Ginseng peptide with anti-inflammatory activity as well as screening method and application thereof

Abstract

The invention belongs to the field of biological medicine, and discloses a ginseng peptide with anti-inflammatory activity and a screening method and application thereof. The amino acid sequence of the ginseng peptide is DGGW, SHCGW, MSHCGW or GGW. Experiments prove that the ginseng peptide has anti-inflammatory activity, and the invention provides technical support for development and application of the ginseng peptide.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and mainly relates to anti-inflammatory active peptides and their applications. Specifically, this invention relates to an anti-inflammatory peptide derived from ginseng and its applications. Background Technology

[0002] Ginseng is the tuberous root and rhizome of a perennial herbaceous plant belonging to the Araliaceae family. It possesses rich nutritional value and significant biological activity. For example, ginseng exhibits various functional activities such as antioxidation, antitumor activity, antidepressant activity, anti-aging, blood sugar reduction, and liver damage protection. Therefore, the applications of ginseng and its products extend to food, health products, pharmaceuticals, and cosmetics.

[0003] The pharmacologically active substances in ginseng include saponins, polysaccharides, peptides, sterols, proteins, amino acids, flavonoids, nucleotides, organic acids, and vitamins. Macromolecular ginseng peptides are polypeptides derived from ginseng, and they have been shown to possess anti-inflammatory, hypoglycemic, lipid-regulating, anti-cell proliferation, and antioxidant physiological activities. However, research on macromolecular ginseng peptides is still limited, particularly the discovery and identification of the biological activities of small-molecule monomeric peptides. Summary of the Invention

[0004] The purpose of this invention is to provide a ginseng peptide, wherein all ginseng peptide monomers have certain anti-inflammatory activity.

[0005] To achieve the above objectives, the present invention provides a ginseng peptide with anti-inflammatory activity, wherein the amino acid sequence of the ginseng peptide is DGGW, SHCGW, MSHCGW or GGW.

[0006] In a second aspect, the present invention provides a method for screening the ginseng peptide, comprising the following steps: All ginseng proteins were screened from the protein database; The screened ginseng protein was digested into peptides through in vitro digestion; The obtained peptides were scored for bioactivity potential using the PeptideRanker tool, and peptides with scores greater than 0.9 were selected. Peptides with a prediction score greater than 0.9 were screened for anti-inflammatory activity to obtain peptides with anti-inflammatory activity. Based on the anti-inflammatory activity experiment, the peptides obtained from the above screening were subjected to anti-inflammatory activity determination and verification, thus obtaining ginseng peptides with anti-inflammatory activity.

[0007] In a third aspect, the present invention provides the application of the ginseng peptide in the preparation of anti-inflammatory products.

[0008] In a fourth aspect, the present invention provides an anti-inflammatory preparation comprising any one or a combination of several ginseng peptides having the amino acid sequences DGGW, SHCGW, MSHCGW, and GGW; or their respective repeating sequences.

[0009] In a preferred embodiment of the present invention, the anti-inflammatory agent is a liquid preparation composed of the ginseng peptide and a culture medium, wherein the culture medium is a DMEM medium containing fetal bovine serum and penicillin-streptomycin, and the volume fraction of fetal bovine serum in the culture medium is 10% and the volume fraction of penicillin-streptomycin is 1%. This anti-inflammatory agent can be prepared into an injectable formulation for anti-inflammatory applications in animal husbandry.

[0010] More preferably, the concentration of ginseng peptide in the anti-inflammatory preparation is 3.75 μg / mL to 62.5 μg / mL.

[0011] More preferably, the concentration of ginseng peptide in the anti-inflammatory preparation is 62.5 μg / mL.

[0012] In a preferred embodiment of the present invention, the anti-inflammatory preparation further includes acceptable excipients. After adding suitable excipients, the anti-inflammatory preparation can be prepared into an oral or injectable formulation according to conventional pharmaceutical methods.

[0013] These clinical dosage forms include, but are not limited to, tablets, capsules, granules, powders, pills or other oral liquid preparations, and injections. These pharmaceutically acceptable excipients include, but are not limited to, diluents, wetting agents, binders, disintegrants, lubricants, regulators, solubilizers, cosolvents, emulsifiers, antioxidants, preservatives, pH adjusters, isotonic or isotropic regulators, etc.

