screening of the obtained peptide sequence; Peptide ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to score the potential biological activity of the alternative polypeptides; GRAVYCALCULATOR (https: / / www.gravy-calculator.de / ) was used to calculate the hydrophobicity; PepDraw (https: / / www.pepdraw.com / ) was used to calculate the relative molecular mass, net charge, and isoelectric point; the toxicity was determined by ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html). According to the docking scores of the peptide segments, the peptide segment NNYNLPILR with good docking effect with alcohol dehydrogenase was screened out, with a docking score of -19.5 kcal / mol, a molecular weight of 1116.30 Da, a polypeptide score of 0.621789, a hydrophilicity of -0.46, a net charge of 1, an isoelectric point of 9.1, and no toxicity.
[0028] The crystal structure of alcohol dehydrogenase (PDB ID: 1U3T) was obtained from the RCSB Protein Data Bank database (http: / / www.rcsb.org / ). The NNYNLPILR was semi-flexibly docked with alcohol dehydrogenase using Discovery studio 2019 software to determine the key amino acid residues and interaction forces involved in its interaction with alcohol dehydrogenase (see Figure 2) The docking results with alcohol dehydrogenase showed that the NNYNLPILR peptide could form 7 hydrogen bonds with ADH1A, involving the residues of ASN1, ASN2, TYR3, ARG9 in the peptide NNYNLPILR and GLU107, GLY321, ASN185, VAL186, ILE318, TYR110, ASP297 in ADH1A. And the interactions included van der Waals forces, hydrogen bonds, hydrophobic interactions, salt bridge interactions, and π-alkyl interactions.
[0029] 4. Solid-phase synthesis, separation, purification and identification of hemp seed liver-protecting peptide
[0030] A high molecular weight resin (Bio-Engineering (Shanghai) Co., Ltd.) was selected. According to the characteristics of the amino acid sequence Asn-Asn-Tyr-Asn-Leu-Pro-Ile-Leu-Arg, the hydroxyl group of Arg was connected to the resin, and then the amino group of Leu and the carboxyl group of Arg underwent a condensation reaction. After treatment, Ile was added, and the amino group of Ile reacted with the carboxyl group of Leu. Amino acids were added sequentially from right to left. After adding the last Asn amino acid, the resin was removed to obtain the target polypeptide.
[0031] Purification was carried out by high performance liquid chromatography. The chromatographic column was NanoChromcore TM120C18 (4.6*250mm*5um). Mobile phase A: 0.1% trifluoroacetic acid in 100% water; Mobile phase B: 0.1% trifluoroacetic acid in 100% acetonitrile; B increased from 23% to 43% within 20 min, the flow rate was 1.0 mL / min, and the detection wavelength was 214 nm. It was quickly frozen in liquid nitrogen and freeze-dried (vacuum degree: 50 Pa, temperature: -80 °C, time: 16 hours) to obtain the final product, with a required purity of over 98%, and the structure was identified by MS (as Figure 3 、 Figure 4 shown).
[0032] 5. Verification of the in vitro activity of solid-phase synthesized polypeptides
[0033] Study on the activation rate of ADH by Asn-Asn-Tyr-Asn-Leu-Pro-Ile-Leu-Arg at different concentrations. Using the above-mentioned method for measuring the ADH activation rate, active peptide solutions with concentrations of 0, 1, 3, 5, 7, and 9 mM were respectively prepared, and the in vitro activation activity of ADH of active peptides at different concentrations was measured. The results are shown in Figure 5 .
Claims
1. A nine - peptide for relieving hangover and protecting the liver, characterized in that: The abbreviation of the nonapeptide is NNYNLPILR, with a molecular weight of 1116.30, and the sequence is: Asn-Asn-Tyr-Asn-Leu-Pro-Ile-Leu-Arg.
2. A preparation method of the nine-peptide for relieving hangover and protecting liver according to claim 1, characterized in that: The method is as follows: Select a high molecular resin. According to the characteristics of the amino acid sequence Asn-Asn-Tyr-Asn-Leu-Pro-Ile-Leu-Arg, the hydroxyl group of Arg is connected to the resin, and then the amino group of Leu and the carboxyl group of Arg undergo a condensation reaction. After treatment, Ile is added, and the amino group of Ile reacts with the carboxyl group of Leu. Amino acids are added sequentially from right to left. After adding the last Asn amino acid, the resin is removed to obtain the target polypeptide.
3. The preparation method of a nine-peptide for relieving hangover and protecting liver according to claim 2, characterized in that: The target polypeptide is purified by high performance liquid chromatography. The chromatographic column is NanoChromcore TM120 C18 (4.6*250mm*5um). Mobile phase A: 0.1% trifluoroacetic in 100% water; Mobile phase B: 0.1% trifluoroacetic in 100% acetonitrile; Mobile phase B rises from 23% to 43% within 20 min, the flow rate is 1.0 mL / min, and the detection wavelength is 214 nm; It is quickly frozen in liquid nitrogen and freeze-dried to obtain the final product, and the required purity is above 98%.
4. The preparation method of a nine - peptide for relieving hangover and protecting liver according to claim 3, characterized in that: The vacuum degree of the freeze-drying is 50 Pa, the temperature is -80 °C, and the time is 16 h.