Anti-fatigue liver peptide and application thereof

Abstract

The application discloses an anti-fatigue liver peptide and application thereof, and relates to the technical field of anti-fatigue liver peptides. The liver peptide comprises an amino acid sequence of LVF. The liver peptide provided by the application can effectively relieve fatigue, and the effect is remarkable.

Description

Technical Field

[0001] This invention relates to the field of liver peptide technology, and more specifically to an anti-fatigue liver peptide and its applications. Background Technology

[0002] Fatigue is a physiological state in which the body's physiological processes are unable to sustain their functions at a specific level and / or maintain a predetermined exercise intensity. It is divided into two categories: exercise-induced fatigue and chronic fatigue. With the accelerated pace of modern life, factors such as long-term overwork and mental stress have led to a year-on-year increase in the incidence of chronic fatigue syndrome, seriously affecting people's quality of life and work efficiency. At the same time, excessive fatigue can exacerbate metabolic disorders, increase the burden on liver function, and form a vicious cycle of "fatigue-liver damage-more fatigue".

[0003] Currently, anti-fatigue products mainly include chemically synthesized drugs and plant extract health supplements. While chemically synthesized drugs have a rapid onset of action, they also have significant side effects and are prone to causing dependence. Plant extract products, such as ginseng and wolfberry extracts, have a relatively high safety profile, but their anti-fatigue effects are not ideal.

[0004] Liver peptides, as active peptides extracted from liver tissue or obtained through biosynthesis, have the function of regulating liver metabolism and repairing liver cell damage, but their application in the field of anti-fatigue has not yet been seen.

[0005] Therefore, developing an anti-fatigue liver peptide is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] In view of this, the present invention provides an anti-fatigue liver peptide and its application, which overcomes the shortcomings of the prior art where the anti-fatigue effect of anti-fatigue products is not significant and liver peptides have not yet been applied in the field of anti-fatigue.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an anti-fatigue liver peptide, wherein the amino acid sequence of the liver peptide is LVF.

[0008] Furthermore, the amino acid sequence of the hepatic peptide also includes a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the LVF.

[0009] Secondly, the present invention provides a nucleic acid molecule that encodes the hepatic peptide, or is reverse complementary to the nucleotide sequence encoding the hepatic peptide.

[0010] Thirdly, the present invention provides a biomaterial containing the aforementioned nucleic acid molecules and capable of expressing the aforementioned liver peptides.

[0011] Furthermore, the biological material includes recombinant plasmids, recombinant vectors, or recombinant host cells.

[0012] Fourthly, the present invention provides the application of the aforementioned hepatic peptide, the aforementioned nucleic acid molecule, and the aforementioned biomaterial in the preparation of anti-fatigue products.

[0013] Furthermore, the anti-fatigue measures include: improving the zebrafish's movement distance and increasing the percentage of behavioral improvement.

[0014] Furthermore, the products include food or pharmaceuticals.

[0015] Fifthly, the present invention provides a medicament for combating fatigue, the medicament comprising the aforementioned hepatic peptide.

[0016] In a sixth aspect, the present invention provides a food for combating fatigue, the food comprising the aforementioned liver peptide.

[0017] As can be seen from the above technical solution, the present invention uses LVF liver peptides as an anti-fatigue component. It has high purity and small molecular weight, is easily absorbed and utilized by the body, can directly participate in the regulation of liver metabolism, reduce the burden on the liver, and promote energy metabolism, thereby relieving fatigue from the root cause. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 This is the mass spectrum of hepatic peptide LVF obtained by protease digestion. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 The amino acid sequence of this invention is LVF (Leu-Val-Phe). The preparation steps are as follows: I. Raw material pretreatment 1. Select healthy pig liver (which has passed quarantine, is free from disease and drug residues), remove the fascia, blood vessels and connective tissue, rinse 3 times with 4℃ pre-cooled physiological saline, drain the surface water, and cut into small pieces of about 1cm³. 2. Add liver tissue to pre-cooled physiological saline at a ratio of 1:2 (mass-volume ratio, g / mL) into a high-speed tissue homogenizer and homogenize for 2 minutes under ice bath conditions to prepare a uniform liver tissue homogenate. 3. Add EDTA (ethylenediaminetetraacetic acid) to the homogenate at a final concentration of 0.05 mol / L and PMSF (phenylmethylsulfonyl fluoride) at a final concentration of 0.1 mmol / L, stir well, and let stand at 4°C for 30 min to inhibit protease activity and prevent liver peptide degradation.