[0014] The diluents are selected from starch, sucrose, cellulose, inorganic salts, etc.; the wetting agents are selected from water, ethanol, etc.; the binders are selected from starch paste, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, etc.; the disintegrants are selected from starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium bicarboxymethyl cellulose, crospovidone, surfactants, and flaking disintegrants, etc.; the lubricants are selected from talc, calcium stearate, magnesium stearate, magnesium dodecyl sulfate, micronized silica gel, polyethylene glycol, etc.; the modifiers are selected from pigments, fragrances, sweeteners, adhesives, and odorants, etc.; the solubilizers are selected from Tween compounds, polyoxyethylene fatty alcohol ethers, soaps, sulfates, sulfonates, etc.; the co-solvents are selected from organic acids and their salts, amides and amines, inorganic salts, polyethylene glycol, glycerin, etc.; and the emulsifiers are selected from... The active ingredients are selected from Span derivatives, Tween derivatives, maltose derivatives, benzyl esters, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, astragalus gum, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxides, silica, bentonite, etc.; antioxidants are selected from sulfites, metabisulfites, bisulfites, ascorbic acid, gallic acid and its esters, etc.; preservatives are selected from parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols and volatile oils, etc.; pH adjusters are selected from hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, acetates, citric acid, citrates, etc.; isotonic or isotonic regulators are selected from glucose, sodium chloride, sodium citrate, sorbitol and xylitol, etc.

[0015] It is understood that the drugs involved in this invention can be prepared in different dosage forms based on different excipients, and correspondingly, the administration methods can also be diverse.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes in vitro simulated digestion technology of ginseng protein to obtain a ginseng peptide fragment library. Furthermore, through online virtual screening and in vitro cell model anti-inflammatory experiments, four ginseng peptide monomers with anti-inflammatory inhibitory activity were identified.

[0017] This invention further validated the ginseng peptides with potential anti-inflammatory activity obtained from the initial screening using an in vitro cell model anti-inflammatory experiment. It was found that four ginseng peptide monomers with amino acid sequences of DGGW, SHCGW, MSHCGW, and GGW have certain anti-inflammatory activities. Attached Figure Description

[0018] Figure 1 The process for discovering and identifying ginseng peptides with anti-inflammatory activity.

[0019] Figure 2 Chemical structure diagram of ginseng peptide DGGW monomer.

[0020] Figure 3Chemical structure diagram of ginseng peptide SHCGW monomer.

[0021] Figure 4 Chemical structure diagram of ginseng peptide MSHCGW monomer.

[0022] Figure 5 Chemical structure diagram of ginseng peptide GGW monomer.

[0023] Figure 6 Anti-inflammatory effects of ginseng peptide DGGW monomer.

[0024] Figure 7 Anti-inflammatory effects of ginseng peptide SHCGW monomer.

[0025] Figure 8 Anti-inflammatory effects of ginseng peptide MSHCGW monomer.

[0026] Figure 9 Anti-inflammatory effects of ginseng peptide GGW monomer. Detailed Implementation

[0027] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0028] Ginseng is the tuberous root and rhizome of a perennial herbaceous plant belonging to the Araliaceae family. It possesses various functional activities, including antioxidant, antitumor, antidepressant, anti-aging, hypoglycemic, and liver-protective effects. The pharmacologically active substances in ginseng include saponins, polysaccharides, peptides, sterols, proteins, amino acids, flavonoids, nucleotides, organic acids, and vitamins. Ginseng peptides are polypeptides derived from ginseng and possess physiological activities such as anti-inflammatory, hypoglycemic, lipid-regulating, anti-cell proliferation, and antioxidant effects. Although ginseng peptides are important bioactive substances in ginseng, research on them is limited, particularly regarding the discovery and identification of small-molecule monomeric peptides.

[0029] Based on this, the present invention provides a ginseng peptide with anti-inflammatory activity, wherein the amino acid sequence of the ginseng peptide is DGGW, SHCGW, MSHCGW, or GGW. The ginseng peptide provided by the present invention, or its respective repeating sequences, can be used to prepare anti-inflammatory products.

[0030] Example 1 A method for screening a novel ginseng peptide with anti-inflammatory activity using computer virtual technology includes the following steps: (1) Search the Uniprot protein database and screen out all verified ginseng proteins; (2) Using the bioinformatics tool Peptide Cutter, the digestive enzymes selected are pepsin, trypsin and chymotrypsin. In vitro digestion simulation is performed to digest the ginseng protein retrieved in step (1) into peptides to obtain a ginseng polypeptide fragment library. (3) The peptides obtained in step (2) were scored for bioactivity potential using the PeptideRanker tool, and peptides with a score greater than 0.9 were selected as candidate active peptides. (4) Submit the peptides selected in step (3) to the BIOPEP-UWM database and use the BIOPEP-UWM bioactivity prediction module to predict the anti-inflammatory activity of the peptides. (5) The ginseng peptides with anti-inflammatory activity selected in step (4) are sorted and synthesized to obtain ginseng peptides with anti-inflammatory activity.

[0031] The amino acid sequence of ginseng peptide is DGGW, SHCGW, MSHCGW, and GGW. The monomeric chemical structures of ginseng peptide represented by DGGW, SHCGW, MSHCGW, and GGW are as follows: Figures 2-5 As shown.