[0022] II. Liver peptide extraction 1. Transfer the pretreated liver homogenate to a centrifuge tube, centrifuge at 4℃ and 8000r / min for 20min, collect the supernatant (crude extract), and discard the precipitate (tissue fragments, large molecular proteins, etc.). 2. Slowly add ammonium sulfate to the supernatant until the saturation is 60%, stir at 4°C for 3 hours to allow the impurities and proteins to precipitate fully; 3. Continue centrifuging at 4℃ and 10000r / min for 25min, collect the supernatant (containing the target hepatic peptide), and discard the impurity protein precipitate; 4. Use an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to ultrafilter the supernatant at 4°C and 0.3 MPa pressure. Collect the filtrate (to remove impurities and polymers with a molecular weight greater than 10 kDa) and retain the filtrate containing small peptides.

[0023] III. Preliminary purification (gel filtration chromatography) 1. Sephadex G-15 gel chromatography was used. The column (2.6 cm × 100 cm) was equilibrated with 0.02 mol / L, pH 7.0 phosphate-buffered saline (PBS) at a volume of 3 times the column volume. 2. Load the ultrafiltration filtrate into the chromatography column, with a loading volume of 5% of the column volume; 3. Elute with the same PBS buffer at a flow rate of 1 mL / min. Monitor the elution peak at 220 nm using a UV detector and collect the eluent corresponding to the molecular weight range (300-500 Da) (the collection range is preset according to the molecular weight of LVF). 4. Combine the collected eluents and freeze-dry them initially using a freeze dryer to obtain crude liver peptide powder.

[0024] IV. Purification (Reversed-phase high-performance liquid chromatography, RP-HPLC) 1. Dissolve the crude hepatin powder in 0.1% trifluoroacetic acid aqueous solution to prepare a sample solution of 10 mg / mL, and filter it through a 0.22 μm microporous membrane; 2. A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with a column temperature of 30 °C; mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was acetonitrile; 3. Gradient elution program: 0-20 min, the volume fraction of mobile phase B increases from 10% to 30%; 20-35 min, the volume fraction of mobile phase B increases from 30% to 45%; 35-40 min, the volume fraction of mobile phase B returns to 10% to equilibrate the column. 4. Elution flow rate 1.0 mL / min, detection at 220 nm wavelength using a UV detector, and collection of the target elution peak corresponding to LVF based on retention time (retention time determined by standard reference: LVF approximately 25.3 min). 5. The collected target eluents were freeze-dried to obtain high-purity liver peptide powder.

[0025] V. Purity Identification and Sequence Confirmation 1. Purity test: The purity of LVF was repeatedly tested by HPLC and the purity was ≥98% with no obvious impurity peaks. 2. Sequence confirmation: The molecular weight was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and the amino acid sequence was determined by Edman degradation method. The obtained product was confirmed to be LVF (molecular weight 339.5 Da), which was completely consistent with the preset sequence. 3. Impurity detection: The residue of heavy metals (lead, mercury, cadmium) is ≤0.1mg / kg, the microbial limit meets the requirements of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition), and there are no toxic or harmful substances residues.

[0026] The anti-fatigue performance of the liver peptide prepared in Example 1 was tested. Animal fatigue experiment: Animal sample selection: Wild-type AB strain zebrafish were selected. The zebrafish were raised in fish tank water at 28℃ and reproduced by natural pair mating. The zebrafish were selected as experimental animal samples at 4 days after fertilization (4dpf).

[0027] Animal fatigue experiment: Experimental Group 1: The anti-fatigue liver peptide prepared in Example 1 was dissolved in a 10% methanol-water standard solution to prepare an experimental stock solution with a concentration of 100 μg / mL. Experimental animal samples were randomly selected into 6-well plates, with 30 zebrafish treated in each well. Five parallel experiments were set up. For each parallel experiment, the corresponding concentration of the experimental stock solution was administered, with a volume of 3 mL per well. After treatment at 28℃ for 1 day, anhydrous sodium sulfite was administered in water to establish a zebrafish fatigue model.