[0032] Experimental Example 1 Ginseng peptide DGGW anti-inflammatory activity test RAW264.7 cells in good logarithmic growth phase were taken and diluted to 1×10⁻⁶ cells. 5 Cell concentration / mL; Add 100 μL of cell dilution buffer to each well of a 96-well plate and incubate at 37°C and 5% CO2 for 24 h. Different amounts of DGGW peptide were added to multiple DMEM culture media containing 10% fetal bovine serum and 1% penicillin-streptomycin (standard commercial solution: 10,000 U / mL penicillin + 10,000 µg / mL streptomycin) to achieve DGGW peptide concentrations of 0, 3.75, 7.5, 15, 31.25, and 62.5 μg / mL. Different concentrations of DGGW peptide medium and 1µg / mL LPS were added to the above 96-well plates to stimulate cells. Each group had 3 replicates and the cells were cultured at 37℃ and 5% CO2 for 24 h. Mix 50 μL of cultured cell culture medium with an equal volume of Griess reagent and incubate at room temperature for 10 min. Measure the absorbance (OD) of the resulting reaction solution at 540 nm using a microplate reader. Calculate the NO inhibition rate (R) using the following formula. In the formula, OD2, OD1, and OD0 are the OD values ​​of the model group, the drug-treated group, and the blank control group, respectively.

[0033] The blank control group consisted of no treatment, the model group consisted of treatment with only 1 µg / mL LPS, and the positive control group consisted of dexamethasone (L-NMMA) instead of peptides. The IC50 was calculated using Graphpad software. 50 .

[0034] The results are as follows Figure 6 As shown, the anti-inflammatory and inhibitory effects were best when the peptide concentration reached 62.5 μg / mL, with an inhibition rate of 33.62%.

[0035] Experiment Example 2 The anti-inflammatory activity of ginseng peptide SHCGW was determined using the same method as in Example 1. The results are as follows: Figure 7 As shown, the anti-inflammatory and inhibitory effects were best when the peptide concentration reached 62.5 μg / mL, with an inhibition rate of 35.29%.

[0036] Experimental Example 3 The anti-inflammatory activity of ginseng peptide MSHCGW was determined using the same method as in Example 1. The results are as follows: Figure 8 As shown, the anti-inflammatory and inhibitory effects were best when the peptide concentration reached 62.5 μg / mL, with an inhibition rate of 43.10%.

[0037] Experiment Example 4 The anti-inflammatory activity of ginseng peptide GGW was determined using the same method as in Experimental Example 1. The results are as follows: Figure 9 As shown, the anti-inflammatory and inhibitory effects were best when the peptide concentration reached 62.5 μg / mL, with an inhibition rate of 41.10%.

[0038] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A ginseng peptide with anti-inflammatory activity, characterized in that, The amino acid sequence of the ginseng peptide is DGGW, SHCGW, MSHCGW, or GGW.

2. A method for screening ginseng peptides according to claim 1, characterized in that, Includes the following steps: All ginseng proteins were screened from the protein database; The screened ginseng protein was digested into peptides through in vitro digestion; The obtained peptides were scored for bioactivity potential using the PeptideRanker tool, and peptides with scores greater than 0.9 were selected. Peptides with a prediction score greater than 0.9 were screened for anti-inflammatory activity to obtain peptides with anti-inflammatory activity. Based on the anti-inflammatory activity experiment, the peptides obtained from the above screening were subjected to anti-inflammatory activity determination and verification, thus obtaining ginseng peptides with anti-inflammatory activity.

3. The use of the ginseng peptide of claim 1 in the preparation of anti-inflammatory products.

4. An anti-inflammatory agent, characterized in that, It includes any one or a combination of several of the ginseng peptides with amino acid sequences of DGGW, SHCGW, MSHCGW, and GGW as described in claim 1; or their respective repeating sequences.

5. The anti-inflammatory agent according to claim 4, characterized in that, The anti-inflammatory agent is a liquid preparation composed of the ginseng peptide and a culture medium. The culture medium is a DMEM medium containing fetal bovine serum and penicillin-streptomycin, and the volume fraction of fetal bovine serum in the culture medium is 10% and the volume fraction of penicillin-streptomycin is 1%.

6. The anti-inflammatory agent according to claim 5, characterized in that, The concentration of ginseng peptide in the anti-inflammatory preparation is 3.75 μg / mL to 62.5 μg / mL.

7. The anti-inflammatory agent according to claim 6, characterized in that, The concentration of ginseng peptide in the anti-inflammatory preparation is 62.5 μg / mL.

8. The anti-inflammatory agent according to claim 4, characterized in that, The anti-inflammatory preparation also includes acceptable excipients.

9. The anti-inflammatory agent according to claim 8, characterized in that, The anti-inflammatory agent is an oral or injectable preparation.

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