[0028] Experimental Group 2: The anti-fatigue liver peptide prepared in Example 1 was dissolved in a 10% methanol-water standard solution to prepare an experimental stock solution with a concentration of 200 μg / mL. Experimental animal samples were randomly selected into 6-well plates, with 30 zebrafish treated in each well. Five parallel experiments were set up. For each parallel experiment, the corresponding concentration of the experimental stock solution was administered, with a volume of 3 mL per well. After treatment at 28℃ for 1 day, anhydrous sodium sulfite was administered in water to establish a zebrafish fatigue model.

[0029] Experimental Group 3: The anti-fatigue liver peptide prepared in Example 1 was dissolved in a 10% methanol-water standard solution to prepare an experimental stock solution with a concentration of 125 μg / mL. Experimental animal samples were randomly selected into 6-well plates, with 30 zebrafish treated in each well. Five parallel experiments were set up. For each parallel experiment, the corresponding concentration of the experimental stock solution was administered, with a volume of 3 mL per well. After treatment at 28℃ for 1 day, anhydrous sodium sulfite was administered in water to establish a zebrafish fatigue model.

[0030] Normal control group: Experimental animal samples were randomly selected into 6-well plates, with 30 zebrafish treated in each well. No experimental stock solution was given to establish a normal control group, and five parallel experiments were conducted.

[0031] Model control group: Experimental animal samples were randomly selected into 6-well plates, with 30 zebrafish treated in each well. No experimental stock solution was given. After treatment at 28℃ for 1 day, anhydrous sodium sulfite was administered in water to establish a zebrafish fatigue model control group. Five parallel experiments were conducted.

[0032] Positive control group: Rhodioloside was prepared as a stock solution with a concentration of 4000 μg / mL. Experimental animal samples were randomly selected into 6-well plates, with 30 zebrafish treated in each well; the stock solution was administered in 3 mL volumes per well. After treatment at 28℃ for 1 day, anhydrous sodium sulfite was administered in water to establish a zebrafish fatigue model. Five parallel experiments were conducted.

[0033] Maximum tolerable concentration (MTC) determination: After maintaining the established zebrafish fatigue model at 28℃ for 1 hour, the mortality rate of the zebrafish was measured. The results are shown in Table 1.

[0034] Table 1 As shown in Table 1, the liver peptide of the present invention is non-toxic, has no side effects, and is highly safe.

[0035] Ten zebrafish from each of the above treatment groups were randomly selected and transferred to a 96-well plate, then placed in a behavior analyzer. The total distance (D) of movement of the zebrafish within 1 hour was measured using the behavior analyzer. The statistical analysis results of this index were used to evaluate the effectiveness of the behavioral improvement. The formula for calculating the effectiveness of behavioral improvement is as follows: The detection and calculation results are shown in Table 2.

[0036] Table 2 Results of Anti-fatigue Efficacy Measurement As shown in Table 2, the liver peptide of the present invention has a significant anti-fatigue effect.

[0037] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0038] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A liver peptide for combating fatigue, characterized in that, The amino acid sequence of the liver peptide is LVF.

2. The anti-fatigue liver peptide according to claim 1, characterized in that, The amino acid sequence of the hepatic peptide also includes a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the LVF.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the liver peptide of claim 1 or 2, or is reverse complementary to the nucleotide sequence encoding the liver peptide of claim 1 or 2.

4. A biomaterial, characterized in that, It contains the nucleic acid molecule as described in claim 3 and is capable of expressing the liver peptide as described in claim 1 or 2.

5. A biomaterial according to claim 4, characterized in that, The biomaterials include recombinant plasmids, recombinant vectors, or recombinant host cells.

6. The use of the liver peptide according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, and the biomaterial according to claim 4 in the preparation of anti-fatigue products.

7. The application according to claim 6, characterized in that, The anti-fatigue measures include: improving the movement distance and behavioral improvement rate of zebrafish.

8. The application according to claim 6, characterized in that, The products include food or medicine.

9. A drug for relieving fatigue, characterized in that, The drug comprises the liver peptide as described in claim 1 or 2.

10. A food product for combating fatigue, characterized in that, The food product includes the liver peptide as described in claim 1 or 2.

Images